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• v.589(Pt 5); 2011 Mar 1

Neo-Darwinism, the Modern Synthesis and selfish genes: are they of use in physiology?

This article argues that the gene-centric interpretations of evolution, and more particularly the selfish gene expression of those interpretations, form barriers to the integration of physiological science with evolutionary theory. A gene-centred approach analyses the relationships between genotypes and phenotypes in terms of differences (change the genotype and observe changes in phenotype). We now know that, most frequently, this does not correctly reveal the relationships because of extensive buffering by robust networks of interactions. By contrast, understanding biological function through physiological analysis requires an integrative approach in which the activity of the proteins and RNAs formed from each DNA template is analysed in networks of interactions. These networks also include components that are not specified by nuclear DNA. Inheritance is not through DNA sequences alone. The selfish gene idea is not useful in the physiological sciences, since selfishness cannot be defined as an intrinsic property of nucleotide sequences independently of gene frequency, i.e. the ‘success’ in the gene pool that is supposed to be attributable to the ‘selfish’ property. It is not a physiologically testable hypothesis.

Denis Noble is Emeritus Professor of Cardiovascular Physiology in the Department of Physiology, Anatomy and Genetics at Oxford University. Fifty years ago he published the first mathematical model of the electrical activity of the heart based on experimental measurements of ion channels. This has since been developed into the virtual heart project within the Human Physiome Project of the International Union of Physiological Sciences (IUPS). He is currently the President of IUPS. He is author of The Music of Life (Oxford University Press, 2006), the first popular book on systems biology, now translated into seven foreign languages.

Introduction

Interpreting molecular genetic information in terms of higher level functions in the organism is a major current goal in the physiological sciences, as is the reverse strategy of bottom-up reconstruction: they complement each other. Computational systems biology is one of the tools being used ( Kohl & Noble, 2009 ; Hunter et al. 2011 ). Achieving this goal could also be a route through which physiology can reconnect with developmental and evolutionary biology. I will explain why some central aspects of neo-Darwinism (or the Modern Synthesis – in this article I am not always distinguishing between them), and their most popular expression in The Selfish Gene ( Dawkins, 1976, 2006 ), form a barrier to the new synthesis required between physiology and evolutionary theory. The barrier can be removed by taking an integrative, multilevel approach in which genes and many other components of organisms that are inherited are viewed as co-operating in networks to express what we call the phenotype ( Kohl et al. 2010 Fig. 2 , reproduced here as Fig. 1 below). In this paper, ‘co-operative genes’ carries this sense, which should be clearly distinguished from the idea of genes ‘for’ co-operative behaviour used widely in ecology, animal behaviour and economics. Attributes like ‘selfish’ and ‘cooperative’ have different meanings when applied to objects or ensembles at different levels. Cooperation at the level of protein networks, for example, may occur even if the organism in which they cooperate is ‘selfish’ at the level of the phenotype, and vice versa. The concept of level in evolutionary theory requires careful analysis ( Gould, 2002 ; Okasha, 2006 ). Concepts and mechanisms do not necessarily carry through from one level to another – an important point to bear in mind also in multi-level physiology.

The nucleus of a common carp, Cyprinus carpio (middle), was transferred into the enucleated egg cell of a goldfish, Carassius auratus (left). The result is a cross-species clone (right) with a vertebral number closer to that of a goldfish (26–28) than of a carp (33–36) and with a more rounded body than a carp. The bottom illustrations are X-ray images of the animals in the top illustration. Figure kindly provided by Professor Yonghua Sun from the work of Sun et al. (2005) .

This diagram represents the interaction between genes (DNA sequences), environment and phenotype as occurring through biological networks. The causation occurs in both directions between all three influences on the networks. This view is very different from the idea that genes ‘cause’ the phenotype (right hand arrow). This diagram also helps to explain the difference between the original concept of a gene as the cause of a particular phenotype and the modern definition as a DNA sequence. For further description and analysis of the ideas behind this diagram see Kohl et al. (2010) from which the diagram is reproduced. Reprinted by permission from Macmillan Publishers Ltd: Clinical Pharmacology and Therapeutics 88, 25–33; ©2010.

I start with a clarification of the relationship between neo-Darwinism, the Modern Synthesis and the selfish gene idea. Neo-Darwinism (a term introduced by the physiologist Georges Romanes (1883) ) and its development (see Pigliucci & Muller, 2010 a for the relevant history) into the Modern Synthesis ( Huxley, 1942 ) as a gene-centred view of evolution can of course be stated without reference to the selfish gene idea. Neo-Darwinism is the term popularly used, even today, for the synthesis between Darwin's theory of evolution by natural selection and the assumption that the variations on which selection acts are produced solely or primarily by gene mutations, though the term Modern Synthesis is more correct since Romanes coined the term neo-Darwinism before Mendel's work on genetics was rediscovered. The Modern Synthesis adds discrete (Mendelian) inheritance to neo-Darwinism. Alternatives to the Modern Synthesis include: symbiogenesis, the idea that major steps in evolution, such as the formation of eukaryotes and multicellular organisms, resulted from cooperation and/or fusion between different organisms; horizontal gene transfer within and between organisms ( Woese & Goldenfeld, 2009 ; Goldenfeld & Woese, 2011 ), a process now known to extend beyond prokaryotes ( Keeling & Palmer, 2008 ); and the inheritance of acquired characteristics, commonly but mistakenly ( Noble, 2010 b ) called ‘Lamarckism’. For further examples see Pigliucci & Muller (2010 a , particularly their Fig. 1.1; 2010 b ) and Jablonka & Lamb (2005) .

In the rest of this article reference to neo-Darwinism should be taken to include the Modern Synthesis. The selfish gene idea ( Dawkins, 1976, 2006 ) is a popularization of neo-Darwinism which goes beyond it to characterise genes as elements in organisms with specific (selfish) behaviour. As we will see later, it was originally formulated as a literal scientific hypothesis. The question of its status is a major focus of this paper.

Another way of stating the claims of this article is that they are twofold: first, that neo-Darwinism is, at the least, incomplete as a theory of evolution. Second, that the selfish gene idea adds nothing since it is essentially empty. These are separate claims, even though in the minds of many biologists neo-Darwinism and the selfish gene idea are not always clearly distinguished. Neo-Darwinism is capable of falsification. Indeed, in its original form as a complete theory, it has already been falsified. We now need to admit processes outside its remit, so that it needs to be extended ( Woese & Goldenfeld, 2009 ; Pigliucci & Muller, 2010 b ). As I will show in this paper, the selfish gene idea is not even capable of direct empirical falsification; it has to be judged by different criteria.

The concept of a gene has changed, and is still changing, so what version do we use?

A serious problem in assessing the nature and utility of the selfish gene story in physiological research is that the concept of a gene has changed (see Fig. 1 ) in fundamental ways ( Pichot, 1999 ; Keller, 2000 ; Beurton et al. 2008 ). We are dealing with a moving target. From being the (hypothetical allelic) cause of each phenotype character, such as eye colour or number of limbs, the developments in molecular biology have led to its being defined more narrowly and specifically as a DNA sequence that is used by the cell as a template for the synthesis of a protein or RNA. These are not at all the same thing when it comes to questions like ‘what do genes do?’ and ‘what kind of causation is involved?’ When Johannsen (1909) introduced the term ‘gene’ it was defined as the (necessary) cause of a phenotype, since it was defined as an inherited phenotype that could be attributed to an allele. But now it has to be shown to be a cause, and the nature of that causation needs clarification. The full implications of this difference are explained elsewhere ( Noble, 2008 ). They are reinforced by the fact that most changes at the level of DNA do not have a measurable phenotypic effect under normal physiological conditions (see, for example, Hillenmeyer et al. 2008 ). By the original definition, these would not even have been identified as genes, since a gene was an entity that necessarily had a phenotypic manifestation.

In this article, I frequently refer to the selfish gene idea as a story since one of the questions I am addressing is whether it is more than a story or viewpoint. Colourful metaphorical stories can be highly influential: no-one can deny that the selfish gene idea has had a huge impact on the way in which both lay people and scientists view genetics, including the social implications ( Midgley, 2010 ). Most of the time, people accept its implied scientific basis. It is important therefore to ask whether the idea could be interpreted as an empirical scientific hypothesis, particularly since Dawkins's own initial interpretation was that it was not metaphorical; in reply to Midgley (1979) he wrote: ‘that was no metaphor. I believe it is the literal truth, provided certain key words are defined in the particular ways favoured by biologists’ ( Dawkins, 1981 ). But a metaphor does not cease to be a metaphor simply because one defines a word to mean something other than its normal meaning. Indeed, it is the function of metaphor to do precisely this. So, we must first clarify what the idea means.

Is the ‘selfish gene’ story metaphor or empirical science or both?

Genes, as DNA sequences, do not of course form selves in any ordinary sense. The DNA molecule on its own does absolutely nothing since it reacts biochemically only to triggering signals. It cannot even initiate its own transcription or replication. It cannot therefore be characterised as selfish in any plausible sense of the word. If we extract DNA and put it in a Petri dish with nutrients, it will do nothing. The cell from which we extracted it would, however, continue to function until it needs to make more proteins, just as red cells function for a hundred days or more without a nucleus. It would therefore be more correct to say that genes are not active causes; they are, rather, caused to give their information by and to the system that activates them. The only kind of causation that can be attributed to them is passive, much in the way a computer program reads and uses databases. The selfish gene idea therefore has to be interpreted not only as a metaphor, but as one that struggles to chime with modern biology. That is where the difficulties begin.

Ideas that incorporate or are based on metaphors have a very different relationship to empirical discovery than do standard scientific hypotheses with clear empirical consequences that ensure their falsifiability. There are several ways in which this is evident.

First, different or even opposing metaphors can both be ‘true’. This is because metaphors highlight different aspects of the target to which they are applied, a fact that has long been familiar to metaphor theorists ( Lakoff & Johnson, 1980 ; Kittay, 1987 ). Metaphors can correspond to different, even incompatible, aspects of reality. That is why, when comparing ‘selfish’ genes with ‘prisoner’ or ‘cooperative’ genes, as I do in chapter 1 of The Music of Life ( Noble, 2006 ), there is no empirical test that will unequivocally show which is correct, a point which was conceded long ago by Richard Dawkins at the beginning of his book The Extended Phenotype : ‘I doubt that there is any experiment that could prove my claim’ ( Dawkins, 1982 , p. 1). This point is analogous to the sense in which no experiment could ever disprove a geometry, whether Euclidean or not ( Poincaré, 1902, 1968 ). Significantly, Dawkins uses a geometric illusion (the Necker Cube) to illustrate his point.

( The Extended Phenotype was an even stronger statement of the selfish gene idea since it argued that “the phenotypic effects of a gene…may extend far outside the body in which the gene sits” ( Dawkins, 1982 , p. vi) Even effects “at a distance” are seen as being “for the benefit” of the selfish gene.)

Second, metaphors often appear circular if interpreted like a scientific theory. I will show that the selfish gene metaphor shows this circularity.

Finally, even though there may be no single empirical fact that will distinguish between very different metaphors, this does not mean that empirical discovery has no impact on our choice of metaphor. The relationship is more nuanced than it may be for most scientific theories. It will usually require a judgment based on a large set of empirical facts to arrive at a conclusion. Much of the meaning associated with metaphorical statements is determined by viewpoints that are a matter of personal choice, even though influenced by empirical facts. I will illustrate this later in this paper.

What does ‘selfish’ mean in the selfish gene story?

First we must decide whether ‘selfish’ defines a property that is universal to all genes (or even all DNA sequences) or whether it is a characteristic that distinguishes some DNA sequences from others. This is not as easy as it may seem. I suspect that the original intention was that all genes could be represented as ‘seeking’ their own success in the gene pool, regardless of how effective they might be in achieving this. One reason for thinking this is that so-called junk DNA is represented in the selfish gene story as an arch-example of selfishness: hitching a ride even with no function.

But on that interpretation, the demonstration that the concept is of no utility in physiological science is trivially easy. Interpreted in this way, a gene cannot ‘help’ being selfish. That is simply the nature of any replicator. But since ‘selfishness’ would not itself be a difference between successful and unsuccessful genes (success being defined here as increasing frequency in the gene pool), nor between functional and non-functional genes, there would be no cashable value whatsoever for the idea in physiology. Physiologists study what makes systems work. It matters to us whether something is successful or not. Attributing selfishness to all genes therefore leaves us with nothing we could measure to determine whether ‘selfishness’ is a correct attribute. As metaphor, it may work. But as a scientific hypothesis it is empty.

Could we rescue the idea for physiological science? I doubt whether anyone would want to do that ab initio , but we live in a scientific culture that is now thoroughly permeated by the idea, and in a way that has strongly disfavoured physiology. The idea has either to be rejected or assimilated. One option would be to re-interpret selfishness to include reference to effectiveness. We could, for example, say that genes whose numbers of copies increase are selfish, or more selfish than their competitors. This move would give us an empirical handle on the idea.

It is a standard move in science to unpack a metaphor or simile in this way. Physicists make similar moves when they give empirical criteria for black holes, quarks, strings and many other strange new entities in their theories. Without an empirical handle they might as well not exist. Indeed, one of the arguments about string theory, for example, is precisely whether it has satisfied this fundamental criterion.

Moreover, including reference to effectiveness, which in evolutionary theory could be interpreted to be fitness, is surely the most relevant way to gain empirical leverage. We can measure changes in gene copies in a population. Now the question becomes whether we can develop the theory a bit further to become predictive. What, in a gene, could tell us whether or not it is selfish in this sense?

On the original definition of a gene as a hypothetical cause of a particular phenotype, this would have been fairly straightforward. We could look, at the functional level of the phenotype, for the reasons why a particular function would be adaptive. This is in practice what defenders of the selfish gene idea do. They refer to the gene (more strictly an allele) as ‘the gene for’ X or Y, where these are functional, phenotype characters. The phenotype view creeps back in through the terminology. Any ‘selfishness’ lies at least as much in the phenotype as in the genes.

But since we now define genes as particular DNA sequences, what in a DNA sequence could possibly tell us whether or not it is selfish? The answer is obvious: the sequences of Cs, Gs, As and Ts could never, by themselves, give us a criterion that would enable us to predict that the frequency of that sequence will increase in the gene pool. A DNA sequence only makes sense in the context of particular organisms in which it is involved in phenotypic characteristics which can be selected for. A sequence that may be very successful in one organism and/or environment, might be lethal in another. This is evident in the fact that almost all cross-species clones do not form an adult (see later for an important exception). The same, or similar, DNA sequence may contribute to different, even unrelated, functions in different species. The sequence, intrinsically, is neutral with regard to such functional questions.

The price therefore of giving the selfish gene idea some empirical leverage is to reveal yet again, though in a different way, that it is an empty hypothesis. There is no criterion independent of the only prediction that the hypothesis makes, i.e. that selfish genes increase their number. It is a strange hypothesis that uses its own definition of its postulated entity as its only prediction.

At this point, I suspect that a defender of the concept would shift back to referring to genes as hypothetical entities, defined as the cause(s) of particular phenotypes. Note, though, that this is to abandon the purely ‘genes-eye’ view since it shifts the focus back to the phenotype. As a physiologist, naturally I would say ‘so it should’. I will discuss the consequences of that shift in a later section.

How is the selfish gene story related to the central dogma?

In one of the central paragraphs of The Selfish Gene (page 21), Dawkins writes:

Now they swarm in huge colonies, safe inside gigantic lumbering robots, sealed off from the outside world, communicating with it by tortuous indirect routes, manipulating it by remote control. They are in you and me; they created us, body and mind; and their preservation is the ultimate rationale for our existence.

The phrase ‘sealed off from the outside world’ is a colourful statement of the idea that genes are uninfluenced by their environment, a view that was strongly buttressed by the central dogma of molecular biology, originally formulated by Crick (1958 , 1970) and taken to exclude information flow other than from genes to proteins. In fact, of course, what the molecular biology showed was simply that amino acid sequences are not used as templates for forming nucleic acid sequences. The unjustified extension was to think that information cannot pass from proteins to nucleic acids, whereas this is precisely what must happen for genes to be activated and for expression patterns to be formed. This extension (which can be seen in phrases like “the inheritance of instructively acquired adaptation would violate the ‘central dogma’ of embryology” ( Dawkins, 1982 , p. 173) was a godsend to the neo-Darwinists since it provided a basis, right down at the level of DNA itself, for regarding genes as ‘sealed off’ from the outside world. The original experimental basis for this idea was the Weismann (1893) barrier.

A godsend, except that it is not correct in the relevant sense, and never has been. Even at the time the dogma was formulated, it was sufficient to ask the question how do different cells in the body, with exactly the same genome, end up as different as bone cells and heart cells? The answer of course is that the way in which the genome is read leads to completely different patterns of gene expression. This requires flow of information onto the genome itself, which, as Barbara McClintock (1984) said, should be regarded as an ‘organ of the cell’, not its dictator. There are feedbacks and restraints, not only between the products of the genes (which might be consistent with a genes-eye view), but right down onto the genome itself, determining when, where and how much of each gene product is formed. As Beurton et al. (2008) comment ‘it seems that a cell's enzymes are capable of actively manipulating DNA to do this or that. A genome consists largely of semistable genetic elements that may be rearranged or even moved around in the genome thus modifying the information content of DNA.’

The central dogma, as a general principle of biology, has therefore been progressively undermined. The only aspect of it still left intact is its original strictly chemical sense, i.e. that protein sequences are not used as templates for forming DNA or RNA sequences. All other aspects of the way in which the dogma has been extended to buttress neo-Darwinism have been deconstructed – by molecular biology itself. Shapiro's (2009) article is the best account of the demolition from a biochemical viewpoint, while Werner (2005) does so from an informatics perspective.

Are genes the only immortals?

A central distinction in the selfish gene story is that between replicators and vehicles. The distinction is based on considering inheritance only of changes . While the vehicle is also ‘inherited’ (genes on their own do nothing and certainly are not sufficient to ‘make’ an organism – since we must also inherit a complete fertilised egg cell), the story goes that changes in the vehicle are not inherited (so no inheritance of acquired characteristics) while changes in the replicator (e.g. mutations) are inherited. This approach is what enables the wholesale inheritance of the vehicle to be ignored.

Yet, the vehicle (the cell, or each cell in a multicellular organism) clearly does reproduce (indeed, it is only through this reproduction that DNA itself is transmitted), and in doing so it passes on all the phenotype characteristics for which there are no nuclear DNA templates and which are necessary to interpret the inherited DNA. An obvious example is the transmission of mitochondria, chloroplasts and other organelles, which almost certainly originated as symbionts (‘invading’ or ‘engulfed’ bacteria) at an early stage of evolution when eukaryotes were first formed. Many other transmitted cytoplasmic factors also exist ( Sun et al. 2005 ; Maurel & Kanellopoulos-Langevin, 2008 ). All these replicate and, in the selfish gene story would have to be given the status of ‘honorary genes’.

The existence of such cellular inheritance requires the selfish gene theory to distinguish between replication and reproduction. The next step in the story is to claim that replicators are potentially immortal, whereas reproducers are not.

Biologically speaking, this is evident nonsense. Through germline cells I am connected via many reproductions to the earliest cells, even to those without genomes . In some sense, the cell as a whole has achieved at least equivalent immortality to that of its DNA. Cells, even those without genomes in the postulated pre-DNA world of RNA enzymes ( Maynard Smith & Szathmáry, 1999 ), clearly reproduce themselves, and in doing so they also pass on any differences among them ( Sonneborn, 1970 ; Sun et al. 2005 ). Any difference between replication and reproduction (which, after all, are just synonyms; the distinction is a linguistic confusion) does not entitle one to say that one is immortal and the other is not. What were all those cells without genomes doing in early life on earth? We wouldn't be here to tell the story if they did not also form an ‘immortal line’. As I have argued elsewhere ( Noble, 2008 ) the main difference between DNA and non-DNA inheritance is simply that one is digital, the other analog. In developing the organism the 3D analog information is just as necessary as the 1D digital (DNA) information. Neither is sufficient by itself. They are mutually dependent. The amount of analog information can also be calculated to be comparable to that of the genome ( Noble, 2011 ). Moreover, organisms are not in fact digital machines ( Shapiro, 2005 ; Noble, 2010 a ).

The genetic differential effect problem

Clearly, many of the problems with the selfish gene story arise from unusual or imprecise use of the language of genetics, leading to untestable ideas. Another central muddle, both in neo-Darwinism and in the selfish gene story, is what I have called ‘The genetic differential effect problem’ ( Noble, 2008 , 2011 ), the idea that genetics is only about differences. This view is now unsustainable, since defining genes as DNA sequences clearly does identify a specific chemical entity whose effects are not merely attributable to differences in the sequence. We can say precisely for which proteins or RNAs the sequence acts as a template and analyse the physiological effects of those proteins or RNAs. The arguments for abandoning the difference perspective are overwhelming (see also Longo & Tendero, 2007 ).

Differences in DNA do not necessarily, or even usually, result in differences in phenotype. The great majority, 80%, of knockouts in yeast, for example, are normally ‘silent’ ( Hillenmeyer et al. 2008 ). While there must be underlying effects in the protein networks, these are clearly buffered at the higher levels. The phenotypic effects therefore appear only when the organism is metabolically stressed, and even then they do not reveal the precise quantitative contributions for reasons I have explained elsewhere ( Noble, 2011 ). The failure of knockouts to systematically and reliably reveal gene functions is one of the great (and expensive) disappointments of recent biology. Note, however, that the disappointment exists only in the gene-centred view. By contrast it is an exciting challenge from the systems perspective. This very effective ‘buffering’ of genetic change is itself an important systems property of cells and organisms.

Moreover, even when a difference in the phenotype does become manifest, it may not reveal the function(s) of the gene. In fact, it cannot do so, since all the functions shared between the original and the mutated gene are necessarily hidden from view. This is clearly evident when we talk of oncogenes. What we mean is that a particular change in DNA sequence predisposes to cancer. But this does not tell us the function(s) of the un-mutated gene, which would be better characterised in terms of its physiological function in, e.g., the cell cycle. Only a full physiological analysis of the roles of the protein it codes for in higher-level functions can reveal that. That will include identifying the real biological regulators as systems properties. Knockout experiments by themselves do not identify regulators ( Davies, 2009 ).

So, the view that we can only observe differences in phenotype correlated with differences in genotype both leads to incorrect labelling of gene functions and falls into the fallacy of confusing the tip with the whole iceberg. We want to know what the relevant gene products do in the organism as a physiological whole, not simply by observing differences. Remember that most genes and their products, RNA and proteins, have multiple functions.

To see the poverty of the view that we can only observe differences, just ask the question what engineer would be satisfied simply to know the difference between the cement he used this time to construct his building compared to what he used previously, or to know just the differences between two electronic components in an aircraft? Of course, he might use the difference approach as one of his experimental tools (as genetics has in the past, to good effect), but the equations and models of an engineer represent the relevant totality of the function of each component of a system. So does physiological analysis of function, which is why physiology cannot be restricted to the limitations of the ‘difference’ approach.

Second, accurate replication of DNA is itself a system property of the cell as a whole, not just of DNA. DNA on its own is an extremely poor replicator. It requires a dedicated set of proteins to ensure correction of transcription errors and eventual faithful transmission. Both in ensuring faithfulness of DNA replication and in creating robustness against genetic defects, systems properties are the important ones. The cell as a whole ‘canalises’ the way in which DNA is interpreted, making it robust and reproducible. The famed ‘immortality’ of DNA is actually a property of the complete cell.

The distinction between replicator and vehicle is therefore out of date from a physiologist's viewpoint. It stems from the original ‘genetic program’ idea, in which organisms are viewed as Turing machines with the DNA being the digital tape of the computer (tape–computer is much the same distinction as replicator–vehicle – this was the basis of Jacob and Monod's concept of the ‘genetic program’; Jacob, 1970 ). Organisms are interaction systems, not Turing machines ( Shapiro, 2005 ; Noble, 2008 ). There is no clear distinction between replicator and vehicle ( Coen, 1999 ).

Finally, the story implies that the ‘vehicles’ do not themselves evolve independently of their DNA. There is no reason why this should be true. In fact it is certainly false. Egg cells from different species are different. So much so that cross-species hybrids using nuclear transfer usually do not survive, and those that do, as in the elegant experiments of Sun et al. (2005) – see Fig. 2 – transferring nuclei between different fish species, reveal precisely the influence of the species-specific cytoplasmic factors on development (see also Jaenisch, 2004 ; Yang et al. 2007 ). Crossing a common carp nucleus with a goldfish enucleated egg cell produces an adult fish that has an intermediate shape and a number of vertebrae closer to that of the goldfish. These factors can therefore determine a phenotype characteristic as fundamental as skeletal formations. Over 50 years ago, McLaren & Michie (1958) showed a similar phenomenon as a maternal effect in mice. The number of tail vertebrae (4 or 6 in the different strains) was determined by the surrogate mother, not the embryo. Of course, such cytoplasmic influences are dependent on the DNA of the mother, but these influences will necessarily include patterns of gene expression that are also dependent on other influences. There is interplay here between DNA and non-DNA inheritance, as there must always be. Moreover, maternal and paternal effects in response to the environment have been shown to be transmitted down two generations (grandparents to grandchildren) in humans ( Pembrey et al. 2006 ) and could therefore be a target for natural selection.

Conclusions

As physiological and systems biological scientists, we need to reconnect to evolutionary theory. It was difficult to do this during most of the 20th century because the neo-Darwinist synthesis more or less excluded us, by relegating the organism to the role of a disposable vehicle. It also, unjustifiably, excluded Lamarck ( Noble, 2010 b ). Darwin himself was not so sure; in the first edition of The Origin of Species ( Darwin, 1859 ) he wrote ‘I am convinced that natural selection has been the main, but not the exclusive means of modification’, a statement he reiterated with increased force in the 1872, 6th edition. As many evolutionary biologists now acknowledge, the Modern Synthesis (neo-Darwinism) requires extending ( Jablonka & Lamb, 2005 ; Pigliucci & Muller, 2010 b ).

If physiology is to make the contribution it should to the fields of evolution and development, we need to move on from the restrictions of the differential approach. The integrative approach can achieve this by reverse engineering using computational modelling, as I have shown elsewhere ( Noble, 2011 ). The genes-eye view is only one way of seeing biology and it doesn't accurately reflect much of what modern biology has revealed. In fact, its central entity, the gene, ‘begins to look like hardly definable temporary products of a cell's physiology’ ( Beurton et al. 2008 ).

Finally, I want to return to the role of metaphor and the selfish gene idea.

When I first read Richard Dawkins's acknowledgement in The Extended Phenotype (‘I doubt that there is any experiment that could be done to prove my claim’) I was strongly inclined to agree with it (both in relation to the original selfish gene idea and its development in The Extended Phenotype ) since, if you compare the selfish gene metaphor with very different metaphors, such as genes as prisoners, it is impossible to think of an experiment that would distinguish between the two views, as I argued earlier in this paper. For any given case, I still think that must be true. But I have slowly changed my view on whether this must be true if we consider many cases, looking at the functioning of the organism as a whole. There are different ways in which empirical discovery can impact on our theoretical understanding. Not all of these are in the form of the straight falsification of a hypothesis, a point that has been well-understood in theoretical physics for many years ( Poincaré, 1902, 1968 ). Sometimes it is the slow accumulation of the weight of evidence that eventually triggers a change of viewpoint. This is the case with insights that are expressed in metaphorical form (like ‘selfish’ and ‘prisoners’), and that should not be intended to be taken literally. The first mistake of the differential approach was to interpret the selfish gene idea as literal truth. It is clearly metaphorical metaphysics, and rather poor metaphysics at that since, as we have seen, it is essentially empty as a scientific hypothesis, at least in physiological science. But in social evolution also, the idea is simply one of several viewpoints that can account for the same data ( Okasha, 2010 ).

The weight of evidence in the physiological sciences is now much more favourable to the metaphor of ‘co-operation’ than of ‘selfishness’. Gene products all co-operate in robust networks one of whose functions is precisely to insulate the organism from many of the vagaries of gene mutation, and stochasticity at lower levels. Investigating these networks and their mechanisms is the way forward.

It is therefore time to move on and remove the conceptual barriers to integrating modern physiological science with evolutionary and developmental theory. The integrative approach can achieve this since it avoids the simplistic fallacies of the gene-centred differential approach and it is essentially what successful systems physiology has employed for many years.

Further reading

This article has been written for a physiological readership that may not be very familiar with the current debates in evolutionary and genetic theory. If you learnt evolutionary biology and genetics a decade or more ago you need to be aware that those debates have moved on very considerably, as has the experimental and field work on which they are based. Amongst the references cited, the following may help the reader to catch up: Margulis (1998) ; Jablonka & Lamb (2005) ; Noble (2006) ; Okasha (2006) ; Beurton et al. (2008) ; Shapiro (2009) ; Pigliucci & Müller (2010 b ) . For those interested in the philosophical and social impacts of the metaphors used, Midgley (2010) gives a very readable account.

Acknowledgments

I should like to acknowledge long and deep discussions with the organisers of the Balliol College, Oxford seminars on conceptual foundations of Systems Biology: Jonathan Bard, Tom Melham and Eric Werner; and the organisers and participants of the ‘Homage to Darwin’ debate ( http://www.voicesfromoxford.com/homagedarwin_part1.html ) held in Oxford in May 2009: Stephen Bell, Martin Brasier, Richard Dawkins and Lynn Margulis. I received criticism of early drafts of this paper from David Vines, David Cleevely, Nicholas Beale and Stig Omholt. I also acknowledge discussions with Peter Kohl, Ray Noble and James Shapiro. Providing valuable input and feedback does not of course signify assent to the claims of my paper. I consulted on a wide range of opinion. Work in the author's laboratory is funded by the PreDiCT project of the European Union under FP7.

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Neo-Empiricism

Barsalou, Glenberg, Prinz, Damasio and other neo-empiricists have theoretically and experimentally challenged the once dominant view that representations in higher cognitive processes are amodal (eg, Fodor, Pylyshyn). They have renewed a  century-old perspective on the mind, according to which representations in perceptual processes and representations in higher cognitive processes are, if not identical, at least qualitatively similar. Additionally, they have proposed empirical evidence for their views.

In my mind, neo-empiricism is a positive development, if only because it invites skeptics and proponents of amodal theories of the mind to specify their views and to provide evidence for them.

However, I have criticized at length neo-empiricism in two recent papers (also available on my site ):

Machery. 2006 Two dogmas of neo-empiricism. Philosophy Compass 1:398-412. Machery. In press Neo-empiricism: A methodological critique . Cognition (Barsalou is supposed to reply to this paper).

I think that there are at least three problems with neo-empiricism:

• First, there is a theoretical question of distinguishing amodal representations from modal representations.
• Second, there are several methodological issues in neo-empiricists’ experimental work (see particularly my Cognition paper). I have identified three main issues. In brief,
• It is impossible to sort out the predictions made by neo-empiricism in general and the predictions made by amodal theories in general; Rather, specific amodal and specific neo-empiricist models of cognitive processes make specific predictions;
• It is necessary to find some tasks that are not obviously best solved by using perceptual representations; Neo-empiricist psychologists have too often focused on tasks that are maybe best solved by means of perceptual representations;
• Psychologists must be cautious in generalizing some findings about some processes in some domains to other processes and other domains; the issue might not be whether representations are amodal or perceptual, but which processes in which domains in which contexts use amodal/perceptual representations.
• Third, there is a large body of evidence for amodal representations used in higher cognitive processes (eg, the representations of cardinality).

I think that issues of this kind are fruitfully dealt with by philosophers. Philosophers who are interested in these theoretical and methodological issues can have a real impact on the development of psychology.

11 Comments

Your point number 3 is especially good (about the need for caution in generalizing from one particular task).

Operationally, is an amodal representation different from a multimodal representation? To the point, could purported evidence for amodal representations be interpreted as supporting a multimodal representation?

I’d be curious to know more about the motivation for neo-empiricism. As I understand things, classical empiricism was motivated by epistemological considerations: establishing the foundations of knowledge in experience. But by now, I suppose this is no longer a motivation for empiricism in psychology (e.g., because we have abandoned the myth of the given and believe that knowledge may be justified in experience even if not all representations are perceptual, etc.). So what’s the motivation for resurrecting empiricism in psychology? Empirical evidence?

I ask because having being raised on a Chomskian diet, I find empiricism prima facie empirically implausible.

Eric writes: “Operationally, is an amodal representation different from a multimodal representation?”

I suppose that by “multimodal representation”, you have in mind those neurons or brain areas that are activated by the inputs belonging to different modalities (e.g., the left inferior parietal sulcus).

To counter the idea that the existence of these multimodal neurons or brain areas is evidence for amodal representations, Jesse Prinz has argued that amodal representations should be distinguished from multimodal representations. Multimodal representations are a kind of modal representations. (Claim 1)

At the same time, Jesse characterizes modal representations as being those representations that belong to a perceptual system. Additionally, he proposes a definition of a perceptual system that does not hang on the notion of a modal representation (Claim 2). (Barsalou has another definition, based on the contrast between linguistic representations and analog representations. I have argued that Barsalou’s way of drawing the distinction between amodal and modal representations won’t do.)

I might be that Jesse can’t have it both ways. Suppose that Jesse sticks to claim 1. Then, the location of these multimodal neurons and brain areas is such that it is not the case that representations are modal by virtue of beloning to a perceptual system, given Jesse’s definition of a modal system. But if Jesse sticks to claim 2, then the multimodal representations are not modal after all, given their location.

I note that Dan Weiskopf discusses Prinz’ reply in a short, unpublished paper.

As far as I am concerned, I think that being multimodal is evidence for amodality, though not an operational definition.

Gualtiero wonders about the *motivation* behind neo-empiricism. I suspect that this varies from psychologist to psychologists and from philosopher to philosopher.

A motivation is that at least some psychologists (e.g., Glenberg) believe that neo-empiricism can solve what is called “the grounding problem”–which is, in fact, the problem of providing a semantics for mental representations. I think that this approach leads to a naive empiricist semantics. (I note that this is not Jesse’s view.)

Another motivation is a growing body of evidence that prima facie suggests that our perceptual systems are used in numerous tasks that were believed to involve amodal representations. Barsalou’s empirical work illustrates this approach.  My discussion of Barsalou targets this motivation. I tend to believe that most of his (very interesting) findings are predicted by some amodal theories of representations together with some models of the processes defined over these representations (see the Cognition paper).

Another naive question: what do these neo-empiricists say about language learning and linguistic cognition, not to mention abstract concepts (such as justice and truth) and mathematics? What do they say about the poverty of the stimulus arguments?

Not all neo-empiricist  endorse the anti-nativism of their forerunners. Barsalou is very happy to endorse innate perceptual representations. Prinz, on the other hand, is a staunch anti-nativist.

What makes them empiricist is their commitment (1) to the view that conceptual representations and perceptual representations are not qualitatively distinct and (2) to the view that cognitive processes are defined over perceptual representations.

Now, it remains unclear how perceptual representations could be sufficient to account for syntactic understanding, if anything like the Comskyan account (use of a tacitly know grammar to parse sentences) is correct. Neo-empiricists usually have little to say about this.

They have more to say about abstract concepts. They recognize that it is a challenge for their views. They typically propose that entertaining an abstract concept consists in entertaining a complex, dynamic set of perceptual representations. It is however striking that most of their examples of such sets of perceptual representations are much too thick-grained to individuate abstract concepts. Typically, their examples could correspond to numerous abstract concepts (see my Two dogmas of neo-empiricism).

Concept empiricists don’t say much about language acquisition, although Jesse does gesture hopefully towards recent data suggesting that children are exposed to a wider array of constructions than nativists have traditionally supposed, as well as towards statistical (e.g., connectionist) models of language acquisition. I don’t see that a concept empiricist needs to believe an empiricist language-learning story, though. It’s a strange feature of neo-empiricism that, if language is a perceptual system (as many maintain), there can be lots of ‘abstract-looking’ representations available to form thoughts with, since concepts can potentially be composed from any perceptual vehicles.

On abstract concepts, there are a range of strategies:

(i) For logical concepts: these might be operations, not representations; so, e.g., a P v Q thought might be a P thought and a Q thought that stand in a certain functional relation to one another (and to other thoughts), rather than containing an abstract logical constituent.

(ii) For mathematical concepts: these might be representations of concrete numerals, or abstract ‘quantity’ mechanisms (e.g., accumulators), following Dehaene’s _Number Sense_ view.

(iii) For ‘lofty’ concepts: these are either images that are associated with their referents (e.g., polling booths for DEMOCRACY), or linguistic vehicles (e.g., the word ‘democracy’).

A major difference between neo-empiricists and classical ones is in the theory of content. Informational semantics allows us to refer to abstract properties no matter what sort of vehicles we use to do so. So all empiricists usually do is find some reasonably plausible candidate vehicle (a word, a numeral, an image of an associated perceivable scene), and claim that it realizes the abstract concept.

One problem with this approach is that while it shows how possibly these concepts might be perceptual, there often isn’t evidence that these images and words are actually used in thinking about these categories. (This is in contrast to the excellent work on concrete object concepts, where there is such evidence.) So insofar as it just shows ‘how possibly’, it’s not as compelling.

Also, I’d add that the linguistic vehicles strategy strikes me as pretty implausible. It’s not enough to use the _noise_ ‘democracy’ to think about democracy. That noise could stand for lots of things (compare genuinely ambiguous terms like ‘bank’). You might represent democracy as something _called_ ‘democracy’, but this involves the concept of being called something. That’s an intentional notion, hence another abstract concept. Looking at the details, it’s pretty hard to see how to make this linguistic strategy work within the confines of empiricism.

why do you think that (ii) is of any help for neo-empiricists? Dehaene and colleagues think of the representation of numerosity as amodal , though analog, representations . The main reason is that these representations are activated by the inputs to different modalities.

It is true that Jesse does not endorse the semantics developed by the traditional empiricists such as Hume. However, many psychologists do, eg. Glenberg. Barsalou is unclear on this issue.

I don’t think that Dehaene’s account is really a boon to empiricists. I think Jesse’s presentation of it fudges a bit. He notes that there is some analog mechanism for representing quantity non-linguistically, but proposes that this can be thought of as a _visual_ representation, e.g., a number line (presumably an unmarked one). Jesse suggests that number lines give analogical ways of understanding quantity. Of course, as you point out, this is different from Dehaene’s interpretation of the results, on which the accumulator is amodal.

For over 30 years I have published empirically supported arguments for a multimodal dual coding theory of cognition as compared to abstract, amodal (single code) theories like Pylyshyn’s. As early as 1983 I summarized over 60 empirical findings that were consistent with predictions from dual coding but not single code theories. There are now many more such findings, especially ones from brain studies showing functional dissociations between modality specific perceptual and memory representations. Comprehensive reviews of the phenomenal domains covered by the empirical-theoretical dual coding approach are available in several books, the most recent being: A. Paivio (2007). Mind and its evolution: A dual coding theoretical approach. Mahwah, NJ: Lawrence Erlbaum Associates.

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Experimental Philosophy

Experimental philosophy is an interdisciplinary approach that brings together ideas from what had previously been regarded as distinct fields. Specifically, research in experimental philosophy brings together two key elements:

• the kinds of questions and theoretical frameworks traditionally associated with philosophy;
• the kinds of experimental methods traditionally associated with psychology and cognitive science.

Though experimental philosophy is united by this broad approach, there is a diverse range of projects in experimental philosophy. Some use experimental evidence to support a “negative program” that challenges more traditional methods in analytic philosophy, others use experimental data to support positive claims about traditional questions, and still others explore questions about how people ordinarily think and feel insofar as these questions are important in themselves.

This entry provides a brief introduction to the core aims of contemporary experimental philosophy. It then reviews recent experimental work on the negative program, free will, moral judgment and epistemology. We conclude with a discussion of major objections to the field of experimental philosophy as a whole.

1. Overview

2.1.1 the argument from diversity, 2.1.2 the argument from sensitivity, 2.2 free will and moral responsibility, 2.3 impact of moral judgment, 2.4 epistemology, 2.5 other topics, 3.1.1 philosophers don’t rely on intuitions, 3.1.2 philosophers shouldn’t rely on intuitions, 3.2 defending privileged intuitions rather than those of ordinary experimental participants, 3.3 but is it philosophy, other internet resources, related entries.

Experimental philosophy is a relatively new approach, usually understood as beginning only in the early years of the 21st century. At the heart of this new approach is the idea of pursuing philosophical questions using methods more typically associated with the social sciences.

Within the broad banner of experimental philosophy research, one finds work using an enormous variety of methods and aims (see, e.g., Schwitzgebel & Rust 2014; Meskin et al. 2013; Bartels & Urminsky 2011). Nonetheless, most research in experimental philosophy makes use of a collection of closely connected methods that in some way involve the study of intuitions. The remainder of this section aims to characterize the different projects experimental philosophers have pursued using these methods and their relevance for broader questions in philosophy.

The practice of exploring intuitions has its origins in a more traditional philosophical approach that long predates the birth of experimental philosophy (see the entry on intuition ). Research within this more traditional approach often relies on the idea that we can make progress on one or another topic by looking at intuitions about that topic. For example, within epistemology, it has been suggested that we can make progress on questions about the nature of knowledge by looking at intuitions about whether certain states count as knowledge. Similarly, within moral philosophy, it has been suggested that we can make progress on questions about moral obligation by looking at intuitions about what actions certain agents are obligated to perform. Similar approaches have been advocated in numerous other areas of philosophy.

There is a complex literature within the analytic tradition about how to understand this traditional method. Some argue that the study of intuitions gives us insight into concepts (Jackson 1998), others argue that the study of intuitions gives us a more direct sort of insight into the actual properties or relations those concepts pick out (Sosa 2007), and still others argue that this whole way of conceiving of the project is a mistaken one (e.g., Cappelen 2012).

It is commonplace to divide existing research in experimental philosophy into distinct projects in accordance with their different relationships to this prior tradition. Dividing things up in this way, one arrives at three basic kinds of research in experimental philosophy.

First, some experimental philosophy research has a purely ‘negative’ relationship to this more traditional use of intuitions. Such research aims to provide evidence that the method used in the more traditional work is in some way flawed or unreliable. For example, it has been argued that intuitions differ across demographic factors such as gender or ethnicity, or that they are subject to order effects, or that they can be influenced by incidental emotion (e.g., Weinberg et al. 2001; Buckwalter & Stich 2014; Swain et al. 2008; Cameron et al. 2013). To the extent that intuitions show these effects, it is argued, we should not be relying uncritically on intuition as a method for addressing substantive philosophical questions. This first project is called ‘negative’ in that it is not intended to make progress on the original philosophical question (e.g., about the nature of knowledge) but only to argue against a specific method for addressing that question (appeal to intuition).

This project has triggered a large and multi-faceted literature among philosophers interested in its metaphilosophical implications (Brown 2013b; Cappelen 2012; Deutsch 2015; Weinberg 2007; Weinberg et al. 2010; Williamson 2007). This literature has explored the question as to whether empirical facts about the patterns in people’s intuitions could give us reason to change our philosophical practices. Much of this work is quite closely tied to prior philosophical work about the role of intuition in philosophy more generally.

Second, some research in experimental philosophy aims to make further progress on precisely the sorts of questions that motivated prior work within analytic philosophy. Thus, this research looks at epistemic intuitions as a way of making progress in epistemology, moral intuitions as a way of making progress in moral philosophy, and so forth. Experimental philosophers pursuing this second project have offered various different accounts of the way in which facts about intuitions could yield progress on these philosophical issues, but the most common approach proceeds by advancing some specific hypothesis about the underlying cognitive processes that generate intuitions in a particular domain. The suggestion is then that this hypothesis can help us assess which intuitions in this domain are worthy of our trust and which should simply be dismissed or ignored (Gerken 2017; Leslie 2013; Greene 2008; Nagel 2010).

Work within this second project has inspired a certain amount of metaphilosophical debate, but its main impact on the philosophical literature has been not at the level of metaphilosophy but rather in discussions of individual philosophical questions. Thus, philosophers interested in epistemic contextualism discuss experiments on people’s intuitions about knowledge (DeRose 2011), philosophers interested in incompatibilism discuss experiments on people’s intuitions about free will (Björnsson & Pereboom 2014; Vargas 2013), and philosophers interested in interventionist accounts of causation discuss experiments on people’s intuitions about causation (Woodward 2014). Work in this vein typically does not focus primarily on more abstract theories about the role of intuitions in philosophy. Instead, it draws more on theories about the particular topic under study (theories of knowledge, free will, causation).

The third type of research being conducted in experimental philosophy is not concerned either way with the kind of project pursued in more traditional analytic philosophy; it is just doing something else entirely. Specifically, in many cases, experimental philosophers are not looking at people’s thoughts and feelings about some topic as a way of making progress on questions about that topic; they are instead trying to make progress on questions that are directly about people’s thoughts and feelings themselves . For example, much of the experimental philosophy research in moral psychology is concerned with questions that truly are about moral psychology itself.

Research in this third vein tends to be highly interdisciplinary. Thus, work on any particular topic within this third vein tends to be at least relatively continuous with work on that same topic in other disciplines (psychology, neuroscience, linguistics, etc.), and the impact of such work is often felt just as much in those other disciplines as in philosophy specifically.

The distinction between these projects has proven helpful within metaphilosophical work on the significance of experimental philosophy, but it should be noted that the metaphilosophical distinction between these three projects does not correspond in any straightforward way to the distinctions between the different concrete research programs experimental philosophers pursue (on free will intuitions, on moral intuitions, on epistemic intuitions, etc.). Each of these concrete research programs can be relevant to a number of different projects, and indeed, it often happens that a single paper reports a result that seems relevant to more than one of these projects. Thus, as we review the actual experimental research coming out of experimental philosophy, we will need to turn away from the metaphilosophical distinction between projects and turn instead to distinctions between concrete research topics.

2. Research in Experimental Philosophy

The best way to get a sense of what experimental philosophy is all about is not just to consider it in the abstract but to look in detail at a few ongoing research programs in the field. Accordingly, we proceed in this section by reviewing existing research in four specific areas: the negative program, free will, the impact of moral judgment, and epistemology.

We focus on these four areas because they have received an especially large amount of attention within the existing experimental philosophy of literature. We should note, however, that experimental philosophers have explored an enormous range of different questions, and work in these four specific areas comprises only a relatively small percentage of the experimental philosophy literature as a whole.

2.1 The Negative Program

In the Theaetetus, Socrates asks, “Herein lies the difficulty which I can never solve to my satisfaction—What is knowledge?” (146a). The subsequent philosophical discussion often proceeds by setting out various hypotheses, e.g., that knowledge is true belief, and considering possible counterexamples to the hypothesis. So, for instance, Socrates argues that knowledge isn’t simply true belief because a skilled lawyer can persuade a person to have a belief that is true, but that belief wouldn’t actually be knowledge [see the entry on the Theaetetus ]. Socrates typically expects, and receives, agreement from his interlocutor. Nor does Socrates ask his interlocutor, “What is your conception of knowledge” or “What counts as knowledge for Athenians?” Rather, he seems to expect a global answer about what knowledge is. In addition, he seems to expect that knowledge has a single nature, as suggested by his telling Theaetetus, “I want you… to give one single account of the many branches of knowledge” (148d).

Work in the negative program of experimental philosophy uses empirical work to challenge this traditional philosophical project. Two somewhat different challenges have been developed.

One challenge arises from the prospect of systematic diversity in how different populations of people think about philosophical questions. The possibility of such diversity had been raised before (e.g., Stich 1990), but experimental philosophers have sought to provide evidence of such diversity. For instance, an early study reported differences between East Asian students and Western students on famous cases from epistemology (Weinberg et al. 2001). Another early study provided evidence for cultural differences in judgments about reference. East Asians were more likely than Westerners to have descriptivist judgments about the reference of proper names (Machery et al. 2004). Some studies have also found gender differences in intuitions about philosophical cases (see, e.g., Buckwalter & Stich 2014; Friesdorf et al. 2015). In addition, there are systematic individual differences in philosophical intuition; for example, people who are more extraverted are more inclined to compatibilism about responsibility (Feltz & Cokely 2009).

This apparent diversity in intuitions about philosophical matters has been used to challenge the use of intuitions in philosophy to tell us about the nature of things like knowledge and reference. If intuitions about knowledge turn out to exhibit diversity between populations, then this looks to put pressure on a traditional philosophical project. In rough form, the worry arises from the following claims:

• D1. The philosophical tradition uses intuitions regarding philosophically important categories or kinds like knowledge in an effort to determine the nature of those categories
• D2. Knowledge (like many other philosophical categories) has a single nature. It’s not the case that knowledge is one thing in Athens and another thing in Sparta.
• D3. Intuitions about philosophical categories systematically vary between populations (by culture, for instance)
• D4. The diversity in intuition cannot be dismissed by privileging the intuitions of one population.

Each of these claims has been challenged. Some argue that philosophers do not—or should not—rely on intuitions (thus rejecting D1) (see section 3.1 ); others hold, contra D4, that certain populations (e.g., professional philosophers) are specially positioned to have reliable intuitions (see section 3.2 ).

Another way to defuse the challenge is to argue (contra D2) that we needn’t suppose that knowledge has a single nature, but instead allow for a kind of pluralism. For instance, “knowledge” might pick out different epistemic notions in different communities. A pluralist might allow, or even celebrate, this diversity. Even if other communities have different epistemic values than we do, this need not undermine our valuing knowledge, as it is construed in our community (e.g., Sosa 2009: 109; also Lycan 2006). For a pluralist, empirical demonstrations of diversity needn’t undermine traditional philosophical methods, but might instead reveal important epistemic features that we have missed.

A more conservative response to the challenge, which leaves traditional philosophy largely untouched, is to question whether there really is diversity between populations in intuitions about philosophical categories. One way to develop this response is to claim that participants in different populations might simply interpret the scenarios in different ways; in that case, we could explain their different answers by saying that they are responding to different questions (e.g., Sosa 2009).

More importantly, a growing body of empirical evidence has called into question the claim that there really are large differences in philosophical intuitions across populations. Some of the original findings of culture differences have not replicated (e.g., Nagel et al. 2013; Kim & Yuan 2015); similarly, many of the original findings of gender differences haven’t replicated (e.g., Seyedsayamdost 2015; Adleberg et al. 2015). These findings provide strong reason to believe that some of the effects suggested by early experimental philosophy studies do not, in fact, exist at all. Moreover, experimental philosophers have also uncovered robust cross-cultural uniformity. For instance, one recent cross-cultural study examined intuitions about Gettier cases across four very different cultures (Brazil, India, Japan, and the USA), with participants in all groups tending to deny knowledge to the protagonist in Gettier cases (Machery et al. 2015). This suggests that there might be a universal “core folk epistemology” (Machery et al. 2015). In any case, these kinds of results suggest that there is less diversity than had been suggested.

The foregoing argument is based on diversity between populations. But experimental philosophers in the negative program have also used intra-individual diversity to undermine traditional philosophical methods (Swain et al. 2008; Sinnott-Armstrong 2008; Weinberg 2016). Experimental philosophers have found that people’s judgments about philosophical cases are sensitive to various kinds of contextual factors that seem to be philosophically irrelevant. The same person will give different responses depending on apparently irrelevant factors of presentation. People’s judgments about cases are affected by the induction of irrelevant emotions (Cameron et al. 2013), the order in which cases are presented (Petrinovich and O’Neill 1996; Swain et al. 2008; Wright 2010), and the way an outcome is described (e.g., Petrinovich and O’Neill 1996; Schwitzgebel & Cushman 2015).

Sensitivity to contextual factors has been used to challenge the philosophical use of intuition in a way that is somewhat distinct from the diversity argument. The challenge begins with the same assumption about the role of intuitions in philosophy, but then draws on somewhat different considerations:

• S1. The philosophical tradition uses intuitions regarding philosophically important categories or kinds like knowledge in an effort to determine the nature of those categories
• S2. A person’s judgments about philosophical cases are sensitive to contextual factors like the order of presentation.
• S3. Sensitivity to these factors is epistemically inappropriate
• S4. This inappropriate sensitivity cannot be dismissed by privileging the intuitions of one population (e.g., philosophers)
• S5. We can’t tell, from the armchair, which of our judgments are inappropriately sensitive in this way.

This set of claims presents a challenge because it seems that even philosophers are susceptible to these epistemically inapt influences, and we can’t tell which of our intuitions are to be trusted. Thus, philosophers are on shaky epistemic ground when they rely on their intuitions to try to glean philosophical truths.

Obviously, the argument from sensitivity is developed in different ways depending on the category in question and the evidence of sensitivity, but it’s useful to see how the general claims (S1–S5) might be questioned. (See section 3.1 for the rejection of intuition in philosophy (S1) and section 3.2 for a defense of privileged populations [contra S4]).

Although there are replicable effects on the influence of contextual factors, pace S2 many of these effects seem too small to threaten the practice of relying on intuitions (see, e.g., Demaree-Cotton 2016; May 2014). The effect might amount to the difference between 2.2 and 2.5 on a 7 point scale. It’s hard to see how such a difference threatens the practice of relying on the operative intuitions.

In some cases, contextual factors have more pronounced effects, and do lead to changes in participants’ verdict about a case. For instance, judgments about certain moral dilemmas and judgments about certain epistemic cases are changed depending on previously seen cases (e.g., Petrinovich & O’Neill 1996; Swain et al. 2008). However, it’s possible that participants respond differently to a case because the contextual differences actually provide an epistemically appropriate basis for changing one’s judgment. For instance, in the order effect studies, seeing one case can provide evidence about the appropriate response on another case (Horne & Livengood 2016). On this view, we can grant that participants change their judgment, but deny that they are doing so in a way that is epistemically inappropriate.

Finally, even if people’s judgments do change in epistemically inappropriate ways, people might be able to recognize which judgments are especially trustworthy. For instance, only some thought experiments are susceptible to order effects, and it turns out that for these thought experiments, people have lower confidence in their responses (e.g., Wright 2010; Zamzow & Nichols 2009). This suggests (contra D5) that there might be an internal resource—confidence—that can be used to discern which judgments are epistemically unstable.

Research in experimental philosophy has explored many aspects of lay beliefs regarding free will. Experimental philosophers have designed improved scales for measuring belief in free will (Nadelhoffer et al. 2014; Deery et al. 2015), they have investigated the role of the desire to punish in attributing free will (Clark et al. 2014), and they have examined the impact of the belief in free will on moral behavior (Baumeister et al. 2009). But the most intensively studied issue concerns intuitions about whether free will is compatible with determinism.

Experimental philosophers have argued that the philosophical defense of incompatibilism depends on intuitions (e.g., Nahmias et al., 2006). The question about whether incompatibilism is true depends on a wide variety of factors, but experimental philosophers have argued that one factor that plausibly matters is the alleged intuitiveness of the thought that determinism is incompatible with free will (Murray & Nahmias 2014, but see Sommers 2010). This then generates a question that invites an empirical inquiry: is incompatibilism intuitive? (Nahmias et al. 2006).

One of the first experimental studies on free will found that people seemed to have compatibilist intuitions. Participants were presented with a scenario describing a deterministic universe, and then asked whether a person in the scenario was free and morally responsible (Nahmias et al. 2006). In one case, participants were asked to imagine a future scenario in which there is a supercomputer that is capable of predicting all future human behavior when provided with a complete description of the universe along with the laws of nature. In this scenario, a man robs a bank, and participants are asked whether the man is morally responsible for his action. Somewhat surprisingly, most participants gave compatibilist answers, saying that the person was morally blameworthy. This basic finding held across a number of scenarios.

In these early studies on intuitions about free will and moral responsibility, the description of determinism focused on the fact that in a deterministic universe, every event is in principle predictable from the past and the laws. In addition, the scenarios involved particular agents in our world doing bad things. Later studies emphasized the causal nature of determinism—that what happens at a given point is completely caused by what happened previously—and stressed that what happens in a deterministic universe is inevitable given the past. Even with this description of determinism, participants still tend to say that a specific concrete individual in such a universe who commits a heinous crime is free and responsible (Nichols & Knobe 2007; Roskies & Nichols 2008). However, when asked a more abstract question about whether it is possible in general for people in such a deterministic universe to be free and responsible, participants tend to say that morally responsibility is not possible in a deterministic universe. This incompatibilist response was also found in a cross cultural sample with participants from India, Hong Kong, Colombia, and the United States (Sarkissian et al. 2010). In addition to the abstract nature of the question, another important element seems to be whether one is considering an alternate deterministic universe or contemplating the possibility that our own universe is deterministic. When led to consider our own universe as deterministic, participants were more likely to say that people would still be morally responsible (Roskies & Nichols 2008).

Thus, it seems like people give compatibilist responses under some conditions and incompatibilist responses under others. One reaction to this apparent inconsistency is to treat one set of responses as defective. Some experimental philosophers maintain that it’s the incompatibilist responses that don’t reflect people’s true judgments. The best developed version of this view maintains people aren’t affirming incompatibilist responses at all (Nahmias & Murray 2011; Murray & Nahmias 2014). Instead, when people deny free will and responsibility it’s because they misunderstand the description of determinism. In particular, people mistakenly interpret the description of determinism to mean that our mental states lack causal efficacy, that the production of our behavior “bypasses” our mental states. That is, on this view, people wrongly think that determinism means that a person will behave as she does regardless of what she thinks, wants, or intends (Murray & Nahmias 2014).

Of course, if people’s mental states have no impact on their behavior, that is an excellent reason to think that people aren’t morally responsible for their behaviors. So, if people interpret determinism to mean bypassing , it is perfectly rational for them to infer the lack of free will and responsibility from bypassing. However, it seems to be a flat-out confusion to interpret determinism as bypassing. Even if determinism is true, our behavior might be caused (not bypassed) by our mental states. Thus, if people give incompatibilist responses because they confuse determinism with bypassing, then people’s responses don’t reflect a real commitment to incompatibilism.

Surprisingly, people do make bypassing judgments when given a description of causal determinism. For instance, when presented with a description of a determinist universe, many participants agreed that in that universe, “what a person wants has no effect on what they end up doing” (Murray & Nahmias 2014). This suggests that people go through the following confused process: determinism means bypassing, and bypassing means no free will. If that’s right, then the incompatibilist response really is a confusion. However, another explanation is that people think that determinism means no free will, and it’s the denial of free will that leads to the bypassing judgments. The idea would be roughly that if we don’t have free will, then in some way our mental states don’t lead to our behavior in the way we had thought. Some experimental philosophers have used statistical causal modeling to try to tease these two possibilities apart, arguing that it’s the latter explanation that is the right one (Björnsson 2014; Rose & Nichols 2013). That is, people take determinism to entail that there is no free will, and it is this judgment that there is no free will that leads to the bypassing judgment.

Thus, there is some reason to think that incompatibilist responses do reflect many people’s intuitions. What about the compatibilist responses? Some experimental philosophers maintain that it is these judgments that are distorted. On one view, the distortion is caused by emotional reactions (e.g., Nichols & Knobe 2007). However, a meta-analysis indicates that there is very little evidence that emotions play a critical role in generating compatibilist judgments (Feltz & Cova 2014). A different argument for demoting compatibilist judgments holds that many people who affirm free will in deterministic scenarios lack any sensitivity to compatibilist considerations, but instead will affirm free will even under fatalistic conditions in which it is explicitly stipulated that John’s behavior is inevitable “regardless of the past events in John’s life and the laws of nature”. (This view is dubbed “free will no matter what”; Feltz & Millan 2015.) One line of argument based on these results is that if people’s attributions of free will are so insensitive, it can hardly be said that people appreciate the consistency of free will and determinism. However, subsequent studies found that in these fatalistic scenarios, subjects who affirmed free will still tended to think that the source of the action was in the agent, in harmony with “source compatibilism” (Andow & Cova 2016).

Thus, the state of the evidence currently suggests that people do have both incompatibilist and compatibilist intuitions. Future empirical work might uncover more clearly what factors and processes draws people in one direction or the other. There are also open questions about whether the role of different psychological mechanisms in intuitions about free will has implications for philosophical questions for whether we are truly free and responsible.

It is common to distinguish between two kinds of judgments that people make about morally significant situations. On one hand, people can make straightforwardly moral judgments (e.g., judgments about moral wrongness, about obligation, about blameworthiness). On the other, they can make judgments that might be morally relevant but that still appear to be in some important sense non-moral judgments (about whether the agent acted intentionally, whether she caused certain outcomes, whether she knew what she was doing). A question now arises as to how to understand the relationship between these two different kinds of judgments.

One possible view would be that the relationship is entirely unidirectional. Thus, it might be thought that (a) people’s moral judgments depend on prior non-moral judgments, but (b) people’s non-moral judgments do not depend on prior moral judgments. We can illustrate this view with the example of the relationship between people’s moral judgments and their intentional action judgments. It seems clear that people’s moral judgments about whether an agent is deserving of blame might depend on prior non-moral judgments about whether this agent acted intentionally. However, one might think that things do not go in the opposite direction. It is not as though your non-moral judgment that the agent acted intentionally could depend on a prior moral judgment that her action was wrong.

Although this view might seem intuitively compelling, a series of studies in experimental philosophy have called it into question. These studies suggest that people’s moral judgments can impact their judgments even about what might appear to be entirely non-moral questions. Such results have been obtained for a wide variety of different apparently non-moral judgments.

• When an agent knows that she will bring about an outcome but is not specifically trying to bring it about, people are more inclined to say that she brought it about intentionally when it is morally bad than when it is morally good (Knobe 2003).
• When an agent correctly believes that an outcome will arise but is only correct in this belief as the result of a coincidence, people are more inclined to say that she has knowledge when the outcome is morally bad than when it is morally good (Beebe & Shea 2013; Buckwalter 2014).
• When an agent has a lot of positive emotion and a high opinion of her life, people are less inclined to say that she is truly happy when her life is morally bad than when it is morally good (Phillips, Nyholm & Liao 2014).
• When a number of different factors are each individually necessary for an outcome to arise, people are more inclined to regard one of the factors as a cause when it is morally bad than when it is morally good (Alicke 1992; Hitchcock & Knobe 2009).

Effects of moral judgment have also been observed on numerous other judgments, including everything from action individuation (Ulatowski, 2012) to attributions of weakness of will (May & Holton 2012) to the semantics of gradable adjectives (Egré & Cova 2015).

These findings might be philosophically relevant at two different levels. On one hand, each individual effect might be relevant to philosophical work that aims to understand the corresponding concept or property. Thus, the findings about intentional action judgments might be relevant to philosophical work about intentional action, those about happiness judgments might be relevant to philosophical work about happiness, and so forth. At the same time, the general finding that moral judgment has this pervasive influence might be relevant to philosophical work that focuses on the human mind and the way people make sense of the world. For example, these findings could help us to understand the nature of folk psychology or the relationship between our ordinary folk theories and more systematic scientific theories.

To make progress on these two issues, research has focused on trying to understand why these effects arise. That is, researchers have aimed to provide hypotheses about the precise cognitive processes that give rise to the patterns observed in people’s judgments. These hypotheses then, in turn, have implications for philosophical questions both about specific concepts and properties and about the human mind.

Existing research has led to a proliferation of hypotheses, drawing on theoretical frameworks from a variety of fields (see Cova 2016 for a review of seventeen hypotheses about the intentional action effect). Still, although there are numerous distinct specific hypotheses, it seems that the basic approaches can be grouped into four broad families.

First, it might be that the effect is not truly driven by moral judgment . Existing studies show that people make different judgments depending on whether the agent is doing something helpful or harmful, but of course, there are many differences between helpful and harmful actions other than their moral status. For example, a number of researchers have argued that the effect is in fact driven by people’s beliefs about the mental states of the agents in the vignettes (Sloman, Fernbach & Ewing 2012; Sripada & Konrath 2011). Agents will tend to have different sorts of mental states when they are doing something helpful than when they are doing something harmful, and it might be that this difference in mental states is driving all of the observed effects.

Second, it might be that the effect is indeed driven by moral judgment but that it is the result of an error . On this view, moral considerations do not play any real role in the concepts at work here (people’s concepts of intentional action, of happiness, etc.). Rather, people’s judgments are being biased or distorted by some further process which gets in the way of their ability to correctly apply their own concepts. For example, some researchers have argued that the effect is due to a process of motivated cognition (Alicke, Rose & Bloom 2011). People believe the agent to be blameworthy and want to justify that belief. This desire to justify blame then distorts their judgments about what might seem to be purely factual matters.

Third, it might be that the effect is driven by moral judgment and doesn’t involve an error but nonetheless simply reflects a fact about how people use words , rather than a fact about their application of the corresponding concepts. Researchers often make inferences from facts about how people use certain words (‘intentionally,’ ‘happy,’ ‘knows’) about how people apply the corresponding concepts (the concept of intentional action, of happiness, of knowledge). However, it is also possible for factors to influence the use of our words without influencing the use of these concepts, and some researchers have suggested that this is the process at work in the present effects. For example, it has been suggested that these effects arise as a result of conversational pragmatics, with people trying to avoid the pragmatic implicatures that would be generated by making certain claims that are in fact literally true (Adams & Steadman 2004). Alternatively, it has been suggested that the relevant words (e.g., ‘intentionally’) are actually associated with more than one different concept and that the impact of morality arises not because morality plays a role in any of these concepts but rather because it plays a role in the way people resolve the ambiguity of the word itself (Nichols & Ulatowski 2007). On these sorts of views, people are not necessarily making a mistake when their use of language is impacted by moral judgment, but all the same, moral judgment is not playing a role in their more basic capacities to make sense of the world.

Fourth, it might be that moral judgment actually plays a role in people’s basic capacities to apply the relevant concepts . For example, it has been argued that the concept of happiness is itself a value-laden concept (Phillips et al. 2014). Similarly, it has been suggested the concepts of intentional action and causation make use of a form of counterfactual thinking in which moral judgments play a key role (Icard, Kominsky & Knobe 2017; Phillips, Luguri & Knobe 2015). On this last view, the effects observed in these experiments point to a genuine role for moral judgment in the most basic capacities underlying people’s application of the relevant concepts.

Debates between these rival views remain ongoing. Within the more recent literature, discussion of these questions has become increasingly interdisciplinary, with many of the key contributions turning to methods from cognitive neuroscience, developmental psychology, or computational cognitive science.

Within experimental work in epistemology, the primary focus of research has been on the patterns of people’s ordinary attributions of knowledge . As we’ve seen ( section 2.1 ), evidence on epistemic intuitions plays a prominent role in the negative program. But work in experimental epistemology has not been dominated by any one single issue or question. Rather, it has been divided among a number of different strands of research, which have each been pursued separately.

One important topic has been the role of stakes in people’s knowledge attributions. Suppose that Keith considers some available evidence and then concludes (correctly) that the bank will be open on Saturday. Now consider two cases. In the low-stakes case, it is not especially important whether the bank actually is open. By contrast, in the high-stakes case, Keith’s whole financial future depends on whether the bank is open or not. The key question now is whether this difference in stakes has any impact on whether it is correct to say: “Keith knows that the bank will be open”.

Within the non-experimental literature, philosophers have appealed to a wide variety of arguments to help resolve this question. Although many of these arguments do not directly involve people’s intuitions about cases (Brown 2013a; see also Fantl & McGrath 2009; Hawthorne 2004), some specifically rely on the empirical claim that people would be more willing to attribute knowledge when the stakes are low than when the stakes are high (DeRose 1992). Among philosophers who accept this empirical claim, there has been considerable debate about precisely how to explain the purported impact of stakes (DeRose 1992; Hawthorne 2004; Rysiew 2001; Stanley 2005).

Surprisingly, a number of early findings from the experimental epistemology literature suggested that people’s ordinary knowledge attributions actually don’t depend on stakes. For example, people seem to say that Keith knows the bank will be open on Saturday not only in the low-stakes case but also in the high-stakes case (Buckwalter 2010; Feltz & Zarpentine 2010; May et al. 2010). This experimental finding threatens to undermine the entire debate within the non-experimental epistemology literature. After all, if there is no effect of stakes, then there is no question as to how to understand this effect.

Subsequent experimental work in this area has therefore focused on the question as to whether the stakes effect even exists at all. Some have criticized the early experiments that did not find an effect (DeRose 2011). Others have shown that although the effect does not emerge in the experimental paradigms used by those early experiments, it does emerge in other paradigms (Pinillos 2012; Sripada & Stanley 2012; but see Buckwalter & Schaffer 2015, for a critique). Regardless of how these debates are resolved, recent experimental work seems to have established, at a very minimum, that the pattern of people’s epistemic intuitions is not quite the way it was assumed to be within the previous non-experimental literature.

A second question concerns the relationship between knowledge and belief. Clearly, a mental state can only count as knowledge if it satisfies certain conditions that go beyond anything that would be required for the state to count as belief. Thus, there can be cases in which a person believes that p but does not know that p . A question arises, however, as to whether the converse also holds. That is, a question arises as to whether a mental state must satisfy certain conditions to count as a belief that go beyond what would be required for it to count as knowledge. Can there be cases in which a person knows that p but does not believe that p ?

Strikingly, a series of studies suggest that people do attribute knowledge in certain cases in which they would not be willing to attribute belief (Myers-Schulz & Schwitzgebel 2013; see also Murray et al. 2013; Rose & Schaffer 2013; Buckwalter et al. 2015; Shields 2016). In one study, participants were given a vignette about a student taking a history test who faces the question: “What year did Queen Elizabeth die?” She has reviewed this date many times, but at that one moment, she is flustered by the pressure and can’t recall the answer. She therefore decides just to guess, and she writes down ‘1603.’ In fact, this is the correct answer. When given this vignette, experimental participants tended to say that (a) the student knows that Queen Elizabeth died in 1603 but to deny that (b) she believes that Queen Elizabeth died in 1603 (Myers-Schulz & Schwitzgebel 2013, drawing on a vignette from Radford). Similar effects have been obtained for numerous other cases (Murray et al. 2013; Rose & Schaffer 2013; Buckwalter et al. 2015; Shields 2016).

Research in this area aims to understand why this effect arises and what implications it has for epistemology. One view is that people’s concept of belief truly does involve certain conditions that are not required by their concept of knowledge (Myers-Schulz & Schwitzgebel 2013). An alternative view is that there is more than one sense of ‘belief,’ such that knowledge requires the mental state picked out by one of the senses but not the other. Within work that adopts this latter approach, there have been a number of more specific suggestions about how to spell out the difference between the two senses and what relation each has to the ordinary concept of knowledge (Rose & Schaffer 2013; Buckwalter et al. 2015).

Experimental epistemology has also explored numerous other issues. A series of studies indicate that people actually do attribute knowledge in ‘fake barn’ cases (Colaço et al. 2014; Turri 2017). Others show that judgments about whether a person’s mental state counts as knowledge depend on whether that person’s evidence comes from facts about an object itself or from statistical base rates (Friedman & Turri 2015). Still others have explored issues at the intersection of formal semantics and epistemology, exploring the impact of specific linguistic factors on knowledge attributions (Schaffer & Szabó 2014).

We have been focusing in on four specific areas in which there have been especially prominent contributions from experimental philosophy, but we should emphasize that it is not as though the majority of experimental philosophy research falls into one or another of these areas. On the contrary, research in experimental philosophy is highly diverse, and it has actually been getting steadily more heterogeneous in recent years.

First, experimental philosophers have been pursuing an ever more diverse array of topics. On one hand, there has been a surge of experimental research using more formal, mathematical tools, including work on causation using Bayes nets (e.g., Livengood & Rose 2016). and work in formal semantics on everything from gradable adjectives to conditionals to epistemic modals (Liao & Meskin 2017; Cariani & Rips 2017; Khoo 2015). On the other, there has been a proliferation of work addressing core topics in the humanities, including art, religion and even questions at the intersection of experimental philosophy and the history of philosophy (De Cruz & De Smedt 2016; Liao et al. 2014; Nichols 2015).

Secondly one finds an ever-growing diversity of experimental methods. There are still plenty of studies that proceed by giving participants vignettes and asking for their intuitions, but in contemporary experimental philosophy, one also finds studies using corpora (Reuter 2011), reaction times (Philips & Cushman 2017), neuroimaging (Greene et al. 2001), even studies that look at whether ethics professors actually behave ethically (Schwitzgebel & Rust 2014).

Finally, and perhaps most noticeably, there is an increasingly close connection between research in experimental philosophy and research in psychology. For example, the experimental research program on intuitions about trolley problems has been dominated by contributions from psychology (e.g., Cushman et al. 2006; Wiegmann et al. 2012), but there have also been important contributions from philosophers (e.g., Mikhail 2011; Kahane & Shackel 2008). Conversely, there have been numerous recent papers in psychology that aim to contribute to research programs that originated in experimental philosophy (Samland & Waldmann 2016; Feldman & Chandrashekar forthcoming; Starmans & Friedman 2012).

3. Challenges to Experimental Philosophy

As is the case with any healthy research area, there is lots of dispute about issues within experimental philosophy. There are disagreements about particular studies, the implications of different kinds of results, and so on. But there are also broad challenges to the very idea that experimental philosophy research could prove helpful in addressing the philosophical questions. We focus here on three of the most prominent of these challenges.

3.1 Disputing the Role of Intuitions in Philosophy

As we’ve seen, much work in experimental philosophy presupposes that intuitions play an important role in philosophical inquiry. Work in the negative program characteristically starts with the assumption that intuitions play a central role in the philosophical tradition. Outside of the negative program, experimental philosophers want to understand what people’s intuitions are about philosophical matters and why they have these intuitions. Several philosophers, however, challenge the role of intuitions in philosophy in ways that also pose a challenge to the philosophical significance of much experimental philosophy.

One way to reject the role of intuitions is simply to deny that philosophers use intuitions as justification for their views (Williamson 2007; Cappelen 2012; Deutsch 2009, 2010, 2015). According to such “intuition deniers”, the experimental investigation of intuitions is thoroughly irrelevant to philosophy (e.g., Cappelen 2012: 1; for discussion, Nado 2016). Obviously if this is right, then the negative program is arguing against a thoroughly mistaken conception of philosophy.

Although work in metaphilosophy often assumes that philosophers use intuitions as evidence, this is exactly what is challenged by intuition deniers. It is granted on all sides that philosophers sometimes mention intuitions, but according to the intuition deniers, intuitions are not integral to the philosophical work. In particular, intuition deniers maintain that a careful inspection of philosophical practice reveals that philosophers don’t rely on intuitions to justify philosophical views; rather, philosophers rely on arguments (see, e.g., Cappelen 2012: 170; Deutsch 2009: 451).

There have been several responses to the intuition deniers, but perhaps the most prominent response to is that the arguments of intuition deniers depend on an implausibly strong conception of the notion of intuition (e.g., Chalmers 2014; Devitt 2015; Weinberg 2014). Once we focus on a less demanding notion of intuition, it’s plausible that philosophers often rely on intuitions as evidence for philosophical theses (Devitt 2015). Indeed, some have argued that for classic examples like Gettier cases, it’s hard to see how the argument works if it doesn’t rely on intuitions (see, e.g., Brown 2017; Sytsma & Livengood 2015: 92–93) Experimental philosophers have also argued against intuition deniers on experimental grounds, noting that a recent study found that over 50% of philosophers agree with the statement “intuitions are useful for justifying philosophical claims” (Kuntz & Kuntz 2011; see Sytsma & Livengood 2015: 91).

A rather different way to challenge the study of intuitions in experimental philosophy is to deny that the study of intuitions is an apt subject matter for philosophical inquiry. On this view, we can grant that it’s a fact that philosophers rely on intuitions, but it’s a lamentable fact. The use of intuitions in philosophy is misguided for reasons that have nothing in particular to do with experimental philosophy—the appeal to intuitions is a relic, which should be rejected because it doesn’t actually answer the philosophical questions. This conclusion threatens positive applications of experimental philosophy (see, e.g., sections 2.2–2.4 ), but is of course, perfectly consistent with the conclusion urged by the negative program in experimental philosophy ( section 2.1 ).

One influential argument against the use of intuitions builds on the rejection of descriptivist theories of reference, according to which concepts refer to kinds via a set of associated descriptions. In place of descriptivism, some maintain that concepts refer in virtue of the function of the concept (e.g., Millikan 2000). Other views maintain that concepts refer in virtue of a causal chain connecting the concept to the kind (Putnam 1973). On these anti-descriptivist views, people can have wildly mistaken intuitions regarding the application of their concepts. As a result, probing lay intuitions might be an ineffective way to investigate the kinds of things to which our concepts refer (e.g., Fischer 2015; Kornblith 2002).

Anti-descriptivism itself doesn’t entail that appeal to intuitions is philosophically irrelevant. Indeed, some of the most influential arguments against descriptivist theories of reference seem to depend on intuitions (Devitt 2015). However, some argue that rather than relying on intuitions about kinds, we should investigate the kinds themselves. So, if the concept knowledge picks out a natural kind, we can consult the distribution and characteristics of knowledge as it is instantiated in the world. Using intuitions to understand knowledge would be like using intuitions to understand gold. The way we come to understand the nature of gold is to examine samples of gold rather than people’s intuitions about gold. Similarly, the way to understand knowledge is to examine samples of knowledge as it presents in animals, rather than people’s intuitions about knowledge (Kornblith 2002). To examine knowledge by intuitions is at best inefficient, and at worst a complete distraction from the task of understanding what knowledge is. This objection is primarily directed at traditional forms of conceptual analysis, but insofar as experimental philosophy focuses on intuitions, it is in the same leaky boat (Kornblith 2013: 197).

The claim that philosophers shouldn’t rely on intuitions constitutes a broad attack on conceptual analysis, in both its traditional and experimental guises. Not surprisingly, there have been several defenses of the importance of intuitions for doing philosophy. For instance, some philosophers argue that in order even to pick out the kind of interest, we need to rely on our intuitive sense of what belongs in the category (e.g., Goldman 2015). To determine the characteristics of knowledge, we need to have a way of picking out which items are genuine members of the kind, and for this we must rely on our intuitive understanding of knowledge. In addition, if we reject outright the appeal to what intuitively belongs to a category, it’s hard to make sense of the intelligibility of eliminativism (e.g., Bermúdez 2006: 305), since eliminativists typically argue that there is a mismatch between intuitive notions of, e.g., free will, and the kinds of things in the world. To give up on the significance of characterizing our intuitive commitments is to preemptively exclude eliminativist views, which have long been regarded as of central philosophical interest.

A second objection would be that even if intuitions do matter, we should not be concerned with just any old kind of intuition. Rather, our concern should be with a distinctive class of intuitions. For example, research in philosophy has traditionally been conducted by trained philosophers who spent years thinking about difficult problems. There is good reason to suspect that the intuitions generated by this type of process will have a special sort of epistemic status, and perhaps these sorts of intuitions can play a legitimate role in philosophy. By contrast, the intuitions explored within experimental philosophy research tend to be those of ordinary folks, with no prior background in philosophy, and one might think that intuitions of this latter type have no real philosophical significance.

One way of spelling out this concern is in terms of what has come to be known as the expertise objection . The key contention here is that trained philosophers have a distinctive type of expertise. Thus, if we want to understand the process at the core of traditional philosophical practice, we need to study people who have this type of expertise. It is no good just looking at the judgments of people who have never taken a single philosophy course. A number of philosophers have developed objections along more or less these lines though with important differences (Williamson 2007; Ludwig 2007).

This is an important objection, and to address it, experimental philosophers launched a major effort to study the intuitions of trained philosophers. The results show that trained philosophers still show order effects (Schwitzgebel & Cushman 2012), actor/observer effects (Tobia et al. 2013), and effects of temperament (Schulz, Cokely, & Feltz 2011). Thus, existing work provides at least some evidence against the claim that trained philosophers have a distinctive expertise that allows them to escape the sorts of biases that plague the judgments of ordinary folks.

Of course, there are numerous ways of defending the objection against this type of response. It could be argued that although philosophers do not have an ability to avoid biases of the type studied within experimental philosophy, their judgments do differ from those of ordinary folks in some other important respect. Similarly, it could be argued that what gives certain intuitions their privileged epistemic state is not the fact that they come from a particular type of person (trained philosophers) but rather the fact that they are the product of a particular way of approaching the question (sustained reflection) (see, e.g., Kauppinen 2007).

Finally, it might be objected that experimental philosophy simply isn’t philosophy at all. On this view, there are certain properties that differentiate work in philosophy from work in other disciplines. Research in experimental philosophy lacks these properties and is therefore best understood as falling outside the philosophical tradition entirely. Note that this last objection is not concerned with the question as to whether experimental philosophy has any value but rather with the question as to whether it should be considered part of a particular discipline. As one recent paper puts it,

… what is at issue is not whether there is room for such empirical study, but whether there is room for it now as a branch of philosophy . (Sorell forthcoming: 6)

In actual practice, debate over this objection has tended to focus on questions in the history of philosophy. Clearly, numerous philosophers from Aristotle through Nietzsche were deeply concerned with empirical questions about human nature, so it might seem that the default view, at least in the absence of any counterarguments, should be that work on these issues can indeed count as philosophy. The key question, then, is whether there are any legitimate counterarguments.

One possible argument would be that although the people we now regard as philosophers did work on these issues, this aspect of their work should not be regarded as falling within the discipline of philosophy. Anthony Appiah questions this gambit:

You would have a difficult time explaining to most of the canonical philosophers that this part of the work was echt philosophy and that part of their work was not. Trying to separate out the “metaphysical” from the “psychological” elements in this corpus is like trying to peel a raspberry. (Appiah 2008: 13)

According to this response, there is a well-established practice within the history of philosophy of exploring empirical and psychological questions, and it is actually the idea of carefully separating the psychological from the philosophical that should be regarded as a departure from philosophical tradition.

More recent work on these issues has been concerned especially with the early modern period. It has been noted that some of the most prominent philosophers in this period actually conducted experimental studies (Sytsma & Livengood 2015), and some explicitly referred to themselves as ‘experimental philosophers’ (Anstey & Vanzo 2016). Though contemporary experimental philosophy obviously differs in certain respects from these historical antecedents, one might argue that the work of contemporary experimental philosophers is best understood as a continuation of this broad historical tradition.

On the other side, it has been argued that this historical continuity picture fails to take account of a change in the use of the word ‘philosophy’ (Sorell forthcoming). In the Renaissance, physics was referred to as ‘philosophy,’ but we would not say that all research in contemporary physics belongs in the discipline of philosophy. Similarly, even if work on the psychology of moral judgment was historically classified as philosophy, one might think that it should not be regarded today as falling into the discipline of philosophy but rather into a distinct discipline.

Certainly, partisans on both sides of this debate should agree that the boundaries of a discipline can change over time, but this point cuts both ways. Just as the boundaries of a discipline may have changed in the past, they can change in the future. It will therefore be interesting to see how the boundaries of the discipline of philosophy evolve over the course of the next few decades and how this evolution impacts the status of experimental philosophy.

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Neo-Behaviorism and Learning Theory

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• Edwin R. Guthrie ,
• Clark L. Hull ,
• B. F. Skinner ,
• Edward C. Tolman ,
• Gregory Razran ,
• John Dollard ,
• Neal E. Miller ,
• O. Hobart Mowrer &
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The most persistent advocate of conditioning is undoubtedly E. R. Guthrie. Guthrie considers association by contiguity in time, or “simultaneous conditioning,” the most general law in psychology. “Stimuli acting at the time of a response tend on their recurrence to evoke that response,” he says. Any other type of behavior can be derived from simultaneous conditioning. Especially the processes of learning represent the general law of stimulaneous conditioning or association by contiguity in time of stimuli and responses. “The outstanding characteristics of learning which have been expressed in forms of frequency, intensity, irradiation, temporary extinction, conditioned inhibition, forgetting, forward and backward conditioning, and so on, are all derivable from this more general law,” 1 Guthrie believes.

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Theoretical Behaviorism

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Skinner, B. F. The verbal summator and a method for the study of latent speech. J. Psychol, 1936 2 , 71–107.

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Skinner, B. F. The behavior of organisms: An experimental analysis. New York: Appleton-Century-Crofts, 1938.

Skinner, B. F. The alliteration in Shakespeare’s sonnets: A study of literary behavior. Psychol Rec., 1939, 3 , 186–192.

Skinner, B. F. A method of maintaining an arbitrary degree of hunger. J. comp. Psychol, 1940, 30 , 139–145.

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Skinner, B. F. Contingencies of reinforcement: A theoretical analysis. New York: Appleton-Century-Crofts, 1969.

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Tolman, E. C. Instinct and purpose. Psychol. Rev., 1920, 27 , 217–233.

Tolman, E. C. Can instincts be given up in psychology ? J. abnorm. soc. Psychol., 1922, 17 , 139–152.

Tolman, E. C. A new formula for behaviorism. Psychol. Rev., 1922, 29 , 44–53.

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Tolman, E. C. The acquisition of string-pulling by rats-conditioned response or sign-gestalt? Psychol Rev., 1937, 44, 195–211.

Tolman, E. C. An operational analysis of “demands. ” Erkenntnis, 1937, 6, 383–392.

Tolman, E. C. The action of punishment in acceleration learning. J. comp. Psychol, 1938, 26, 187–200.

Tolman, E. C. The determiners of behavior at a choice point. Psychol Rev., 1938, 45, 1–41.

Tolman, E. C. A reply to Professor Guthrie. Psychol Rev., 1938, 45, 163–164.

Tolman, E. C. The law of effect. Psychol Rev., 1938, 45, 200–203.

Tolman, E. C. Prediction of vicarious trial and error by means of the schematic sowbug. Psychol Rev., 1939, 46, 318–336.

Tolman, E. C. Spatial angle and vicarious trial and error. J. comp. Psychol., 1940, 30, 129–135.

Tolman, E. C. Discrimination vs. learning and the schematic sowbug. Psychol. Rev., 1941, 48, 367–382.

Tolman, E. C. Motivation, learning and adjustment. Proc. Amer. philos. soc., 1941, 84, 543–563.

Tolman, E. C. A drive-conversion diagram. Psychol. Rev., 1943, 50, 503–513.

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Tolman, E. C. Studies in spatial learning. II. Place learning vs. response learning. J. exp. Psychol, 1946, 36, 221–229.

Tolman, E. C. Studies in spatial learning. IV. The transfer of place learning to other starting paths. J. exp. Psychol., 1947, 37, 39–47.

Tolman, E. C. Studies in spatial learning: VII. Place and response learning under different degrees of motivation. J. exp. Psychol., 1949, 39, 653–659.

Tolman, E. C. Discussion: Interrelationships between perception and personality. J. Pers., 1949, 18, 48–50.

Tolman, E. C. The nature and functioning of wants. Psychol. Rev., 1949, 56, 357–369.

Tolman, E. C. The psychology of social learning. J. soc. Issues, 1949, 5, Suppl. No. 3, 5–18.

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Tolman, E. C., & Davis, F. C. A note on the correlations between two mazes. J. comp. Psychol, 1924, 4, 125–135.

Tolman, E. C., & Geier, F. M. Goal distance and restless activity. I. The goal gradient of restless activity. J. comp. Psychol, 1943, 35, 197–204.

Tolman, E. C., & Gleitman, H. Studies in learning and motivation: I. Equal reinforcements in both end-boxes, followed by shock in one end-box. J. exp. Psychol, 1949, 39, 810–819.

Tolman, E. C., Hall, C. S., & Bretnall, E. P. A disproof of the law of effect and a substitution of the laws of emphasis, motivation, and disruption. J. exp. Psychol, 1932, 15, 601–614.

Tolman, E. C., & Honzik, C. H. Degrees of hunger, reward and non-reward, and maze learning in rats. Univer. Calif. Publ. Psychol, 1930, 4, 241–257.

Tolman, E. C., & Honzik, C. H. Introduction and removal of reward and maze performance of rats. Univer. Calif. Publ Psychol, 1930, 4, 257–275.

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Tolman, E. C., & Minium, E. VTE in rats: Overlearning and difficulty of discrimination. J. comp. Psychol, 1942, 34, 301–306.

Tolman, E. C., & Ritchie, B. F. Correlation between VTE’s on a maze and on a visual discrimination apparatus. J. comp. Psychol, 1943, 36, 91–98.

Tolman, E. C., Ritchie, B. F., & Kalish, D. Studies in spatial learning. V. Response learning vs. place learning by the non-correction method. J. exp. Psychol, 1947, 37, 285–292.

Tolman, E. C., & Robinson, E. W. The effect of degrees of hunger upon the order of elimination of long and short blinds. Univer. Calif. Publ Psychol, 1930, 4, 189–202.

Tolman, E. C., & Sams, C. F. Time discrimination in white rats. J. comp. Psychol, 1925, 5, 255–263.

Tolman, E. C., & White, A. E. A note on the elimination of short and long blind alleys. J. comp. Psychol, 1923, 3, 327–332.

Voeks, V. W. Formalization and clarification of a theory of learning. J. Psychol, 1950, 30, 341–362.

Walker, E. L. The acquisition of a response to food under conditions of food satiation. Amer. Psychologist, 1948, 3, 239. (Reported by title only).

Walker, E. L. Drive specificity and learning. J. exp. Psychol, 1948, 38, 39–449.

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White, R. K. The case for the Tolman-Lewin interpretation of learning. Psychol. Rev., 1943, 50, 157–186.

Wilcoxen, H. C., Hays, R., & Jull, C. L. A preliminary determination of the functional relationship of effective reaction potential (sEr) to the ordinal number of Vincentized extinction reactions (n). J. exp. Psychol, 1950, 40, 194–199.

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Williams, S. B. Resistance to extinction as a function of the number of reinforcements. J. exp. Psychol, 1938, 23, 506–522.

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Learning Theory Influenced by Psychoanalysis

Dollard, J., Doob, L. W., Miller, N. E., Mowrer, O. H., Sears, R. R., Ford, C. S., Hovland, C. I., & Sollenberger, R. T. Frustration and aggression. New Haven: Yale University Press, 1939.

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Dollard, J., & Miller, N. E. Personality and psychotherapy. New York: McGraw-Hill, 1950.

Eriksen, C. W. Defense against ego-threat in memory and perception. J. abnorm. soc. Psychol, 1942, 47, 230–235.

Friedman, S. M. An empirical study of the castration and oedipus complexes. Genet. Psychol Monogr., 1952, 46, 61–130.

Hilgard, E. R. Theories of learning. ( Rev. ed.) New York: Appleton-Century-Crofts, 1956.

Hilgard, E. R., Kubie, L. S., & Pumpian-Mindlin, E. Psychoanalysis and science. Stanford, Calif.: Stanford University Press, 1952.

Hull, C. L. Modern behaviorism and psychoanalysis. Trans. N.Y. Acad. Sci., 1939, 1, 78–82.

Maier, N. R. F. Frustration, the study of behavior without a goal New York: McGraw-Hill, 1949.

Miller, N. E. Experiments relating Freudian displacement to generalization of conditioning. Psychol Bull, 1939, 36, 516–517.

Miller, N. E. The frustration-aggression hypothesis. Psychol Rev., 1941, 48, 337–342.

Miller, N. E. Experimental studies in conflict. In J. McV. Hunt (Ed.), Personality and the behavior disorders. New York: Ronald, 1944.

Miller, N. E. Theory and experiment relating psychoanalytic displacement to stimulus- response generalization. J. abnorm. soc. Psych., 1948, 43, 155–178.

Miller, N. E., & Dollard, J. Social learning and imitation. New Haven: Yale University Press, 1941.

Misbach, L. Psychoanalysis and theories of learning. Psychol. Rev., 1948, 55, 143–156.

Mowrer, O. H. Anxiety reduction-and learning. J. exp. Psychol., 1940, 27, 497–516.

Mowrer, O. H. An experimental analogue of “repression” with incidental observations on “reaction formation.” J. abnorm. soc. Psychol., 1940, 35, 56–87.

Mowrer, O. H. On the dual nature of learning: a re-interpretation of “conditioning” and “problem-solving.” Harvard educ. Rev., 1947, 17, 102–148.

Mowrer, O. H. Learning theory and the neurotic paradox. Amer. J. Orthopsychiat., 1948, 18, 577–610.

Mowrer, O. H. Learning theory and personality dynamics. New York: Ronald, 1950.

Mowrer, O. H. Two-factor learning theory: Summary and comment. Psychol. Rev., 1951, 58, 350–354.

Mowrer, O. H. Psychotherapy: Theory and research. New York: Ronald, 1953.

Mowrer, O. H. Learning theory: historical review and re-interpretation. Harvard educ. Rev., 1954, 24, 37–58.

Murray, E. J., & Berkum, M. M. Displacement as a function of conflict. J. abnorm. soc. Psychol., 1955, 51, 47–56.

Sarbin, T. R. Mental age changes in experimental regression. J. Pers., 1950, 19, 221–228.

Sears, R. R. Experimental studies of projection: I. Attribution of traits. J. soc. Psychol., 1936, 7, 151–163.

Sears, R. R. Experimental studies of projection: II. Ideas of reference. J. soc. Psychol., 1937, 8, 389–100.

Sears, R. R. Survey of objective studies of psychoanalytic concepts. Soc. Sci. Res. Counc., 1943.

Sears, R. R. Effects of frustration and anxiety on fantasy aggression. Amer. J. Orthopsychiat., 1951, 21, 498–505.

Sears, R. R. Social behavior and personality development. In T. Parsons & E. A. Shils (Eds.), Toward a general theory of action. Cambridge, Mass.: Harvard University Press, 1951.

Sears, R. R. A theoretical framework for personality and social behavior. Amer. Psychologist, 1951, 6, 476–482.

Sears, R. R., & Wise, G. W. The relation of cup feeding in infancy to thumb-sucking and the oral drive. Amer. J. Orthopsychiat., 1950, 20, 123–138.

Some Problems in the Theory of Learning

Estes, W. K., Koch, S., MacCorquodale, K., Meehl, P. E., Mueller, C. G., Jr., Schoenfeld, W. N., & Verplanck, W. S. Modern learning theory. New York: Appleton-Century-Crofts, 1954.

Hilgard, E. R. Theories of learning. (2nd ed.) New York: Appleton-Century-Crofts, 1956.

Kentucky Symposium. Learning theory, personality theory, and clinical research. New York: Wiley, 1954.

Melton, A. W. Learning. In W. S. Monroe (Ed.), Encyclopedia of educational research. (Rev. ed.) New York: Macmillan, 1950.

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Guthrie, E.R. et al. (1981). Neo-Behaviorism and Learning Theory. In: Contemporary Theories and Systems in Psychology. Springer, Boston, MA. https://doi.org/10.1007/978-1-4684-3800-0_4

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Evolution beyond neo-Darwinism: a new conceptual framework

Affiliation.

• 1 Department of Physiology, Anatomy and Genetics, Parks Road, Oxford OX1 3PT, UK [email protected].
• PMID: 25568446
• DOI: 10.1242/jeb.106310
• Evolution beyond neo-Darwinism: a new conceptual framework. Noble D. Noble D. J Exp Biol. 2015 Apr 15;218(Pt 8):1273. doi: 10.1242/jeb.123125. J Exp Biol. 2015. PMID: 25911737 Free PMC article. No abstract available.

Experimental results in epigenetics and related fields of biological research show that the Modern Synthesis (neo-Darwinist) theory of evolution requires either extension or replacement. This article examines the conceptual framework of neo-Darwinism, including the concepts of 'gene', 'selfish', 'code', 'program', 'blueprint', 'book of life', 'replicator' and 'vehicle'. This form of representation is a barrier to extending or replacing existing theory as it confuses conceptual and empirical matters. These need to be clearly distinguished. In the case of the central concept of 'gene', the definition has moved all the way from describing a necessary cause (defined in terms of the inheritable phenotype itself) to an empirically testable hypothesis (in terms of causation by DNA sequences). Neo-Darwinism also privileges 'genes' in causation, whereas in multi-way networks of interactions there can be no privileged cause. An alternative conceptual framework is proposed that avoids these problems, and which is more favourable to an integrated systems view of evolution.

Keywords: Epigenetics; Genetic program; Lamarck; Modern synthesis; Systems biology.

© 2015. Published by The Company of Biologists Ltd.

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• Neo-Darwinism is just fine. Williams CA. Williams CA. J Exp Biol. 2015 Aug;218(Pt 16):2658-9. doi: 10.1242/jeb.125088. J Exp Biol. 2015. PMID: 26290594 No abstract available.
• Central tenets of neo-Darwinism broken. Response to 'Neo-Darwinism is just fine'. Noble D. Noble D. J Exp Biol. 2015 Aug;218(Pt 16):2659. doi: 10.1242/jeb.125526. J Exp Biol. 2015. PMID: 26290595 No abstract available.

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Introduction: Neo-Avant-Garde, Why Bother?

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The introduction deals with the relevance of the term ‘neo-avant-garde’ for literature. The term is used most typically for experimental art forms from the long sixties (1955-1975), but sometimes extended to present-day pioneering art. The text relates the neo-avant-garde to three other dominant innovations of the twentieth century, namely the historical avant-garde, modernism and postmodernism. First, the introduction looks at influential studies concerning the neo-avant-garde by scholars such as Peter Bürger, Hal Foster, Benjamin Buchloh and Dietrich Scheunemann. It then presents four parameters to define the term neo-avant-garde as it is used in the present volume and it draws attention to the pros and cons of its usage. Finally, the introduction points to the relevance of the neo-avant-garde frame for the study of the various literary traditions that are discussed in this book, especially based on – but not restricted to – American, Austrian, British, Caribbean, Dutch, French and German examples. In this way, the text explores the possible relevance of neo-avant-garde productions for contemporary artists and writers.

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Neoantigens: From Discovery to Cancer Immunotherapy

This article was originally posted on March 16, 2020.|

Neoantigen, also called tumor-specific antigen (TSA), is a repertoire of peptides that arise as the result of DNA mutations in tumor cells. Unlike the other two common types of tumor antigens, named tumor-associated antigens (TAAs) and cancer-germline antigens (CGAs), tumor neoantigen is the truly foreign protein and entirely absent from normal human organs or tissues. As a result, targeting neoantigens could allow the patient’s immune system to exclusively find and attack cancer cells without harming normal healthy cells. Decades of work has increasingly shown their potential for cancer immunotherapy. 

We will systematically introduce the history of understanding and identification of neoantigens, how to develop personalized neoantigen-based vaccines, how neoantigen vaccines work, their role on current cancer immunotherapies and the key players in the industry.

Chapter 1. What are neoantigens and how to identify them?

Genetic instability of tumor cells often leads to a large number of mutations, and expression of non-synonymous mutations can produce tumor-specific antigens, called neoantigens [1]. Some of the neoantigens can be expressed, processed and presented on the cell surface, and subsequently recognized by T cells in the context of major histocompatibility complexes (MHCs) molecules. Because neoantigens are not expressed in normal tissues, neoantigen-specific T cells are not subject to central and peripheral tolerance, and also lack the ability to induce normal tissue destruction. As a result, neoantigens appear to be the ideal targets for T cell-based cancer immunotherapy [2]. 

How are neoantigens discovered?

In the early twentieth century, many findings revealed that the immune system can recognize and eliminate tumor cells. However, the nature of antigens that could trigger antitumor immune response was unclear during that time. Until 1988 De Plaen and colleagues identified the first neoantigen that can be recognized by T cells in a mouse tumor model by using cDNA library screening [3, 4]. They found that only one nucleotide differed between the normal and tumor gene and this mutation produced an amino acid change. After that, a series of neoantigens derived from somatic mutations were identified in various human tumors including melanoma and renal cell carcinoma [3].

Fig. 1 Historical overview of tumor neoantigens. The figure is originally published by Tao Jiang et al [3] and designed by MolecularCloud.

How to identify neoantigens in tumor cells?

According to articles published during 1995-2013, most of the neoantigens were identified by cDNA library screening. In this approach, cDNA library and MHC molecules were over-expressed in cell lines, and then co-cultured with T cells to identify antigens that could induce the T cell activation [2]. However, this method is labor-intensive, expensive and cannot effectively identify all tumor antigens. The rapid development of next-generation sequencing (NGS) technology makes it possible to rapidly compare the DNA sequences of tumor cells and normal cells and identify tumor-specific mutations. In 2012, NGS technology was firstly reported to be applied in identifying neoantigens in mouse tumor models [5, 6] and soon widely used. Currently, based on the whole exome sequencing (WES) technology and constantly optimized bioinformatics algorithms, a lot of neoantigens have been identified with high efficiency, wide coverage, and low false negative rate [1]. Even though sophisticated machine learning methods are used to winnow down candidate numbers returned by prediction algorithms, scientists have to go back to wet lab experiments to validate that the potential neoantigens are active and could be able to trigger bona fide antitumor responses in patients [7].

Fig. 2 Overview of neoantigen identification using tumor WES [8].

Step 1: Identification of tumor non-synonymous mutations (NSM). WES is performed on tumor and normal DNA to identify tumor-specific NSM. When available, RNA-seq is used to select mutations that are expressed. 

Step 2: Selection of candidate neoantigens. Once NSMs are identified, three strategies can be used to select the list of candidate neoantigens that will be assessed for immunogenicity. 

Step 3: Evaluation of immunogenicity of candidate neoantigens. Finally, the immunogenicity of the selected candidate peptides is evaluated with different immunological screening assays. 

Immunotherapy

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• Published: 06 January 2023

Neoantigens: promising targets for cancer therapy

• Na Xie 1   na1 ,
• Guobo Shen 1   na1 ,
• Wei Gao 2 ,
• Zhao Huang 1 ,
• Canhua Huang   ORCID: orcid.org/0000-0003-2247-7750 1 &
• Li Fu   ORCID: orcid.org/0000-0003-2643-6278 3  

Signal Transduction and Targeted Therapy volume  8 , Article number:  9 ( 2023 ) Cite this article

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• Biomaterials
• Predictive markers
• Predictive medicine
• Tumour immunology

Recent advances in neoantigen research have accelerated the development and regulatory approval of tumor immunotherapies, including cancer vaccines, adoptive cell therapy and antibody-based therapies, especially for solid tumors. Neoantigens are newly formed antigens generated by tumor cells as a result of various tumor-specific alterations, such as genomic mutation, dysregulated RNA splicing, disordered post-translational modification, and integrated viral open reading frames. Neoantigens are recognized as non-self and trigger an immune response that is not subject to central and peripheral tolerance. The quick identification and prediction of tumor-specific neoantigens have been made possible by the advanced development of next-generation sequencing and bioinformatic technologies. Compared to tumor-associated antigens, the highly immunogenic and tumor-specific neoantigens provide emerging targets for personalized cancer immunotherapies, and serve as prospective predictors for tumor survival prognosis and immune checkpoint blockade responses. The development of cancer therapies will be aided by understanding the mechanism underlying neoantigen-induced anti-tumor immune response and by streamlining the process of neoantigen-based immunotherapies. This review provides an overview on the identification and characterization of neoantigens and outlines the clinical applications of prospective immunotherapeutic strategies based on neoantigens. We also explore their current status, inherent challenges, and clinical translation potential.

Similar content being viewed by others

Identification of neoantigens for individualized therapeutic cancer vaccines

Targeting public neoantigens for cancer immunotherapy

Immunogenomics in personalized cancer treatments

Introduction.

Accumulating genetic alterations in cancers result in the production of tumor-specific antigens (TSAs) or neoantigens, which can be presented by major histocompatibility (MHC) molecules of tumor cells. 1 , 2 , 3 , 4 , 5 , 6 These tumor-specific peptide-MHC (pMHC) complexes are recognized by T cells and trigger an anti-cancer immune response in patients. However, it has been discovered that cancer cells have evolved resistance to anti-cancer immunity. 7 These immune escape mechanisms can be reversed by cancer immunotherapies, including the use of tumor vaccines to improve antigen presentation, the increase of anti-tumor T cells via adoptive transferring of tumor-infiltrating lymphocytes (TILs) and T cell receptor (TCR)-transduced T cells, restoring the effector capacity of CD8 + T cells by immune checkpoint blockades (ICBs), increasing the immune recognition of tumors with bispecific antibodies (bsAbs) and chimeric antigen receptor (CAR)-transduced T cells, and modulating the tumor immune microenvironment. 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 A variety of clinical studies examined the efficacy of immunotherapies targeting tumor-associated antigens (TAAs), like vaccines against ERBB2, MUC1, and hTERT. TAAs exhibit abnormal expression in malignancies or are only produced during specific stages of differentiation, whereas their expression in normal tissues is extremely limited. The prevalence of TAAs among cancer patients makes them public targets for off-the-shelf immunotherapies. However, as TAAs are non-mutated self-antigens, central T cell tolerance may contribute to the largely poor T cell responses observed in clinical trials. 20 , 21 Nonetheless, the widespread use of tumor immunotherapies has been hindered by a shortage of targetable antigens in various cancers. 22

Neoantigens are self-antigens generated by tumor cells because of genomic mutations. Besides, neoantigens can also derive from unique proteins or peptides produced by dysregulated RNA splicing and disordered post-translational protein modification in non-virus-associated malignancies. For cancers with a viral infection, such as HPV-positive cervical cancer and EBV-associated nasopharyngeal carcinoma, neoantigens can also be created by virally encoded open reading frames (ORFs). 22 , 23 , 24 Compared with other types of tumor antigens, such as cancer-testis antigens (CTAs) and TAAs, neoantigens offer a distinct advantage in their unique tumor-specific and absence in normal tissues, presenting ideal targets for effectively personalized treatment of tumors (Table 1 ). 25 , 26 Notably, T cells specialized for neoantigens can bypass negative selection effects in the thymus due to the highly antigenic neoantigens acquired through somatic tumor mutations. Increasing the pool of neoantigen-specific T cells due to this ability to avoid T cell central tolerance makes it possible to enhance tumor-specific immune responses. 27 , 28 , 29 Furthermore, the capacity of immunotherapy-enhanced neoantigen-specific T cell responses in enduring and giving post-treatment immunological memory offers hope for long-term protection against disease recurrence. 30

Currently, advanced techniques, including tumor gene sequencing, neoantigen discovery, and neoantigen-based immune therapeutic product preparation, play a significant role in the development of personalized cancer vaccines (PCVs) and adoptive cell therapy (ACT). 19 Next-generation sequencing (NGS) has permitted the fast and cost-effective detection of tumor-specific mutations in individual patients. In addition, the development of algorithms for predicting MHC molecules-binding epitopes has made it possible to identify possibly immunogenic neoepitopes. 31 These technological developments have enabled the production of personalized immunotherapy, specifically targeting tumors in individual patients (Fig. 1 ). However, some limitations such as the costs and time in the process of personalized immunotherapeutic products, and the ideal platform for neoantigen identification, need further improvement. With the continuous development and wide cross-integration of biotechnology, immunology, materials science, chemistry, and artificial intelligence, additional neoantigens will be identified and employed in tumor immunotherapy. 13 , 32

Historical overview of tumor-specific neoantigens. Based on keyword searches in the PubMed database using the terms "neoantigen" or "neoepitope", the number of articles from 1965 to 2022 is displayed in the column chart

Herein, we provide a comprehensive summary of the source and biological function of neoantigens, potential neoantigen prediction tools, and clinical applications of neoantigen-based immunotherapy strategies. Moreover, we also discuss the opportunities and limitations associated with the clinical application of immunotherapies based on neoantigens and propose some potential solutions.

The source of neoantigens

Neoantigens are identified as foreign proteins that are absent in normal tissues, but can arise from tumors through various mechanisms, such as genomic mutation, aberrant transcriptomic variants, post-translational modifications (PTMs), and viral ORFs (Fig. 2 ). 27 , 33

Overview of the neoantigen production and presentation. Neoantigens can develop at the genomic level through SNVs, base INDELs and gene fusions, at the transcriptomic level through alternative splicing, polyadenylation (pA), RNA editing and allegedly non-coding regions, and at the proteomic level through dysregulated translation and PTMs. The integrated viral ORF is another source of neoantigens for cancers linked to viruses. The mutant peptides created by the proteasome-mediated breakdown of endogenous proteins are subsequently transported to the endoplasmic reticulum (ER) via transporters associated with antigen processing (TAP), where they may be loaded onto MHC-I. MHC-II dimers are assembled and bound to the invariant chain (Ii) in the ER. The Ii-MHC-II complex can be directly transported or sometimes indirectly internalized from the cell surface to the MHC-II compartment (MIIC), where the degradation of Ii by a series of endosomal proteases releases the MHC-II for binding a specific peptide derived from a mutant protein broken down in the endosomal pathway. These pMHC complexes will then traffic to the cell surface where they are recognized by T cells

Genomic variants

Somatic genomic alterations, including single-nucleotide variants (SNVs), base insertions and deletions (INDELs) and gene fusions, are the main factors that promote the production of tumor neoantigens. 8 , 34 , 35 , 36 , 37

SNVs are the most prevalent type of mutation at the genomic level in tumor cells; they can yield variant peptides distinct from wild-type proteins that are presented by MHC-I as specific antigens. 19 , 38 Up to hundreds of non-synonymous somatic mutations per cancer patient have been recorded, resulting in an average of 150 potential neoantigenic peptides per individual. For example, a total of 231 non-synonymous SNVs, 13 gene fusions and 21 INDELs have been identified in Ph-negative myeloproliferative neoplasms (MPNs). 39 Using The Cancer Genome Atlas (TCGA) database, 933,954 expressed neoantigens in 20 solid tumors have been characterized, which originates from 893,960 somatic mutations with a varied median frequency of neoantigens across cancers. Only 24 of these neoantigens, including those arising from mutations in driver genes like PIK3CA, RAS, and BRAF, are shared by at least 5% of patients with the same or distinct cancers. 38 , 40 Notably, relapsed populations may have greater tumor mutation burden (TMB) and more novel potential neoantigens than newly diagnosed patients. In patients with multiple myeloma, only two potential neoantigens, UBR4 and PRKDC, were detected in both relapsed and newly diagnosed patients. 41 Therefore, the SNV neoantigens landscape is highly variable between different cancer types and different stages of the same cancer type (Table 2 ).

SNVs can also arise in mitochondrial DNA (mtDNA), which is found in most cancer cells and correlates with alterations in tumor metabolic profiles and cancer cell metastatic capacity. 42 , 43 , 44 , 45 Despite the fact that the compact normal human mitochondrial genome, a 16,569 base-pair circular DNA, encodes only 13 protein subunits of the electron transport chain, it may account for around 30% of total mRNA transcripts in certain organs. 46 mtDNA has a 10- to 20-fold greater mutation rate than nuclear DNA. 47 , 48 Both mouse and human immune systems were able to recognize and respond to mtDNA single nucleotide polymorphisms (SNPs)-derived peptides, suggesting that individual SNPs in mtDNA are adequate to generate immunogenic neoantigens. Thus, non-synonymous SNPs in mtDNA may yield a substantial quantity of mutant peptides, offering an additional source of neoantigens. 49 , 50 , 51

INDEL mutations are mainly caused by the insertion or deletion of base pairs in the genome, which frequently lead to non-synonymous novel ORFs, also known as frameshift mutations. 27 , 52 Frameshift INDELs can generate more types of neoantigens with increased MHC-I binding affinity, suggesting a higher immunogenic mutation type compared to SNVs (Table 2 ). 53 , 54 Especially in renal cell carcinoma with a medium-range mutational burden, about 16% of predicted neopeptides are derived from frameshift INDELs, whereas 21% of T cell-recognized neoepitopes are arising from frameshift INDELs, indicating that frameshift-derived neoepitopes have a greater immunogenic potential. 53 , 55

Similar to SNV neoantigens, INDEL neoantigens are more prevalent in cancers with microsatellite instability-high (MSI-H) due to the lack of DNA mismatch repair (MMR) mechanisms. 27 , 56 , 57 , 58 As the evolution of MMR-deficient cancers is mainly triggered by mutations that inactivate tumor suppressor genes (TSGs) containing coding microsatellites, frameshift peptide neoantigens are more frequently shared among MMR-deficient cancers (e.g., endometrial, colorectal, and gastric) than missense mutation-derived neoantigens. 59 , 60 , 61 , 62 , 63 , 64 Frameshift INDEL neoantigen burden has a strong correlation with immunological response. 38 MSI colorectal cancers with frameshift mutations have a larger proportion of TILs than other colorectal cancers. 64 , 65 , 66 Similarly, shared immunogenic frameshift peptide neoantigens can be produced as a result of recurrent frameshift mutations, offering excellent candidates for immunotherapy against MSI cancers. 59 , 61 , 62 , 63 , 64 The combination of four frameshift peptide neoantigens dramatically boosts neoantigens-specific adaptive immunity, decreases intestinal tumor burden, and prolongs the overall survival in the VCMsh2-driven intestinal cancer mouse model, which can be further strengthened by naproxen. 67 , 68 According to a clinical phase I/IIa trial, the frameshift peptide neoantigenic vaccine is well tolerated systemically and triggers immune responses regularly, representing a promising new strategy for the treatment and even prevention of MMR-deficient malignancy. 60 These findings showed that an off-the-shelf vaccine is feasible for treating and preventing cancers with frameshift mutations and neoantigenic peptides because of MSI. 69

The frameshift INDEL neoantigen burden is also a novel biomarker for ICB response. 27 , 38 , 70 , 71 , 72 INDEL frameshift mutations are supposed to produce more immunogenic neoantigens, hence improving response to ICBs. When frameshift mutations are present, the progression-free survival of patients receiving ICBs is significantly prolonged. Further evidence that frameshift mutations may play a predictive role in ICB response comes from the considerable discrepancies in overall response rates and disease control rates observed in non-small cell lung cancer (NSCLC) patients with frameshift mutations. 73 In addition, ICBs can also strengthen the immune response to frameshift neoantigens. The frameshift mutation in CALR elicits both CD4 + and CD8 + T cell responses, which are inhibited by the expression of PD-1 or CTLA4. Importantly, blocking PD-1 and CLTA4 ex vivo and PD-1 in vivo with pembrolizumab restores frameshift neoantigen-specific T cell immunity in myeloproliferative neoplasms. 39 , 74

Gene fusions

Gene fusion is another important type of mutation in tumors that may provide many neoantigens, which can be generated by mesenchymal deletion, chromosomal translocation or chromosomal inversion. 28 , 75 , 76 , 77 Studies have shown that polypeptides derived from the different fusion regions of the proteins can be recognized by the patient’s own T cells, such as the BCR-ABL fusion protein produced by the translocation between chromosomes 9 and 22 in chronic myeloid leukemia (CML) patients and SYT-SSX1 fusion proteins produced by X and 18 chromosomal translocations in synovial sarcoma patients. Even in some tumors with low TMB and limited immune infiltration, neoantigens generated by gene fusion are still able to activate cytotoxic T cells. 21 , 78 , 79 In a comprehensive study of fusion neoantigens in tumors, analyses of three datasets from the TCGA database found that fusion mutations could generate more novel ORFs, yielding 6-fold neoantigens and 11-fold specific candidate neoantigens more than SNVs and INDELs. The fusion neoantigens are more likely to induce stronger immune response than the neoantigens produced by SNVs and INDELs, and the neoantigen produced by frameshift fusion has better immunogenicity than the in-frame fusion neoantigen (Table 2 ). Similar to the candidate neoantigen burden of SNVs and INDELs, fusion neoantigen burden was closely related to fusion mutation burden, especially in microsatellite stable tumors with higher fusion mutation burden. 19 , 80 An expanded study of 30 different tumor types revealed that 24% of fusion protein-expressing cancers contained neoepitopes resulting from the fusion, and these neoantigens were predicted to bind to patient-specific MHC-I. 78 , 81 It is worth noting that the repetition rate of fusion neoantigens between different patients is extremely low. According to statistics, only 5.8% of fusion neoantigens in the TCGA database repeat between patients, and these neoantigens usually have very low immunogenic potential. 80 In addition, malignancies with greater immune-depleted microenvironments or human leukocyte antigen (HLA) loss exhibited fusion neoantigens more frequently. In melanomas treated with anti-PD-1 therapy, the removal of tumor cells carrying fusion-derived neoantigens demonstrated a negative immune surveillance selective pressure on these neoantigens. 78 , 82 According to FACETS analysis of the TCGA exome data, 18.4% of cases had a loss of heterozygosity (LOH) in the HLA, which increased the possibility that a fusion neoantigen would be present. 78 , 83 These results demonstrate the significance of gene fusions as a source of tumor-specific neoantigens. 78 , 81

Gene fusion-derived neoantigens can elicit specific immune responses against tumors. 84 , 85 The fusion neoantigens, such as BCR-ABL, SYT-SSX1/SSX2, PAX3-FOXO1, TPM3/TPM4-ALK, and EWS-FLI1, showed immunogenic potential, providing the possible targets for immunotherapy to treat tumors. 86 , 87 CBFB-MYH11 fusion neoantigen is distributed on acute myeloid leukemia (AML) cells, which activates T cells and induces specific killing against AML, a cancer with low mutation frequency. 88 , 89 , 90 , 91 Two neoantigens, SS393 (GYDQIMPKK) and SS391 (PYGYDQIMPK), are derived from the SYT-SSX fusion neoantigen that is common in synovial sarcoma. These neoantigen peptides successfully induced synovial sarcoma-specific cytotoxic T lymphocytes (CTLs) that specifically killed HLA-A24-positive synovial sarcoma cells containing the SYT-SSX neoantigen as well as the target cells pulsed with these peptides. 92 , 93 , 94 A study on head and neck squamous cell carcinomas (HNSCC) found that the tumor’s immune response to anti-PD-1 therapy was mediated by neoantigens generated by DEK-AFF2 fusion. A DEK-AFF2-derived peptide (DKESEEEVS) enhanced T cell activation depending on MHC class when delivered to autologous peripheral blood mononuclear cells (PBMCs). 78 A comprehensive study of 33 tumor types found that various common recurrent fusion neoantigens, including TMPRSS2-ERG, MYB-NFIB, FGFR3-TACC3, EML4-ALK and CCDC6-RET. 95 TMPRSS2-ERG is the most common recurrent gene fusion that occurred in 38.2% of prostate cancer patients. Several high-affinity HLA-restricted epitopes were identified from the recurrent TMPRSS2-ERG type VI fusion, which could bind to HLA-A*02:01 in vitro and were recognized by CD8 + T cells. 96 The fusion of the proto-oncogene MYB with the transcription factor NFIB serves as a biomarker for adenoid cystic carcinoma, which occurs in 60% of cases. Three MYB-NFIB-derived peptides (QFIDSSWYL, SLASPLQPT and SLASPLQSWYL) and one NFIB-MYB-derived peptide (MMYSPICLTQT) can bind to HLA-A*02:01 to activate the immune system. 78 , 97 The EML4-ALK fusion gene is predominantly found in young, rarely/never smoker NSCLC patients, and ~5% of NSCLC patients have this fusion mutation. The use of EML4-ALK-derived peptides can stimulate specific CTL responses and have the potential to treat EML4-ALK-positive NSCLC. 98 Therefore, the neoantigens generated by fusion mutations greatly increase the capacity of the tumor-specific neoantigen repertoire, providing more potential targets or predictors for cancer immunotherapies. 8 , 78 , 81 , 88 , 96 , 99 , 100 , 101

Structural variants

Structural variant (SVs) is one of the most frequent forms of driver mutations in tumors, which can result in alterations in genome structure and then change the expression or function of genes to promote malignant transformation. SVs generally refer to genetic variants that are larger than 50 base pairs, such as insertions, deletions, inversions, translocations, duplications/amplifications, and chromosomal additions and deletions, as well as chromosomal rearrangements. 5 , 102 , 103 , 104 , 105 , 106 , 107 , 108 , 109 Among them, chromosomal rearrangement is the most complex (such as chromothripsis and chromoplexy), which is a prominent feature of tumors and plays a crucial role in the occurrence and immune recognition of various malignant tumors. 110 , 111 , 112 Chromosomal rearrangements are not easily detected by traditional DNA sequencing techniques but can be screened by WES methods like mate pair sequencing (MPseq). Potential neoantigens generated by chromosomal rearrangements have been identified by a combination of MPseq and RNA sequencing (RNA-seq) in malignant pleural mesothelioma (MPM) patients. Rearrangement-related neoantigens may generate MPM-specific immune responses in a manner similar to frameshift INDEL neoantigens. Specifically, neoantigens produced by SVs are predicted to be presented by tumors on MHC proteins, which are closely related to clonal expansion of TILs, and effector T cells against these neoantigens are found in the circulation of cancer patients. 110 , 113 Therefore, SV-derived neoantigens may also serve as valuable targets for anti-tumor immunotherapy.

Transcriptomic variants

Post-transcriptionally events offer the potential of a broadened neoantigen space. Alternative processing of mRNA, including alternative splicing events, polyadenylation (pA), RNA editing and allegedly non-coding regions, contributes to the diversity of tumor-specific neoantigens. 19 , 81 , 114 , 115 , 116

Transcript alternative splicing

The abnormal alternative mRNA splicing is another potential source of tumor-specific neoantigens. 22 , 117 RNA splicing process the premature mRNA into mature RNA with high efficiency and fidelity in normal cells. However, it may be induced by mutations in RNA cis-regulatory elements, trans-acting regulators or the core spliceosome. 117 , 118 , 119 The highly aberrant splicing events in tumors expand the scope of tumor-specific neoantigens, especially in tumors with low rates of copy number variation and somatic mutations. 23 , 117 , 120

Cis-acting mutations

Mutation at cis-acting elements generates potential neoantigens through altered splicing, including alternative 5’ and 3’ splice site determination, intron retention, exon skipping and mutually exclusive exons. 23 , 33 , 121 , 122 Intron retention is more prevalent in nearly all cancers compared with normal control tissues, even in the absence of mutations in genes encoding splicing factors. Normally, intron retention transcripts will be degraded by nonsense-mediated RNA decay (NMD). Neoantigens can still be generated from intron retention during their pioneer round of translation before being subjected to NMD. 33 , 117 , 123 , 124 Numerous exon-exon junctions that are unique in tumors have been identified through extensive study of the TCGA, most of which can express neoantigens. 117 , 125 , 126 The production of neoantigens from skipped exons, also known as “neojunctions”, occurs more frequently and is more likely to be shared among patients than those generated from SNV mutations. 29 , 117 , 127 , 128 A recent study has identified exitron splicing, a non-canonical splicing mechanism, as a new source of tumor neoantigens. Exitrons are exon-embedded cryptic introns distinguished from conventional introns in that they have both splicing (intron) and protein-coding (exon) potential while lacking stop codons or premature termination codons. Because tumor-specific exitron-spliced transcripts are far more likely to escape NMD than intron retentions, their overall expression is higher than retained introns. Accordingly, exitrons splicing creates more validated neoantigens with higher immunogenicity in malignancies with low TMB. 128 , 129

Trans-acting alterations in splicing factors

Trans-acting alterations, in which a somatic mutation in a splicing factor results in an altered splicing variant, induce the production of neoantigens throughout the genome. 130 In hematological malignancies, common mutations in spliceosomal components, including SRSF2, SF3B1, and U2AF1/2, raise the expression of splice variant mRNAs, resulting in the translation of TSAs and neoantigens. In addition to hematological tumors, a recent reassessment of pan-cancer data in the TCGA database has shown that the somatic alterations of splicing factors, including SF3B1, U2AF1, SRSF2 and Zinc Finger CCCH-Type, ZRSR2, U2AF2, SF1, PRPF8, and SF3A1, leading to the production of splicing variant-derived neoantigens across the genome in solid tumors. 22 , 27 , 117 , 119 , 131 , 132 , 133 , 134 , 135 , 136 , 137 , 138 Moreover, epigenetic alterations and PTMs of splicing factors might promote global splicing dysregulation. 139 Neoantigens derived from splicing variants due to mutation and dysregulated expression of splicing factors have facilitated the development of novel therapeutics for tumors. For example, mutated SF3B1 (a splicing factor in the spliceosome) in uveal melanoma generates tumor-specific neoantigens that activate specific CD8 + T cells to kill tumor cells. 140

Nonsense-mediated RNA decay (NMD)

Another important determinator for tumor-specific splicing variants is NMD, a highly conserved RNA turnover mechanism that preferentially destroys RNAs carrying premature translation termination codons. In cells with normal NMD function, a pioneer round of translation is required for initiating the NMD-mediated degradation of aberrant transcripts, which can lead to the production of small amounts of neoantigens. 23 , 123 , 141 , 142 Moreover, the NMD regulatory mechanism is frequently impaired in tumor cells, enabling aberrant transcripts to avoid degradation and potentially produce large amounts of neoantigens. For example, mutations in the highly conserved core NMD factor UPF1 are prevalent in pancreatic squamous cell carcinoma and lung adenocarcinoma, increasing the frequency of aberrant transcripts and neoantigen production. 143 , 144 , 145 A recent study demonstrated that NMD regulates the mutational profile of malignancies by preferentially suppressing the expression of TSGs rather than oncogenes. Further evidence for the beneficial effect of NMD on tumors comes from the observation that NMD frequently degrades mRNA encoding immunogenic neoantigen peptides. Accordingly, NMD inhibitory therapy may be beneficial in the treatment of a variety of cancers, including those capable of producing large numbers of mutated neoantigens. 146 , 147

Altogether, these studies highlight that alternative splicing of transcripts could promote the production of neoantigens. Even though the application of splicing variant neoantigens in personalized therapies has not yet been thoroughly investigated, screening new alternative splicing-based neoantigens as immunotherapeutic targets will benefit tumor patients. 22 , 23 , 27

Polyadenylation (pA) and RNA editing

Similar to RNA splicing, polyadenylation (pA) and RNA editing can alter the proteomic profile of tumor cells, thereby increasing the pool of potential immunotherapeutic targets. 81 , 148

Polyadenylation plays a critical role in the processing and maturation of most eukaryotic mRNAs, primarily by cleaving and adding a poly(A) tail at the 3’ end. Most alternative polyadenylation (APA) events occur in the 3ʹ untranslated region (UTR) of mRNA. APA can significantly affect post-transcriptional gene regulation in several aspects, including transcript stability, translation, cellular localization, and nuclear export. 149 , 150 , 151 , 152 , 153 , 154 Nonetheless, some APA events occur in the intronic region upstream of the last exon, which is called intronic polyadenylation (IPA). 155 , 156 IPA can result in the production of truncated or non-coding transcripts that have the potential to generate tumor-specific immunotherapeutic targets. A recent study used 3’ end sequencing technology to analyze normal and malignant B cells of 59 patients with chronic lymphocytic leukemia (CLL), the study found that IPA-induced mRNA and protein truncations are prevalent in CLL cells, mainly involving TSGs such as DICER and FOXN3, and even some oncogenes such as CARD11, MGA, and CHST11. 157 It is worth noting that 72% of the 190 TSGs found in hematological tumors are only truncated in solid tumors. 158 In tumors, when a specific IPA event occurs in the coding region, genes upstream of the new pA site and downstream of the closest 5ʹ splice site are translated, creating neoantigens that can be presented by MHC and recognized by the immune system. 81 By comparing RNA-seq data between tumor and normal tissue samples from various cancers, more neoantigens created by IPA might be identified, providing prospective targets for cancer immunotherapy.

RNA editing is an important pre-mRNA processing method that can induce non-synonymous substitutions by altering specific nucleotides in the RNA sequence, resulting in the production of new proteins. 38 , 159 Similar to splicing and polyadenylation, RNA editing events frequently occur in a variety of tumors. 160 , 161 , 162 , 163 , 164 Adenosine-to-inosine (A-to-I) editing is the most prevalent type of RNA editing in mammals, and millions of such sites have been found in human genes. Protein peptides produced by A-to-I editing can be presented by MHC-I molecules, which further induce the activation of specific CD8 + T cells, suggesting that these novel peptides are immunogenic and can activate the immune system. 165 , 166 , 167 , 168 , 169 Nevertheless, it must be emphasized that these peptides are not necessarily tumor-specific, as RNA editing can also occur in normal tissues. Therefore, more in-depth research and advanced prediction methods are needed to identify tumor-specific RNA editing protein products for immunotherapy. 81 , 170

Allegedly non-coding regions

Given that 99% of tumor-specific mutations occur in non-coding regions of genes, and exonic regions account for only 2% of the entire human genome, the screening for neoantigens only derived from mutations in exonic regions is limited. Recent studies showed that many regions previously defined as non-coding are now found to have coding functions. Therefore, by studying these newly defined genes with coding capacity, researchers have discovered many novel antigenic peptides that can be presented by MHC-I, and some antigens have been confirmed as target for TIL immunotherapy. 171 , 172 , 173 These MHC-I-associated peptides (MAPs) derived from genes at non-coding regions expand the range of CD8 + T cell immune surveillance from 2% (the proportion of the human genome in exons) to 75%. 52 , 172 , 174 , 175 According to a proteogenomic profiling of non-canonical proteins, 60% of non-canonical proteins are encoded by genes that were considered to be located at non-coding regions previously. More recently, using the mass spectrometry (MS) methods, many sorts of non-coding regions have been identified to produce large amounts of aberrantly expressed tumor-specific antigens, the bulk of which originate from epigenetic modifications in atypical translation events rather than mutations. 176 These aberrantly expressed tumor-specific neoantigens are more prevalent than neoantigens created by mutations in coding areas and can be shared between tumor patients. 27 , 172 , 177 Numerous such cryptic peptides were found in tumor immunopeptidomes using Peptide-PRISM. The presentation of cryptic peptides is HLA-I allele dependent, with HLA-A*03 and HLA-A*11 showing the largest proportion of cryptic peptides. 178 Critically, cryptic proteins create MHC-I peptides five times more efficiently per translation event than canonical proteins do, due to their more predicted disordered residues and lower stability. 179 No studies have reported that MHC-II-associated neoantigens generated from non-coding regions may activate CD4 + T cells. Compared with other mutations at the genome and transcriptome level mentioned in this review, neoantigens derived from the translation of non-coding regions are rarely clearly understood. Therefore, it is urgent to develop fast and efficient computational algorithms to screen these potential neoantigens and to verify their feasibility for immunotherapy. 38

Proteomic variants

Dysregulated translation that is a characteristic of carcinogenesis offers an important new source of tumor-specific neoantigens. 180 In addition, the proteomic variants also come from the aberrant function of PTMs, proteasome processing, and transporter associated with antigen processing (TAP). 181 , 182 , 183 , 184

Neoantigen presentation by MHC molecules to T cells can maintain specific PTMs. 181 , 182 Aberrant PTMs, including glycosylation, O-linked β-N-acetylglucosamine (O-GlcNAc) and phosphorylation, can create neoantigenic peptides presented by MHC complexes in tumors. 185 For example, the neoantigen arising from post-translationally modified MUC1 was presented by MHC-I and exclusively recognized by a glycoform-specific TCR. 182 Moreover, an unusually large proportion of mutations may enhance the formation of novel N-glycosylation sites, resulting in generation of neoantigens. 186 Five O-GlcNAc modified peptides in leukemia were found to induce multifunctional memory T cell responses in healthy donors. Neoantigens derived from O-GlcNAc modified proteins explain why leukemias are highly immunogenic despite having a low mutational load, thereby offering prospective therapeutic targets. 187 Dysregulated phosphorylation can generate neoantigens by promoting the binding of epitopes to MHC molecules or by altering the antigenic features of presented epitopes. 188 The cancer-associated phosphopeptides derived from insulin receptor substrate 2 (pIRS2) and breast cancer antiestrogen resistance 3 (BCAR3) were immunogenic in vivo in mice, and in vitro in normal human donors. 189 , 190 Several T cell lines have demonstrated a specifically recognition of the post-translationally modified peptide but not the unmodified peptide, indicating that the aberrant PTMs results in a different neoantigen and cognate TCR. 182 , 187 Notably, immunogenic peptides derived from dysregulated PTMs in cancer cells constitute an unexplored class of tumor-specific neoantigens that could serve as off-the-shelf targets for cancer immunotherapy. PTMs can also be employed to produce unique neoantigens to improve the immune recognition of cancer cells. Covalent KRAS-G12C inhibitors, like ARS1620, result in covalently modified peptides, which can be presented on MHC-I to elicit T cell response. These tumor-specific PTMs, which involve the covalent drug-mediated alkylation of mutant cysteine residues on oncoproteins, provide a novel source of neoantigens that can be readily targeted by immunotherapies. 191 , 192

Another repertoire of neoantigenic epitopes is derived from impaired proteasome processing or TAP complexes. The proteasome processes proteins and converts them into peptides, which is particularly critical for transforming proteins into MHC-restricted epitopes. The oxidants like peroxynitrite generated by myeloid cells in tumor microenvironment (TME) inhibit the activity of proteasome, thereby decreasing the production of MHC-I peptides. 193 , 194 Protein splicing significantly increases the proteome complexity of malignancies, which alters the hierarchy of antigenic epitopes. 195 , 196 Studies have also revealed that the proteasome can produce novel immunoreactive spliced epitopes (splicetopes) by fusing with peptide fragments excised by reverse proteolysis during proteasome-catalyzed peptide splicing (PCPS), which differ from the original substrate protein sequence. 196 , 197 , 198 According to preliminary statistical analysis, the proteasome is responsible for splicing around one-third of MHC-I-related immune peptides. 199

There is evidence that neoantigens involving the linkage of existing individual peptides can activate CD4 + T cells in type 1 diabetes (T1D), indicating that proteomic variants processes may generate MHC-II-associated neoantigens. 200 Several studies have reported that the spliced peptides produced by the proteasome are able to activate CD8 + T cells. 198 , 201 , 202 Splicetope-specific CD8 + T cells from TILs isolated from human AML patients inhibited the growth of their corresponding tumor cells in severe combined immunodeficient (SCID) mice model. 203 Combining in vitro confirmation of proteasome-dependent splicetope with screening of specific anti-tumor CD8 + T cells enables monitoring of HLA class I binding and immune recognition processes, which will help to obtain more novel tumor-associated splicetope. 200 Epitopes such as FGF-5, SP110, and gp100-derived splicetope have been identified during in vitro PCPS approach, which could be recognized by CD8 + T cells. However, the current research strategy to discover new tumor-specific splicetope needs to be further developed and refined in the future. Protein splicing-derived neoantigens could provide more yet-to-be-developed or identified neoantigens for anti-tumor vaccines and cancer immunotherapy. 38 , 199

Most tumor antigens require proteasome processing and TAP-mediated peptide transport. However, most tumors eventually acquire drug resistance and immune escape. It has been reported that tumors can avoid recognition by T cells by producing defective HLA-I antigen processing pathways or downregulating related gene expression. Notably, a class of neoantigens called T cell epitopes associated with impaired peptide processing (TEIPP) have been identified in some HLA-I low/TAP-deficient tumors. They are a class of unmutated antigens derived from the tumor’s own housekeeping proteins that activate TEIPP-specific CD8 + T cells and specifically kill these TAP-deficient cancer cells. It is currently believed that TEIPP peptides are immunogenic because they cannot be presented by normal cells, and TEIPP-specific T cells are not negatively selected in the thymus. A TEIPP peptide derived from Lass5 protein, also known as Trh4, was able to activate specific T lymphocytes and inhibit the growth of MHC-I low/TAP-deficient tumors in a TCR transgenic mouse model. In addition, several TEIPP non-mutated tumor epitopes have been identified in humans, including the procalcitonin (ppCT) signal peptide (ppCT16-25, ppCT9-17) regions, and the procalcitonin (pCT) precursor protein (ppCT50-59 and ppCT91-100) regions. Further studies confirmed that these TEIPP-based antigenic peptides can effectively induce anti-tumor CTL effects and inhibit tumor growth. Therefore, targeting these TEIPP neoantigens will potentially provide a promising new immunotherapeutic approach for the treatment of TAP-deficient/HLA-I-low tumors. 27 , 183 , 184 , 204 , 205 , 206 , 207

Viral-derived tumor antigens (Viral ORFs)

Viral proteins may be considered as another class of neoantigens in tumors caused by viruses because they are almost completely different from normal cellular proteins, and they can elicit high-affinity TCR responses. Some solid tumors are directly caused by viral infection, including Merkel cell carcinoma (MCC) caused by Merkel cell polyoma virus (MCPyV) infection and nasopharyngeal carcinoma caused by Epstein-Barr virus (EBV) infection. 208 , 209 , 210 , 211 , 212 , 213 In other tumors, viral genes with oncogenic properties can integrate into the cellular genome, promoting the continuous expression of viral genes and leading to tumorigenesis. For example, the expression of E6 and E7 genes from HPV promotes the development and progression of human papillomavirus (HPV)-related cervical, anal, head and neck cancers. 214 , 215 , 216 , 217

Numerous immunotherapy studies have focused on virus-derived tumor antigens. Two of nine HPV-positive patients with metastatic malignancies achieved sustained tumor regressions in ACT research using TILs chosen for their reactivity against viral antigens. 218 A further investigation revealed that the number of HPV-reactive cells in the reinfused product exceeded those that recognized other types of tumor antigens. 219 In two separate clinical trials, autologous T cells transduced with anti-E7 TCR responded in 4 of 12 patients, while T cells transduced with anti-E6 TCR responded in all 12 patients. 220 , 221 The NCT02280811 and NCT02858310 trials using these TCRs are currently ongoing and should yield more conclusive proof about the value of focusing on HPV epitopes. Treatment of the corresponding tumors with ACT therapy targeting MCPyV and EBV also achieved clinical results, although other effective therapies were also administered in these experimental regimens. Notably, none of these clinical trials occurred with any apparent toxicity to normal tissues. Collectively, these trials demonstrate the safety and efficacy of targeting oncogenic viral proteins to treat related tumors, supporting the development of further comprehensive treatment regimens. Given their critical function in oncogenesis and the fact that patients share them, these neoantigens continue to be desirable targets for cancer immunotherapy. 21 , 221 , 222 , 223

The neoantigens are generated as a result of alterations at genomic, transcriptomic and proteomic levels (Fig. 2 ). Current studies mainly focus on SNVs and INDELs, the most prevalent types of mutations at the genome level in tumor cells. However, the clinical application of neoantigens produced from SNVs and INDELs is limited by their patient specificity and poor immunogenicity, which results in less clinical benefit for cancer patients. Accumulating evidence suggests that alternative sources of cancer neoantigens, such as gene fusions, alternative splicing variants and PTMs, may be attractive novel targets for immunotherapy. The neoantigens produced by gene fusion, particularly the frameshift fusion, have better immunogenicity than the SNV- and INDEL-neoantigens, which were included in numerous clinical trials. Furthermore, neoantigens generated from gene fusion, recurrent mutations in cancer driver genes, non-coding regions and abnormal PTMs have a higher likelihood of being shared among patients, providing readily public neoantigens for immunotherapy. 27 , 35 , 38

Identification, prediction, and validation of immunogenic neoantigens

Identification of immunogenic neoantigens from the numerous sources mentioned above is a crucial step in the development of effective immunotherapies. 177 Neoantigens may now be thoroughly screened across the entire cancer spectrum thanks to the convergence of whole-exome sequencing (WES), RNA-seq, and proteomic data from TCGA. 120 , 224 However, given the wide variations in tumor types, tumor lesions, and patients, customized immune treatments necessitate the detection and prediction of neoantigens based on distinct patient and tumor characteristics. The identification of genome-expressed mutations as well as details on MHC types of patients are required for the prediction of immunogenic neoantigens, as the sequential stimulation of immune response by tumor neoantigens from mutations depends on several variables, including the translation and processing of peptides, the presentation of the mutated peptides by the MHC molecules and the affinity of the pMHC complexes with the TCRs. 177 , 225 , 226 Two main strategies for identifying neoantigen epitopes are developed: the immunogenomic approach can create virtual peptidomes by in silico methods based on NGS, and the immunopeptidomic strategy use MS to analyze the MHC-loaded peptides. 227 Several TCR-guided neoantigen discovery strategies have recently been developed to systematically map the immunogenic neoantigens.

Identification of somatic mutations

The immunogenomic strategies were greatly hastened by comparing the genetic changes between tumor and normal tissue using NGS. Currently, the initial stage in the process of detecting possible neoantigens from NGS data is mapping tumor-specific genetic abnormalities using WES of the tumor and normal DNA. RNA-seq data may be combined with WES to determine whether a mutant gene is expressed in the tumor. In addition, more hidden biological information, such as information about copy number changes, microbial contamination, transposable elements, cell type, and the existence of neoantigens, can be found in RNA-seq. 228 , 229 RNA-seq can also be used to detect alternative splicing events and estimate the relative frequency of the mutant allele’s expression. 230 By using methods like mate-pair sequencing that may detect chromosomal rearrangements, the predictive values of NGS-based TMB measures may be greatly improved. 110 Recent studies have shown that antigenic peptides are produced by transcripts with frameshift mutations and atypical splicing patterns when NMD is assumed to be present. Exact peptide sequences from full-length transcript structures are required in order to fully identify the neoantigens that resulted from frameshift mutations and aberrant isoforms. 231 Using the Oxford Nanopore Technologies nanopore-type sequencer MinION, full-length transcriptome sequencing may cover the whole transcript at the proper sequencing depth with an accuracy of roughly 90%, providing complementary information to the current RNA-seq to identify allele-specific transcription and splicing. 143 , 232

Based on cancer genomic data, the immunogenomic technique predicted millions of possible mutation-derived neoantigens, but the vast majority of them did not manifest in proteomic profiling of HLA-bound peptides. 233 , 234 The high-throughput identification of peptides attached to MHC is made possible by immunopeptidomics techniques, which use MS to directly examine the immunoprecipitated and extracted MHC-bound peptides. 230 , 235 , 236 , 237 , 238 , 239 MS has advanced in verifying in silico predicted neoantigens. 38 Comparing the tandem mass spectra of the sample with that of the synthetic peptide can verify the neoantigens that are predicted by immunogenomic approaches. 240 , 241 Particularly for rare HLA allotypes and HLA-II ligands, mapping the tumor HLA ligandome has helped to uncover targets for the neoantigen-specific cancer immunotherapies in clinical trials. 38 In addition to validate the neoantigens arising from aberrant DNA sequence or RNA expression, MS-based proteomics provide the "gold standard" for neoantigen detection at the protein level, which cannot be discovered from DNA and RNA studies. For instance, MS can be used to detect novel MHC-associated neoantigens resulting from PTMs that are dysregulated during cellular transformation. 127 , 188 , 190 , 242 , 243 , 244 Moreover, MS is also integrated with NGS to further detect the tumor-specific neoantigens created by somatic mutations, non-coding RNA and proteasome splicing, which is omitted by whole-exome or transcriptome-based sequencing technology. 38 , 172 , 236 To allow a deeper knowledge of neoantigens in protein levels, more user-friendly and practical tools that integrates genomic, transcriptomic and proteomic data for immunopeptidomic-based neoantigen detection should be created.

In silico neoantigen prediction

Based on the NGS data, virtual peptidomes have been created and potential neoantigens have been discovered by in silico methods. 177 , 245 Briefly, a typical workflow for neoantigen prediction can be summarized into the following steps: (i) mutation calling, (ii) HLA typing, (iii) neoantigen filtering and prioritization based on HLA binding affinity, and (iv) experimental validation of immunogenic neoantigens using T cell-based assays (Fig. 3 ). 177 , 246 , 247

Computational workflow for neoantigen prediction. Current available bioinformatic pipelines for neoantigen prediction from somatic mutations share four main computational modules: (i) HLA typing from tumor WGS, WES data and RNA-seq; (ii) mutant peptide calling using a set of somatic mutations and splicing variants; (iii) HLA binding prediction; and (iv) T cell recognition prediction. The in silico tools for mutation calling are listed as follows. Mutation calling: INTEGRATE-neo, 561 neoFusion, 80 pVACtools, 562 Epidisco, 563 GATK 564 and Antigen.garnish, 565 , 566 Spliceman, 567 MutPred, 568 REVEL, 569 rMATS, 570 pVACseq, 240 Neopepsee, 571 MuPeXI, 572 RepeatMasker, CloudNeo, 573 Tlminer, MuTect/MuTect2, Strelka/Strelka2, 574 SMUFIN, VarScan2, SomaticSniper, CaVEMan, MuSE, cgpPindel, SvABA, RADIA, NeuSomatic, NeoantigenR, MutPred, JuncBase, Splice, SpliceGrapher, rMATS, SplAdder, ASGAL, REVEL, TSNAD, 575 HERVd, 569 HESAS 576 and EnHERV, 577 hervQuant. 578 HLA typing: Polysolver, 254 OptiType, 253 HLAreporter, 579 PHLAT, 580 HLAScan, 260 , 581 HLAProfiler. 260 HLA binding affinity: NetMHCpan, 265 NetMHCIIpan4.0, 267 MixMHC2pred, 582 MARIA, 268 neomhc2, 583 pVAC-Seq, TIminer, HLAthena, DeepHLApan, TEPITOPEpan, NetMHCIIpan, SYFPEITHI, RNAKPEP, MULTIPRED2, ProPred, MHCPred, MARIA, Neonmhc2, EDGE. 38 , 238 T cell recognition: NetCTL/NetCTLpan, POPISK, PAComplex, CTLPred, EpiMatrix, TCRMatch

Neoantigens are often presented in a cell-specific way by MHC-I for CD8 + T cells and MHC-II for CD4 + T cells, much like other antigens. Humans have more than 24,000 distinct HLA-I (HLA-A, -B, and -C) and HLA-II (HLA-DR, HLA-DQ, and HLA-DP) alleles, and their admixture results in polymorphism diversity. 248 , 249 , 250 , 251 The HLA alleles of the patient determine their tumor-specific neoantigen repertoire that will be presented for T cell recognition. In addition, HLA-LOH, which occurs in 40% of NSCLC, impairs the presentation of neoantigens, facilitating immune evasion. Therefore, one of the most important initial steps in neoantigen prediction is determining the patient’s HLA genotypes. 83 , 252 Several computational methods can now be applied with NGS data to achieve this goal. Most methods rely on DNA-derived NGS data acquired from WES or WGS. For example, Optitype 253 and Polysolver 254 are well performing tools for identification of class I HLA alleles. A bioinformatics tool, LOHHLA, is developed for accurate measurement of allele-specific HLA copy numbers. The tools, including HISAT genotype, 255 ATHLATES, 256 and HLA-HD 257 can be used for both class I and class II typing. 83 RNA-seq data can also be used by tools, such as arcasHLA, 258 seq2HLA 259 and HLAProfiler, 260 to type HLA alleles with advantage of the unbiased dataset that covers both fully expressed parental alleles equally. 260 The newly developed RNA-seq data-based methods bring a new dimension to HLA typing and biomarker investigations, even though Optitype discovered that WES produced superior results for HLA typing than RNA-seq data. 230 , 253

Mutation and variant calling

By comparing NGS data of tumor and normal tissues from the same patient, mutant peptides resulting from somatic mutations can be predicted. 261 WES is the recommended source of NGS data for neoantigen prediction because it offers the highest mutation coverage through focusing on the protein-coding regions of the genome. The computational analysis consists of data pre-processing and quality control, variant calling for somatic mutations, and prediction of the altered proteins and functional impact utilizing public genomic, transcriptomic, and proteomic sequence databases. For various neoantigen sources, a variety of integrated techniques have been developed for neoantigen identification and prioritization. 29 Based on the strategy employed to screen putative neoantigens, these technologies can be classified into two groups: stepwise-analyses-based filtering strategy and integrative-scoring-system-based filtering strategy. The efficient one-stop tools accept WES/WGS and RNA-seq data as input and perform a series of filtering steps based on selected cutoff metrics, such as the binding affinity of peptides and MHC molecules, sequence coverage, variant allele frequency and gene expression, to remove false positives and generate a list of potential neoantigens. An integrated scoring system-based filtering technique assesses the immunogenicity of neoantigens by a quantitative score based on significant neopeptide characteristics, including the rank affinity of the mutant and normal peptides, the frequency of mutant alleles, and the amount of gene expression to experimentally assess the immunogenicity of the discovered neopeptides. 38 , 262 , 263 Recently, a scoring method for evaluating immunogenicity that is based on machine learning models has also been suggested, optimizing the accurate prediction of neoantigens and reducing false positives. 177 For a review and extensive discussion of these methods, we refer to prior literatures. 246 , 249

Prediction of HLA binding and neoantigen presentation

Numerous computer prediction tools have been created for the in silico discovery of neoantigens based on MHC molecule processing and presentation, including NetChop, NetCTL and NetCTLpan 264 , 265 (Fig. 3 ). The prediction capacity is actively improved by incorporating HLA-ligandome data into machine learning algorithms, such as linear regression and artificial neural networks. 230 , 249 In vitro peptide-HLA binding dataset is used to train machine learning models by NetMHCpan 265 and MHCflurry 266 that are the main component of current HLA ligand identification pipelines. 38 It is noteworthy that NetMHCpan, in contrast to state-of-the-art methods, improves the prediction performance of tumor neoantigens by combining information from binding affinity data with MS peptidome data to give a "panspecific" machine-learning strategy for MHC-I alleles. 230 , 264 , 267 Two recent studies created computational frameworks called MSIntrinsic and EDGE that are highly effective in predicting HLA antigens using HLA peptides acquired from RNA-seq and liquid chromatography tandem MS (LC-MS/MS) data. Based on 24,000 HLA-I peptides collected by LC-MS/MS, the neural-network prediction algorithm, MSIntrinsic, outperformed previous affinity-based predictors by an average of 30% in positive predictive value (PPV). 251 Similar findings were made by EDGE, which found that adopting a deep-learning architecture to identify HLA ligands using proteomic and transcriptomic data can improve the accuracy of HLA antigen prediction by up to ninefold. 38 , 238

Emerging evidence has proved the significance of MHC-II neoantigens in anti-tumor immune response. 234 , 268 , 269 , 270 , 271 A wide range of computational techniques for predicting MHC-II binding epitopes have been developed using artificial neural networks, including NetMHCII, NetMHCIIpan, 272 , 273 SYFPEITHI, RNAKPEP, MULTIPRED2, ProPred, and MHCPred. However, compared to MHC-I molecules, computational prediction of the MHC-II-peptide binding affinity are currently less precise. First, compared to MHC-I molecules, MHC-II-binding peptides are more promiscuous in terms of peptide length and binding sequence motifs. Second, the polymorphism of the α and β chains in MHC-II molecules also considerably expands the diversity of peptide binding specificity. 38 , 230 Recently, computational methods based on transcriptome and MS data have been developed. The deep learning model trained by MARIA, which incorporates both sequencing data with naturally occurring MHC-II ligandomes, was demonstrated to outperform the most widely used predictor NetMHCIIpan3.1 in the lymphoma dataset when cross validated against known MHC-II ligands. However, more study using significant datasets is necessary to demonstrate its robustness and effectiveness. 38 , 268

Given multiple processes control the neoantigen presentation, it can be inferred that improving binding affinity alone does not accurately reflect cellular processing and CD8 + T cell responses. Additional properties, including proteasomal cleavage, transportation of peptides into the endoplasmic reticulum, and HLA alleles, are in conjunction with binding affinities between the peptide and the MHC molecules to prioritize possible neoantigens. 230

Evaluation and validation of candidate neoantigens’ immunogenicity

It is well known that an immunogenic neoantigen must satisfy two or more requirements, the main bottlenecks are appropriate MHC molecule presentation and effective TCR recognition. According to recent studies, the majority of predicted neoantigens via MHC molecule presentation do not trigger an immune response. 234 , 274 , 275 Therefore, while assessing the immunogenicity of potential neoantigens, it is crucial to take the TCR recognition of pMHC complexes into account. There are many in silico techniques that can forecast neoantigen-specific T cell recognition. The most used method is NetCTL/NetCTLpan, which generates a composite score rather than predicting T cell binding directly by combining MHC binding, C-terminal cleavage affinity and TAP transport. 38 Recent studies use machine learning or deep learning techniques to predict TCR-peptide/-pMHC binding. The batch of TCR repertoire annotation in several manually curated databases, including McPAS-TCR and VDJdb, allows for the training of TCR specificity predictors and match against TCRs of interest. 276 , 277 , 278 McPAS-TCR provides a list of TCR sequences linked with various pathologies, while VDJdb offers a detailed description of TCR:pMHC interactions based on epitope-centric approach for TCR annotation rather than the underlying biological context. 279 , 280 Besides identification of TCR-pMHC pairings, clustering methods, like pMTnet and GLIPH, can also cluster TCRs that recognize the same epitope and predict their HLA restriction. 281 , 282 , 283 , 284 , 285 Nevertheless, the prediction for binding affinity of TCR and pMHC in silico is still challenging due to the low affinities of TCRs for their pMHC ligands. 230 , 246 , 249 , 286 , 287

For a more precise assessment of the possible application of neoantigens in immunotherapy, experimental validation of their T cell reactivity is essential. Neoantigen-reactive T cells have been validated or screened using T cell-based assays, multicolor-labeled MHC tetramers, the enzyme-linked immunosorbent spot (ELISpot) and T-cell repertoire profiling. 33 , 288 T cell immunogenicity assay is the most direct way to evaluate the immunogenicity of candidate neoantigens. The entire set of possible mutant peptides discovered by cancer exome/RNA-seq can be tested using T cells from either cancer patients or healthy donors. After peptide stimulation, the in vitro expanded neoantigen-specific T cell reactivity is measured by flow cytometric measurement of the T-cell activation markers 4-1BB and OX-40 and IFN-production on the ELISpot assay. 62 , 289 Multicolor-labeled MHC tetramers allow for the highly sensitive and minimally material-required evaluation of T cell reactivity against a wide range of potential epitopes using DNA barcoding, lanthanide coding, or fluorochrome coding of peptides. These technologies rely on epitope predictions and are low throughput since they can only efficiently generate a subset of the human MHC class I alleles. Integrating single-cell RNA sequencing (scRNA-seq) with TCR sequencing of responsive cell groups may be used to boost the sensitivity of detection. The scRNA-seq was used to discover paired TCR sequences linked with cells expressing high levels of IFN- γ and IL-2 in TILs co-cultured with tandem minigene (TMG)-transfected or peptide-stimulated antigen-presenting cells (APCs). 290 , 291 Based on WES-guided prediction of neoantigens and TCR sequencing of short-term peptide-stimulated T cell cultures, the Mutation-Associated Neoantigen Functional Expansion of Specific T cells (MANAFEST) assay sensitively characterizes neoantigen-specific TCR Vβ clonotypes. The MANAFEST assay is compatible with all HLA haplotypes and can track neoantigen-specific T cells in formalin-fixed paraffin-embedded (FFPE) and/or frozen tissues. In addition to assess the tumor specificity of TCR Vβ clonotypes, MANAFEST can also look into the dynamics of the neoantigen-specific T cell response over time and monitor the efficacy of immunotherapy using liquid biopsies obtained before or after treatment. 292

Several unbiased TCR-guided neoantigen discovery strategies have been developed to systematically profile neoantigen-specific TCRs. A yeast-displayed pMHC library can be used to discover neoantigen-specific TCRs. However, it is a time-consuming process to make soluble TCR reagents. Without endogenous processing of neoantigens or functional activation of T cells, the identified random peptides may not represent the physiological TCR-pMHC interaction. 293 To overcome these drawbacks, two innovative strategies make use of different biological processes to mark the target cells in a co-culture system. One approach utilizes the chimeric receptors known as signaling and antigen-presenting bifunctional receptors (SABRs), which can induce a TCR‐like signal following pMHC-TCR interactions. SABRs enable the successful identification of TCR-pMHC interaction, which can be used for both known public TCRs and private neoantigen-specific TCRs. 294 Trogocytosis, a membrane transfer process, is exploited by a cell-based selection platform for TCR ligand discovery. The TCR-pMHC interactions result in specific labeling of cognate target cells, which are then isolated and sequenced to identify the neoantigen-specific TCRs. 295 In addition, putative pMHCs are displayed on spectrally encoded beads in BATTLES, facilitating the investigation of neoantigen-specific T cell responses under physiological force. 296 T-Scan, a method for TCR epitope scanning independent of predictive algorithms, relies on the physiological activity of T cell killing rather than just assessing TCR-pMHC binding affinity, enabling the interrogation of a significantly larger antigen space than previous methods. 297 Thus, these emerging approaches for TCR ligand discovery will be useful for studying the immunogenicity of candidate neoantigens, providing new targets for immunotherapy.

Neoantigens-based therapeutic strategies

As previously mentioned, tumor-specific neoantigens arising from genetic alterations elicit high-avidity T cells due to the absence of thymic selection and central tolerance. Based on their advantages of tumor-specific and immunogenetic, neoantigens may serve as emerging targets for cancer immunotherapies, including tumor vaccines, ACTs and antibody-based therapies, as well as potential predictors for ICBs (Fig. 4 ). 8 , 226 , 298 , 299 The neoantigens consist of either personalized neoantigens found specifically for each patient or shared neoantigens expressed in numerous patient cancers. The off-the-shelf therapies based on public neoantigens are less resource- and time-intensive than individualized neoantigen therapies. Because personalized neoantigens are patient-specific, they cannot be used to target a large number of patients. With the recent advance in high-throughput sequencing, personalized neoantigens enable the immune system to target appropriately immunogenic epitopes on malignancies without predefined public antigens. 300 , 301

Classification of neoantigen-based therapies. Immunotherapies that target neoantigens mainly include ACTs, bispecific antibodies and cancer vaccines. Cancer vaccines stimulate a specific immune response to tumor neoantigens using nucleic acids, peptides and DCs. The ACT utilizes the neoantigen-specific TCR or CAR engineered T cells to selectively recognize and kill tumor cells. The bispecific antibodies have one arm that targets neoantigens presented by tumor cells and one arm that targets CD3 on the surface of T cells

Neoantigen-based therapeutic vaccines

Neoantigen vaccines are an effective approach for stimulating, enhancing, and diversifying anti-tumor T cell responses, with their high feasibility, general safety and easier to manufacture. Various forms of neoantigen-based vaccines, such as peptide, nucleic acid and dendritic cell (DC) vaccines are being evaluated in clinical trials on patients with different types of tumors (Fig. 5 ). 9 , 15 , 245 , 302 Current peptide and nucleic acid vaccines mainly target the predicted neoantigens derived from somatic mutations, including SNVs, frameshift INDELs and gene fusions. DC vaccines can target both selected neoantigens via pulsing with synthetic peptides or nucleic acids and overall TSAs by introducing with whole cell lysates (WCL).

Schematic illustration of neoantigen-based cancer immunotherapy production. The individualized neoantigens are identified using blood cells and tumor tissues from patient. These patient-specific neoantigens are used to develop immunotherapies, such as cancer vaccines and ACTs. Cancer vaccines in the form of peptides, DNA or mRNA, and dendritic cells are generated and administered to the same patient. For ACTs, T cells are extracted from the peripheral blood or tumor tissues of a patient and then induced to proliferate by cytokines, monoclonal antibodies against CD3 and CD28, and other reagents. The development of neoantigen-specific T lymphocytes with neoantigen-specific targeting requires co-culturing T cells with primed APCs and genetic engineering of immune cells with TCRs or CARs. After sufficient T cell expansion, T cell products are injected into lymphodepleted patients with the hope of eliciting an immune response that attacks the tumors

Peptide vaccines

Peptide-based neoantigen vaccines have received most of the attention in the research area of personalized neoantigen vaccines due to their high specificity, economical manufacture and established safety record (Table 3 ). 177 , 303 , 304 The neoantigen peptides can be produced as genetically encoded long peptides or fused polypeptides and chemically synthesized short peptides. The peptides are subjected to affinity chromatography, size-exclusion chromatography (SEC) or high-pressure liquid chromatography (HPLC) to obtain sterile, endotoxin-free products with a purity of >98%. Following verification by MS, the peptides are mixed with appropriate adjuvants for subcutaneous injection immunization. 99 , 305 In a phase I immunotherapy clinical trial in patients with disseminated synovial sarcoma, an SYT-SSX neoantigen peptide-based vaccine prevented disease progression in one patient and successfully induced specific CTL responses in four patients, and no serious adverse reactions or delayed-type hypersensitivity (DTH) reactions occurred throughout the treatment. 305 The peptides, such as KQSSKALQR, produced from the breakpoint of BCR-ABL can be processed in the cytosol and loaded onto MHC molecules, which will be transferred to the CML cell surface for potential T cell recognition. 86 In an initial clinical trial, this neoantigen-based vaccine elicits a BCR-ABL peptide-specific T cell immune response, while has no significant toxic effects. 99 , 306 Selected neoantigens containing T cell epitopes can be produced in the form of single epitopes, polypeptide chains, or peptide pools. To overcome issues like tumor heterogeneity, HLA haplotype diversity and antigen down-regulation, overlapping peptides or long multi-epitope peptides rather than short single-epitope peptides are typically used to stimulate a powerful immune response in T cells. 307 In addition, immunostimulatory adjuvants and multimeric formulation techniques are being developed to boost the immunogenicity of personalized peptide vaccinations. Therefore, personalized neoantigen vaccines based on synthetic peptides have been evaluated in clinical studies on patients with various types of cancers, including lung cancer, breast cancer, bladder cancer, pancreatic cancer, pediatric brain tumor, melanoma, and colorectal cancer (Table 4 ).

Neoantigen peptide vaccines elicit and amplify anti-tumor immune responses in cancers with either high or low mutational burden. A vaccine formulated with the adjuvant poly-ICLC and a synthetic neoantigen long peptide efficiently activates CD8 + T and CD4 + lymphocytes in patients with advanced melanoma, NSCLC, or bladder cancer, all of which have high levels of mutations (NCT02897765). This neoantigen vaccine prevents recurrence for 25 months after treatment in four out of six high-risk melanoma patients. 15 In NSCLC patients who have failed in multiple conventional therapies, personalized neoantigen peptide vaccination triggers specific T cell responses targeting EGFR mutations, including the relatively prevalent mutations L858R and T790M. Accordingly, a large subset of NSCLC patients responding relatively poorly to ICB approaches may benefit from the neoantigen vaccines based on shared immunogenic EGFR mutations. 308 In addition, the peptide-based neoantigen vaccination can potentially modify the immune milieu of immunologically cold tumors with a relatively low mutational burden, inducing neoantigen-specific T cells to infiltrate and destroy tumor cells. For example, administration of neoantigen vaccines induces T cell immune responses in HLA-A*24:02 or HLA-A*02:01-positive glioblastoma patients. These neoantigen-specific T cells are able to cross the blood-brain barrier (BBB) and infiltrate the tumor, thereby altering the immune milieu of glioblastoma and extending the median overall survival of patients to 29.0 months. 309 , 310 , 311 , 312 , 313 , 314

Personalized neoantigen peptide vaccines can expand the durability and repertoire of tumor-specific T cells. 30 According to a retrospective analysis of the circulating immune responses in melanoma patients after vaccination, neoantigen-specific T lymphocytes exhibit a memory phenotype that lasts for an average of ~4 years following vaccination (NCT01970358). The neoantigen-specific T cells have evolved overtime into a variety of clones with different functional avidities. Meanwhile, non-vaccine antigen-directed T cell responses are also detected, suggesting epitope spreading after vaccination. The epitope spreading is associated with prolonged progression-free survival. 15 , 315 , 316 The long-term persistence and diversification of functional neoantigen-specific T cell clones support the neoantigen peptide vaccines as a potent strategy for controlling the continuously evolving metastatic tumors. 317

The immunogenicity of peptide vaccines can be further enhanced through improving the neoantigen presentation and using immunostimulatory adjuvants. 318 , 319 , 320 , 321 , 322 For example, KRAS-G12D mutant peptides are fused to the C-terminal of diphtheria toxin to produce a more immunogenic peptide vaccine. This vaccine boosts CD8 + T cells while decreases T regulatory cells in mice with CT26 tumor. 323 Heat shock proteins (HSPs), like HSP70, have also been complexed with synthetic peptides derived from tumor-specific neoantigens to enhance the presentation and recognition of antigens, which are widely used for treating advanced tumors resistant to conventional therapies (NCT02992977, NCT03673020). 324 , 325 Nanoparticle formation is another technique for improving the immunogenicity of peptide vaccines. B16.F10 and CT26 neoantigens formulated with polyethyleneimine (PEI)-adsorbed mesoporous silicas micro-rod (MSR) can completely eradicate existing lung metastases in tumor-bearing mice. 325 , 326 Another advantage of nanoparticle platform is capable of co-delivering peptides and adjuvants. Self-assembled intertwining DNA-RNA nanocapsules have been used to efficiently deliver tumor-specific neoantigen peptide and synergistic adjuvants, DNA CpG and shRNA to APCs in lymph nodes. These neoantigen vaccines induce peripheral memory neoantigen-specific CD8 + T lymphocyte, suppressing the progression of neoantigen-associated colorectal cancers. 327 , 328 , 329 High density lipoprotein-mimicking nanodiscs elevate the efficient co-delivery of peptides and adjuvants to lymphoid organs and maintain the neoantigen presentation on DCs. In clinical trials, neoantigen-specific CTLs activated by nanodisc vaccines are 31 times more frequencies than the strongest adjuvant and up to 47 times more than soluble vaccines. 330 The formulation of SNP-7/8a derived from charge-modified peptide-TLR-7/8a can effectively activate specific CD8 + T lymphocytes against 50% of neoantigens with high predicted MHC-I affinity binding, thereby enhancing anti-tumor efficacy. 331 Collectively, a generic approach can be utilized to improve the anti-tumor immune response of personalized peptide vaccines.

Nucleic acid vaccines

Like peptide vaccines, nucleic acid vaccines, such as RNA and DNA vaccines, also have the advantage of being low-cost and non-HLA-specific (Table 3 ). Nucleic acid vaccines can deliver multiple tumor neoantigens in a single vaccination, triggering both cellular and humoral anti-tumor immune responses. 245 , 262 , 325

Currently, mRNA technology has been widely used in the clinical treatment of tumors, the prevention of infectious diseases and protein-encoding therapies. The recent success of the COVID-19 mRNA vaccine has revealed the therapeutic potential of mRNA technology. 332 mRNA vaccines offer considerable anti-tumor potential due to their advantages in safety, high potency, rapid and low-cost industrial production, and ability to encode entire antigens. 333 Currently, in vitro transcription (IVT) is the major method used to create mRNA that contains the sequence for neoantigens. A cap structure is added to mRNAs post-IVT to increase their stability and decrease their immunogenicity. After purification through SEC or tangential flow filtration (TFF), appropriate delivery systems, such as liposomes and polymers, are selected to introduce mRNA into cells and tissues to translate the target neoantigens, thereby activating the immune response. 334 , 335 Personalized mRNA vaccines based on tumor-specific neoantigens induce a more potent immune response than shared tumor-associated self-antigens due to the absence of central immune tolerance. For example, neoantigen-specific mRNA vaccines in 13 evaluable melanoma patients activated several neoepitope-specific CD4 + and CD8 + T cells, greatly reducing the cumulative incidence of recurrences and leading to persistent progression-free survival. 302 , 335 , 336 The mRNA-4650 vaccine, which contains defined neoantigens, novel neoantigens derived from driver gene mutations and predicted HLA-I epitopes, elicits both CD8 + and CD4 + T cell response, with a preference for CD4 + T cell responses with no severe side effects. 337 Clinical studies for the personalized mRNA-4157 and BNT122 vaccines are currently underway. mRNA-4157 monotherapy or in combination with the PD-1 inhibitor is well tolerated and induces a neoantigen-specific T cell response in clinical trials (NCT03313778; NCT03897881). 338 A phase I trial of RNA vaccine (NCT02316457) in triple negative breast cancer (TNBC) patients demonstrate a highly effective at eliciting robust poly-epitopic T cell responses, increasing the clinical benefit for TNBC patients following surgery and (neo-)adjuvant chemotherapy. 339 Moreover, the RO7198457 vaccines have been explored by BioNTech to treat various solid tumors, including melanoma, NSCLC and colorectal cancer, in combination with PD-L1 antibody. 340

mRNA-encoded neoantigen vaccines may offer a proper but more potent immunogenic response and therapeutic efficacy when compared with peptide vaccines. This superiority may arise from the biological function of mRNA as a template for protein synthesis. The mRNA vaccine enables post-translational modification of protein products in human, which has the potential to present various epitopes without being constrained to a specific HLA type. In addition, numerous neoantigen epitopes can be incorporated into the same backbone, producing myriad neoantigens that can exist either as independent molecules or as a series of multiple coding sequences. 302 , 337 One such example is the RNA-based poly-neoepitope approach developed by Sahin and colleagues. Ten selected mutations per patient are engineered into two synthetic pharmacologically optimized RNA molecules, each of which encodes five linker-connected 27mer peptides (NCT02035956). 302 Another example is the personalized cancer vaccines in clinical trials, including mRNA-4157 and mRNA-4650, containing an mRNA backbone that can encode up to 30 different neoantigens. 337 As a result, mRNA vaccine can express a variety of neoantigens originating from patient’s own tumor, resulting in a stronger immune response. 177

Effective application of mRNA vaccines in vivo requires maintaining mRNA stability and effective intracellular distribution of the mRNA moiety to target cells. Since RNA is intrinsically unstable, early attempts focused mostly on its stabilization. The 5′ cap structure, the length of 3′ poly(A) tail and regulatory elements in the untranslated regions have all been optimized for this purpose. 177 , 341 Efficient intracellular delivery is also required for effective mRNA therapies in vivo. Nanoformulations, such as lipid, calcium, and phosphate nanoparticles, are one method for shielding RNA from extracellular ribonucleases, resulting in improved delivery efficiency and immunogenicity. 342 , 343 , 344 Clinical studies have been initiated for several personalized cancer vaccines based on lipid nanoparticle-mRNA formulations. 177 The lipid nanoparticle-formulated mRNA-4157 and mRNA-4650 vaccines are used alone in individuals with primary solid tumors or in combination with PD-1 inhibitor (NCT03313778, NCT03897881, NCT03480152). 338 Advanced RNA-lipoplex formulations have been developed and explored as therapeutic cancer vaccines in several clinical studies owing to their advantage in systemic DC targeting and synchronized induction of highly potent adaptive and innate immune responses (NCT02410733, NCT02316457). 345 , 346 Another point worth noting in mRNA vaccine delivery is the various oncology-related administration routes. 177 Intravenous administration is preferable over intradermal or subcutaneous injection for mRNA-lipoplex vaccination, which induces a higher level of T cell responses in syngeneic tumor models. 345 The route of administration mechanically determines the antagonistic effects of IFN on mRNA-lipoplex vaccines-induced T cell response. When mRNA-lipoplex vaccine is delivered subcutaneously, IFN signaling inhibits the antigen-specific T cell response; conversely, IFN increases T cell responses when administered intravenously. 345 , 347 , 348 Intravenous injection has been widely used for the clinical administration of mRNA vaccines, which can deliver mRNA vaccine into direct intratumoral injection-inaccessible malignancies or those without reachable lymph nodes (NCT03897881, NCT03480152, NCT03908671, and NCT03948763). 177 Altogether, neoantigen-based mRNA vaccines benefit from approaches that preserve their stability and improve the delivery efficiency.

In contrast to RNA and peptide vaccines, DNA vaccines are a multifunctional platform with numerous benefits, such as the ability to accommodate any sequence without affecting its stability or solubility, rapid industrial manufacturing at low cost, and easy storage without complicated cold-chain procedures. The DNA sequence encoding the predicted neoantigens is constructed into a suitable expression vector, which is amplified and purified in prokaryotic cells like Escherichia coli . Plasmid DNA is then introduced into cells or tissues via intramuscular or subcutaneous injection in combination with electroporation, where neoantigen is expressed to induce immune responses. 349 DNA vaccines also offer a significant advantage in boosting immunity, including activation of humoral immunity via antigen-induced CD4 + and CD8 + T cell responses and stimulation of innate immune response by recognition of the double-stranded DNA structure. 350 , 351 , 352 , 353 Rational selection of tumor-specific neoantigens can improve the immunogenicity of DNA vaccines by broadening immune responses and overcoming concerns, like antigen loss, modification and tolerance. A DNA vaccine based on polyepitopic neoantigens induces similar therapeutic anti-tumor responses achieved by peptide vaccines in mice bearing mammary tumors E0771 or 4T1. 308 , 350 , 354 Combining a therapeutic DNA vaccine and anti-PD-1 therapy synergistically controls tumor growth in mice. 336 , 355 An optimized polyepitope neoantigen DNA vaccine that encodes long epitopes linked with mutant ubiquitin also elicits strong neoantigen-specific immune responses in patients with pancreatic neuroendocrine tumors when paired with ICB therapy. 308 There are numerous neoantigen-based DNA vaccine clinical trials being conducted for solid tumors, including TNBC, advanced small cell lung cancer, glioblastoma, pancreatic cancer, and pediatric recurrent brain tumor (Table 4 ).

Even though the personalized mRNA and DNA vaccines show less efficacy and success than ICBs and T cell therapies, tremendous improvements are still being made in the formulations and preparations of nucleic acid cancer vaccines, which will further accelerate the clinical application of neoantigen-based personalized nucleic acid vaccines in cancer patients. 156 , 334

Dendritic cell vaccines

APCs like DCs continuously present antigens to the immune system, making them an effective platform for delivering neoantigens. Autologous DCs can be isolated from patients and exposed to neoantigens, which are then injected back into the patient to elicit neoantigen-specific immune responses. Ex vivo loading of blood-isolated monocytes or hematopoietic progenitor cells with tumor neoantigens effectively improves the anti-tumor effects of neoantigen-based vaccines. Neoantigen-loaded DC vaccines can expand the antigenic breadth and clonal diversity of anti-tumor immunity. 9 , 112 , 356 , 357 , 358 , 359 , 360 , 361 Several clinical trials are investigating the efficacy and safety of personalized neoantigen DC vaccines in solid tumors, such as melanoma, bladder cancer, colorectal cancer, esophageal cancer, breast cancer, ovarian cancer, pancreatic cancer, hepatocellular carcinoma, lung cancer, and gastric cancer (Table 4 ).

Neoantigens can be loaded to DCs by a variety of techniques, including pulses with the whole mRNA derived from autologous tumors, pulses with synthetic peptides and pulses with autologous whole tumor lysate (WTL), and fusion with tumor cells. The mRNA transfection is the simplest method for intracellular neoantigen production in DCs. Beyond introducing neoantigens, mRNA electroporation can also deliver functional proteins into the DCs, providing additional activation and maturation signals. 362 The whole tumor mRNA-transfected DC vaccines induce T cell responses in vitro and improve the survival of immune responders with advanced melanoma (NCT01278940). 363 The whole tumor mRNA-loaded DC vaccines also elicit neoantigen-specific T cell responses and exhibit safety in patients with various tumors, including melanoma, renal cancer, prostate cancer, uterine and ovarian cancer, colorectal cancer, pancreatic cancer, multiple myeloma and AML. 335

Direct pulsing with synthetic peptides is another easy technique to load DCs with neoantigen-derived epitopes, which induces the necessary immune responses. This method requires the accurate identification and prediction of existing suitable epitopes in individuals, which are then synthesized into peptides or even full-length proteins to properly trigger an antigen presentation by the patient’s HLA repertoire on DCs. 362 , 364 In several clinical trials, personalized neoantigen peptide-pulsed DCs have been tested against cancers, including melanoma, ovarian cancer, NSCLC and pancreatic cancers. DCs pulsed with synthetic long peptides and adjuvant Poly(I:C) broaden the breadth and diversity of neoantigen-specific T lymphocytes in melanoma. The t(2;13) translocation in 80% of alveolar rhabdomyosarcomas results in a PAX-FKHR fusion protein that is endogenously processed to generate a breakpoint epitope presented by HLA-B7. Stimulation of DCs with the SPQNSIRHNL fusion peptide derived from the PAX-FKHR neoantigen produced a specific CTL effect, resulting in lysis of rhabdomyosarcoma tumor cells. 365 DCs pulsed with AR and ESFT fused neoantigen-specific breakpoint peptides, including EWS/FLI-1, EWS/FLI-2, PAX3/FKHR, and rhIL-2-treated autologous lymphocytes were reinfused to patients, and this regimen produced an immune response rate of 39% against the fusion breakpoint peptide. 100 , 366 A personalized neoantigen peptide-pulsed autologous DC vaccine is also combined with chemotherapy or ICBs to treat patients with advanced lung cancer and pancreatic cancer (NCT05195619, NCT04627246, NCT02956551).

DCs pulsed with autologous WTL are safe and effective at inducing a broad anti-tumor immunity which have been extensively studied in various malignancies. In recurrent ovarian cancer patients, autologous DCs pulsed with oxidized WTL are well tolerated and elicit potent anti-tumor T cell responses. The vaccination amplifies T cell responses against neoepitopes originated from somatic mutations, including T cell clones against novel neoepitopes and clones with significantly higher avidity against known neoepitopes. 367 , 368 , 369 Furthermore, neoantigens can be loaded into DCs by electrofusion technology, which fuses only the cytoplasm of two cell types without damaging the nucleus, thus maintaining the cellular function of these cells. In addition to expressing the tumor antigens, the fusion cells also enhance the co-stimulation ability of DCs. 315 DC-tumor cell fusion vaccines have been tested in renal cancers, breast cancers, multiple myeloma and melanoma. In a subset of patients with renal cancer, the fusion cells induce tumor-specific immune responses and disease regression. 325 , 370 , 371 , 372 Collectively, these preclinical and clinical studies have proven that neoantigen-based DC vaccines can elicit tumor-specific T cell responses, suggesting a feasible, safe, and effective immunotherapy for solid tumors. 373

Neoantigen-based adoptive cell therapies

Neoantigens with high immunogenicity, as described above, provide excellent targets for the ACT, which employs patients’ own naturally existing or genetically engineered anti-tumor lymphocytes. Neoantigen-based adoptive cell therapies, including TILs and genetically engineered immune cells with novel TCRs or CARs, are currently successfully used to treat multiple malignancies. 374

Adoptive transfer of TILs

CD8 + T lymphocytes have the capacity to identify and eradicate cancer cells, as discovered over 50 years ago. 375 It has been demonstrated that adoptive transfer of in vitro expanded autologous TILs without genetic modifications can induce a full remission of certain human cancers. These TILs are taken from the patient, expanded under particular circumstances, and primed to increase their anti-cancer activity. Then, this cell product is reinfused back into the same patient, who have previous non-myeloablative lymphodepleting chemotherapy and subsequent cytokine therapy, like IL-2, thereby stimulating a potent anti-tumor immune response (Fig. 5 ). 376 , 377 TILs enriched in specificity for neoantigens are preferable to unselected TILs at achieving complete and durable tumor regression. Compared to the low avidities of tumor antigen-specific TCRs, the majority of neoantigen-specific TCRs display significantly higher avidities, even towards cognate antigens expressed at relatively lower levels. 378 Even a modest number of T lymphocytes with an affinity for scarcely tumor-specific neoantigens can be expanded for therapeutic application with the proper manufacturing process. Adoptive transfer of TILs enriched in neoantigen-targeted T cells is a promising treatment strategy, even for tumors with a low mutational burden. 379

Neoantigen-reactive TILs mediate a remarkable regression of epithelial cancers, including advanced breast cancer, metastatic cholangiocarcinoma, colorectal cancer, melanoma, and cervical cancers. 28 , 380 , 381 , 382 , 383 , 384 , 385 In the earliest prospective study of neoantigen-reactive T cells in epithelial cancers, metastatic cholangiocarcinoma patients with low TMB showed effective tumor regression lasting up to 35 months, offering the first concrete proof that neoantigen-targeted TILs can induce regression of metastatic epithelial cancer. Retrospective analysis of the infusion product has shown that the CD4 + T-helper 1 cells were reactive to an ERBB2IP mutation, suggesting a potential function of neoantigen-specific CD4 + T cells in the control of a metastatic epithelial cancer. 386 TILs from individuals with metastatic gastrointestinal cancers have CD4 + and/or CD8 + T cells that recognize neoantigens resulting from somatic tumor mutations. Even though no common immunogenic epitopes are shared in these patients, a prevalent hotspot driver mutation KRAS-G12D in numerous patients can be targeted by CD8 + TILs. 271 Similarly, in patients with metastatic colorectal cancer, KRAS-G12D mutant-targeted CD8 + TILs induce an efficient anti-tumor immune response against lung metastases that expressed HLA-C*08:02. 387 The potential anti-tumor effect of neoantigen-reactive T cells has also been supported by retrospective investigations on the infusion of TIL products in patients with solid tumors. Patients with HPV16 + metastatic cervical squamous cell carcinoma have a full response to TILs that were initially selected based on their sensitivity to HPV antigens together with a high-dose of IL-2. 388 Follow-up studies found that nearly 35% of the TILs could recognize the antigens resulting from tumor mutations compared to the 14% of the viral antigen-reactive TILs, indicating that the personalized neoantigen-reactive CD8 + T cells were responsible for tumor regression. 219

TILs have been utilized to treat patients with metastatic malignancies who are refractory to current therapies, including chemotherapies, radiotherapies and anti–PD-1 therapies. 389 , 390 , 391 , 392 , 393 , 394 Adoptive transfer of TILs targeting specific mutations in four genes, CTSB, CADPS2, KIAA0368, and SLC3A2, along with IL-2 and pembrolizumab results in a full durable regression of chemo-refractory HR + metastatic breast cancer, which is still active at the last follow-up, 5.5 years after therapy. 377 Patients with metastatic melanoma who are resistant to current therapies might achieve objective response rates of 50% to 70% with autologous TIL transfer and IL-2 after host lymphodepletion by total-body irradiation or chemotherapy. 395 Patients who have metastatic NSCLC and are refractory to anti–PD-1 therapies showed a clinical response to immunotherapy combining the TILs, IL-2, and anti–PD-1 (NCT03215810, NCT04032847). 389 Altogether, these studies have provided strong evidence that neoantigen-reactive T cells can improve the clinical outcome of epithelial cancers resistant to current therapies.

The frequency and breadth of TILs are key determinants of their therapeutic efficacy. The quantity and quality of tumor-reactive TILs are unambiguous variable across cancers with complex correlations with anti-tumor immune responses. For example, tumor-reactive TILs are limited to a small number of cells, as only about 10% of intratumoral CD8 + T cells can recognize autologous TSAs in ovarian and colorectal cancers, even no tumor-reactive TCRs have been found in some patients with the presence TILs. 396 In contrast, neoantigen-reactive TILs have been detected in infusion products derived from nonresponding patients with metastatic breast cancer, gastrointestinal cancer, and NSCLC. 389 , 397 , 398 Therefore, assessing the proportion of intratumoral T cell repertoires and their ability to recognize autologous tumors is critical for predicting the clinical activity of human cancer immunotherapies. Human CD8 + TILs can recognize a wide range of epitopes other than tumor antigens, such as antigens derived from viruses, forming bystander T cells that may infiltrate the tissue as effector cells. Neoantigen-specific TILs frequently exhibit stronger anti-tumor activity and tumor-specific expansion as compared to blood-emigrant bystander and regulatory TILs at various signatures and phenotypes. 399 , 400 , 401 , 402 , 403 CD39, a marker of T cell reactivity to tumors and T cell exhaustion, can be used to identify the tumor-reactive T cells in a variety of malignancies. The bystander CD8 + TILs have overlapping characteristics with tumor-specific cells but lack CD39 expression and signs of persistent antigen stimulation at the tumor site. 396 Furthermore, the frequency of CD39 expression in CD8 + TILs correlates with several important clinical parameters, such as the mutation burden and survival rate. 396 , 404 Therefore, CD39 may be a promising indicator for evaluating the prognosis of cancer immunotherapy. 405 The expression of CD39 could also be a viable biomarker for identification, isolation and expansion of tumor-reactive T cell populations in cancers. 404 Using Cellular Indexing of Transcriptome and Epitopes by sequencing (CITE-seq) and TCR sequencing based on the signatures, such as CD39 and CXCL13 expression, neoantigen-reactive TCRs in NSCLC TILs can be identified with a success rate of 45% for CD8 + and 66% for CD4 + T cells. 406 The immunomagnetic cell sorting of stem-like, self-renewable and tumor-specific TILs based on CD39 expression increases the median survival of mice by 60%. 407 Collectively, optimizing the quality of the intratumoral TCR repertoire that is tumor-specific will improve the therapeutic potency of ACT. 396

The intrinsic properties of the transferred T cells, including phenotype, avidity and persistent time, also influence the efficiency of neoantigen-directed ACT. 397 High-dimensional analysis of TIL products identified two CD8 + T cell populations: one has a memory-progenitor CD39-negative stem-like phenotype (CD39 - CD69 - ) and the other has a highly differentiated exhausted CD39-positive state (CD39 + CD69 + ) TILs. 375 , 408 The persistent exposure of TILs to antigens within the intratumoral microenvironment markedly shifted their phenotype towards an exhausted cell state (PD1 + CD39 + ), accompanying by a progressive loss of CD8 + T cell activities and overexpression of inhibitory receptors like PD-1. 375 , 378 It has been recently discovered that PD-1 + CD8 + T cells retain a less differentiated subpopulation of stem-like TILs with ability of self-renewal, expansion, persistence, terminally differentiation and superior anti-tumor activity in vivo. These memory-like or progenitor-exhausted PD-1 + CD8 + T cells serve as a source of terminally exhausted T cells that are capable of killing target cells. 375 , 378 , 409 In contrast to the ACT non-responders, ACT responders have a reservoir of stem-like neoantigen-reactive TILs that expand prolifically and supply differentiated subsets, promoting T cell persistence and long-term tumor control. 375 , 408 Consistent with their exhausted status, progenitor exhausted cells displayed inadequate enrichment for a central memory signature as opposed to an effector memory signature, relative to that of true central memory cells. 409 When compared to T cells produced from an effector memory source, those from a central memory population show a stronger replicative potential in response to antigen and a longer in vivo persistence. 410 The disentanglement of TIL exhaustion through isolating and expanding a desirable neoantigen-specific T cells with memory phenotype, engineering T cells to have stem-like properties, boosting the memory specificities outside the tumor with cancer vaccines, could pave the way for the creation of more effective T cell-based immunotherapies.

Genetically engineered anti-tumor immune cells

Immune cells, including T cells, natural killer (NK) cells and macrophages, can be genetically modified in vitro to generate TCRs and CARs that redirect their specificity to neoantigens. These engineered immune cells circumvent the issues such as limited proportion of tumor antigen-reactive TILs. 411 , 412 , 413 , 414 , 415 Since tumor neoantigens encoded by tumor-specific somatic mutations have emerged as primary antigenic targets of CD8 + and CD4 + T cells in ACT therapy without the toxicity of targeting normal tissues, rapid development of neoantigen-based immune cells holds promising effects for the treatment of solid tumors. 416 A number of neoantigen-targeted TCR-T and CAR-T therapies are being actively investigated in early phase clinical studies, which show an intriguing therapeutic prospect (Table 4 ). 417

TCR-T cells

TCR-transduced T cells can target any surface or intracellular antigens. Several groups have proved the viability of an efficient approach from neoantigen identification to the engineering of the neoantigen-targeting cytotoxic TCR-T cells. 416 , 418 , 419 , 420 When neoantigens are identified and predicted, neoepitope-specific T cells are isolated and their TCRs are sequenced. Candidate TCR sequences with known neoantigen reactivity can be introduced into T cells by transposon or CRISPR/Cas9 systems. These engineered cells expressing TCRs that are specific to neoantigens were infused into the patient after being verified for their tumor reactivity. 416

The engineered high-avidity TCRs render CD8 + T cells specifically cytotoxic to neoantigen-containing tumors. The TCRs specifically targeting recurrent fusion genes CBFB-MYH11 confer CD8 + T cells antileukemic activity in vitro and in patient-derived murine xenograft (PDX) models with fusion gene-driven AML. 88 , 419 , 421 Similarly, peripheral blood lymphocytes transduced with TCRs highly reactive to the mutated KRAS variants G12V and G12D could recognize multiple HLA-A*11:01 + pancreatic cell lines bearing the appropriate KRAS mutations in a xenograft model. 419 , 420 The safety and efficacy of autologous T cells that have been engineered to express TCRs particularly targeting the HLA-A*11:01-presented public neoantigens, KRAS-G12V or G12D, are investigated in a clinical trial enrolling patients with advanced pancreatic cancer (NCT04146298, NCT05438667). Moreover, autologous T cells engineered with personalized neoantigen-specific TCRs are also being conducted in solid tumors, such as ovarian cancer, lung cancer, colorectal cancer, pancreatic cancer, cholangiocarcinoma and gynecologic cancer (NCT05292859, NCT05194735, NCT04520711).

In TCR-T therapy, replacing the endogenous TCR with a neoantigen-specific TCR (neoTCR) can precisely redirect the T cells to tumor cells with specific neoantigens presented by HLA. A recently developed non-viral precision genome editing technique can simultaneously knock-out the endogenous TCR or CAR genes and introduce a neoTCR or CAR, allowing a faster production of clinical-grade T cells. 422 , 423 Based on this non-viral precision TCR replacement technology, a variety of T cell products with distinctly personalized neoTCRs for one patient are available to improve the anti-tumor effect. Three TCR-T cell products with unique personalized neoTCRs were administered to each of sixteen patients with refractory solid cancers, five of which had stable disease and the other 11 had disease progression as best response on therapy. 422 Therefore, it is feasible and safe to create a broadly applicable, tumor-specific, and tailored T cell treatment for patients with solid malignancies based on this non-viral precision TCR replacement approach.

CAR-T cells

CAR-T cell approaches have a substantial advantage over TCR-T cells since they do not rely on HLA expression and neoantigen presentation, the loss of which are commonly exploited by cancer cells for immune evasion. The engineered expression of CAR molecules, which contain an intracellular signaling and co-signaling domain and an extracellular antigen-binding domain, enable CAR-T cells to bind any cell surface protein once for which there is an antibody and then activate CAR-T cells independent of MHC. 424 , 425 Early clinical trials using CD19-targeted CAR-T cells for the treatment of B-cell malignancies patients showed outstanding results, while CAR-T cells for the treatment of patients with solid cancer showed poor outcome because of the limited antigens. 424 Tumor neoantigens have inspired creative solutions and given solid tumor patients hope for CAR-T therapy. The limited number of tumor-specific surface neoantigens that are suited for CAR-T can be overcome by integrating a single-chain variable fragment (scFv) that recognizes a neoantigenic pMHC complex on the tumor surface. CAR-T cells with an scFv that recognizes the oncogene nucleophosmin (NPM1c) epitope-HLA-A2 complex demonstrated strong cytotoxicity against NPM1c + HLA-A2 + leukemia cells and AML blasts with no or minimal on-target/off-tumor toxicity. 336 , 376 , 426

CAR-T cells redirected at novel neoantigens are being tested in ongoing clinical trials in hematological and solid tumors. 424 , 427 The most well-known example of neoantigen-based CAR-T therapy in solid tumors is neoantigens from EGFRvIII mutation, which are caused by in-frame deletion of a piece of the extracellular domain spontaneously in 30% of glioblastoma patients, 428 making it a desirable target for CAR-T therapy. A CAR that can recognize the EGFRvIII neoantigen has been created as a part of a lentiviral vector and a truncated EGFR that lacks the ligand binding domain and cytoplasmic kinase domain is incorporated for in vivo tracking and ablation of CAR-T cells in necessary. Human EGFRvIII + xenogeneic subcutaneous and orthotopic models showed that EGFRvIII-directed CAR-T cells could control tumor growth. 429 The safety and effectiveness of autologous anti-EGFRvIII CAR-T cells are also tested in a pilot project in patients with recurrent glioblastoma (NCT02844062). However, only a small portion of tumor cells would be killed by targeting EGFRvIII due to the highly heterogeneous of glioblastoma.

Even if the antigens are imperfectly specific individually, a Boolean logic gate can be used in CAR-T cells to improve the specificity of tumor recognition by priming with tumor-specific neoantigens and boost the eradication efficiency of tumor cells by targeting antigens uniformly expressed by tumors. The T cells can generate CARs that target antigens universally expressed by tumors, like EphA2 and IL13R2, after being primed by a highly tumor-specific neoantigen, like EGFRvIII, and being trained to carry out complete tumor destruction. In addition, a synthetic Notch (synNotch)-regulated CAR activation maintains a significant proportion of T cells in a naive/stem cell memory state, leading to improved anti-tumor immunity. In immunodeficient animals bearing intracerebral PDXs with a heterogeneous expression of EGFRvIII, EGFRvIII synNotch-CAR-T cells outperformed conventional constitutively expressed CAR-T cells in terms of anti-tumor activity and T cell persistence without causing off-tumor damage. T cells engineered with prime-and-kill circuits induce CAR-driven cytotoxicity that is spatially limited only to the proximity of priming cells, preventing off-tumor killing in distant normal tissues that carry the killing antigen but lack the priming antigen. 231 , 430 , 431

In addition to T cells, NK cells can also be engineered to express CARs. NK cells have the same capabilities as CD8 +  cytotoxic T cells but they are not dependent on MHC-I -mediated tumor neoantigen presentation. As a result, the CAR-NK cells have the potential for immunotherapy against tumors with an extremely low mutational load and deficient neoantigen presentation. Arming NK cells with neoepitope-specific CARs remarkably improve their anti-tumor responses to NPM1-mutated AML without causing off-target toxicity. 432 Moreover, NK cells further prime the DC maturation and neoantigen presentation via releasing GM-CSF, and recruit neoantigen-specific CCR5 + CD8 + T cells by producing CCL5. 433 Thus, the variety of cancer types amenable to immunotherapy increases as a result of modified NK cells. 7

Antibody-based therapy against neoantigens

Antibody therapies have been successfully used to treat cancers, such as the anti-PD1/PD-L1/CTLA4 antibody for ICBs. Compared to the conventional antibodies that are incapable of targeting intracellular proteins, TCR-mimic (TCRm) antibodies or mutation-associated neoantigens (MANA)-specific antibodies can recognize the intracellular neoantigens by focusing on pMHC complexes. TCRm antibodies have a greater affinity than TCRs, which has been shown to be essential for minimizing the on-target, off-tumor effects. 434 , 435 , 436 , 437 , 438 , 439 These neoantigen-targeted antibodies are simple to transform into a variety of therapeutic formats, including full-length antibodies, antibody-drug conjugates (ADCs) and BsAbs. As mentioned above, TCRm antibody moieties can also be employed to drive specific activity for neoantigens by CAR-T therapy, which has proved remarkably effective in treating certain cancers. 440 Moreover, these antibody-based immunological strategies have the potential to develop off-the-shelf products for any patient whose tumors exhibit the targeted public neoantigens. 300

Phage display, yeast display and genetic platform are some of the technologies used to determine human TCRm antibodies with exquisite specificity for the neoantigen as presented on HLA. In order to identify scFvs specific for mutant pMHC complex, a phage or yeast display library encoding a vast number of scFv sequences was initially created. Using a competitive selection technique, clones specific for mutant peptides bound to predetermined HLA types were subsequently identified. 441 , 442 A high-throughput genetic platform, PresentER, is comprised of minigenes that encode MHC-I peptide libraries. By assessing the reactivities of TCR-like therapeutic agents against vast libraries of MHC-I ligands, PresentER could be utilized to determine the on-and-off targets of T cells and TCRm antibodies. 443 Combining structural analysis of a reagent with its corresponding pMHC complexes with library screening helps improve TCRm antibody specificity evaluations. 300 According to a crystal structure, a human TCRm antibody called ESK1 attaches to Wilms tumor (WT1)-derived peptide/HLA-A*02:01 in a manner distinct from TCRs. The possible patient pool for ESK1 therapy can be expanded by using the structure to anticipate high-affinity binding of ESK1 with several different HLA-A*02 subtypes and potential off-target binding. 444

Public neoantigens originating from recurrent driver mutations, including oncogenes and TSGs, provide shared targets that could benefit a substantial proportion of patients. The scFvs that target the public neoantigens coming from oncogene mutations, such as EGFR, KRAS, PIK3CA, and CTNNB1, have been identified and transformed into therapeutic formats. 300 , 441 , 445 , 446 , 447 For example, one scFv specific for KRAS mutant-derived peptide and one for EGFR mutant-derived peptide has been identified by phage display. These scFvs recognize the peptides only in complexes with HLA, such as KRAS peptide/HLA-A2 or EGFR peptide/HLA-A3 complexes. The scFv specific for KRAS(G12V)-HLA-A2 is converted to a full-length antibody, which responds with mutant peptide-HLA complexes even when the peptide differs from the normal wild-type form by just one amino acid. 441

In contrast to oncogenes, public neoantigens coming from recurrent mutations in TSGs are unable to trigger an immune response because they are either rendered inactive by non-recurrent mutations or produced at low levels due to nonsense-mediated RNA decay. The well-characterized TSG p53 is a special case due to the identification of TCRm antibodies that target the p53 pMHC complex. Due to MHC-binding restrictions, peptides containing mutant p53 sequences are uncommon; however, tumors expressing mutant p53 may have increased expression and the MHC molecule-mediated presentation of wild-type p53 peptide, which distinguishes the tumors with mutant p53 from healthy cells expressing wild-type p53. 448 , 449 , 450 , 451 Therefore, a TCR-like antibody P1C1TM that is specific for the wild-type p53 125-134 peptide in complex with the HLA-A24:02 (HLA-24) MHC allele can target tumors harboring mutant p53 and HLA-A24. This specificity for the p53 peptide/HLA-A24 complex enables P1C1TM as an antibody-drug conjugate to effectively deliver a cytotoxic payload to tumors with mutant p53, as demonstrated by the lethal effects of PNU-159682-P1C1TM restricted to mutant p53-expressing colorectal cancer cells in in vivo models. 451

BsAbs can be employed to address the issue that the density of the mutant p53 pMHC complex on the cell surface was insufficient to recruit T lymphocytes to the tumor site. Bispecific T cell engager (BiTE) is a bsAb construct that provides an efficient and potent signal for T cell activation through simultaneously binding a neoantigen on tumor cells and a CD3 complex on T cells. Even when the neoantigen-MHC complex is expressed at low levels, the highly powerful bsAb is able to decisively reverse the undruggable reputation of p53. 317 , 440 A peptide produced from the p53 missense mutant (R175H) can be presented by HLA-A*02:01 to form a mutant p53 pMHC complex at the cell surface, which serves as a natural TCR ligand to activate T cells. An H2 antibody fragment with enhanced affinity for the HLA-A*02:01-restricted p53 R175H neoantigen has been discovered by screening utilizing a large phage library. This TCRm antibody fragment was fused with a CD3-specific antibody fragment to create a bsAb that could improve the activation of T cells to recognize and destroy cancer cells and grafts in animal models expressing the p53 R175H pMHC complex. 440

Dimeric T cell engaging bsAbs are also created based on human TCRm antibodies with exquisite specificity for the mutant LMP2A peptide-HLA-A*02:01 and mutant RAS peptide-HLA complexes. These bsAbs were effective in precisely activating T cells and killing target cancer cells that expressed endogenous, incredibly low quantities of the mutant neoantigens and cognate HLA alleles. 452 , 453 In addition, bsAbs were also employed to target public neoantigens originating from dysregulated PTM in malignancies. BsAbs engaging CD3 with TCRm specific for a pIRS2-derived phosphopeptide in complex with HLA-A*02:01 were capable of killing tumor cells in a pIRS2- and HLA-A*02:01-restricted manner. 189 Alternatively, soluble structures guided by monoclonal TCR moieties specific for tumor neoantigens can also be coupled to an anti-CD3 antibody component to generate a group of bispecific molecules, known as immune-mobilizing monoclonal TCRs against cancer (ImmTACs). ImmTACs get over the biophysical constraints that prevent TCR-based immunotherapeutic methods in the past and might make it possible to target any cell based on its proteomic traits. Cancer cells with extraordinarily low surface epitope concentrations were successfully killed by T lymphocytes that had been guided by ImmTACs. 300 , 454 , 455

As a result, TCRm antibodies-based strategy could be used to target neoantigens originating from mutations in both oncogenes and TSGs that are challenging to eradicate using traditional methods, enabling the development of more targeted anti-cancer therapies. 452 , 456 , 457 , 458 , 459 Given TCR mimic antibodies have a much better affinity to peptide-HLA molecules than natural TCRs. To prevent cross-reactivity or binding of the HLA component unrelated to the given peptide, TCR mimic antibodies must be properly screened. Similar to designed TCRs, cross-reactivity can be prevented by using negative selection against off-target peptides. 426 , 441 , 447 At least one instance of synthetic reagents has been developed that exhibits lower cross-reactivity than equivalent natural receptors. 460

Combinational therapies

The therapeutic efficacy of a single immunotherapy for patients with advanced cancer is inadequate due to the heterogeneity of the neoantigen landscape and the continually evolving cancer immune evasion mechanisms. Combining several immunotherapies can improve the efficacy against cancers by simultaneously targeting various stages of the cancer-immunity cycle, including antigen release and presentation, immune cell priming and activation, immune cell trafficking and infiltration into tumors, and recognition and killing of cancer cells. 7 , 461 Another strategy is combining therapies with different mechanisms of action to overcome the resistance induced by tumor heterogeneity. All targeted cancer cells must have the same pattern of neoantigen expression and presentation, otherwise, a resistance clone without the predicted neoantigens can survival and confer a clonal growth advantage. Therefore, precision immunotherapy can be combined with conventional treatments like radiotherapy and chemotherapy that kills cancer cells independent of the neoantigens, achieving a more prominent and durable therapeutic effect (Fig. 6 ).

Combinational neoantigens-based anti-tumor strategies. The “Cancer-Immunity Cycle” refers to the sequential events that must be initiated, proceeded, and expanded to achieve an anti-cancer immune response, resulting in the efficient eradication of cancer cells. Briefly, neoantigens generated by oncogenesis are released and captured by DCs (step 1). DCs convey the collected neoantigens on MHC-I and MHC-II molecules to T cells (step 2), resulting in priming and activation of effector T cell responses against cancer-specific neoantigens (step 3). Subsequently, activated effector T cells migrate to (step 4) and infiltrate into (step 5) the tumor bed, where they recognize and finally destroy their target cancer cells (step 6). The death of cancer cells produces additional tumor-associated neoantigens (step 1 once more), which broadens and intensifies the immune response in subsequent cycles. Therefore, cancer immunotherapies have been designed to reinitiate or amplify a self-sustaining cycle of cancer immunity. Multiple immunotherapies have been developed to target the rate-limiting steps in “Cancer-Immunity Cycle”, including enhancing the neoantigen release by chemotherapy, radiation therapy and oncolytic virus, increasing the quantity and quality of tumor-reactive T cells through cancer vaccine and ACTs, and boosting the infiltration and cytotoxicity efficacy of immune cells via checkpoints inhibitors

Neoantigen-based immunotherapies and ICBs

Checkpoint inhibitor-based immunotherapy has achieved prolonged anti-tumor effects in several malignancies, including renal cell carcinoma, NSCLC and melanoma. Patients, however, do not react to ICB therapy in the absence of tumor-specific effector T cells. 245 Moreover, ICB therapy only affects one or two phases of the anti-cancer immunity pathways, such as anti-CTLA4 antibodies regulate the immune cell priming and activation, while anti-PD-1/PD-L1 antibodies focus on the final negative regulation of T effector cells. Therefore, only a small percentage of patients have anti-tumor response with a single agent. The neoantigen load and intratumor heterogeneity can be predictive biomarkers for the ICB response. 38 It is reasonable to suspect that more effective anti-tumor response would be achieved by combining ICBs with neoantigen-based immunotherapy approaches that boost the tumor-reactive T cells. 8 ICBs enhance specific T cell responses by targeting neoantigens, including PRKDC, EVI2B and S100A9, in a relapsed multiple myeloma patient. 41 Compared to monotherapy, the neoantigen vaccine (PancVAX) in combination with two checkpoint modulators, such as anti-PD-1 and agonist OX40 antibodies, causes an improved and more substantial tumor regression. 462 For patients with solid tumors who are unresponsive to, or relapsed following anti-PD-1 therapy, mRNA-based neoantigen vaccines, such as mRNA-4157, mRNA-5671, and BNT122, are used together with immune checkpoint inhibitors in multiple clinical trials (Table 4 ). 177 Frequently, immunosuppressant regulators, such as PD-1, PD-L1, CTLA-4, and TIM3, are upregulated by neoantigen vaccines. 245 , 433 ICBs could mitigate this negative effect of neoantigen vaccinations, leading to fast and long-lasting CD8 + T cell control of malignancies. 177 , 433 Therefore, the combination of neoantigen vaccines and ICBs can achieve a better expected effect of anti-tumor immune response. 245

The anti-tumor efficacy of CTLs, including those specific for mutation-associated neoantigens, can be further boosted by ICB therapy. TILs often exist in small quantities within a tumor and demonstrate an irreversible hypo-responsiveness as a result of the suppressive microenvironment. Therefore, most cancer patients are not eligible for TIL therapy. 389 , 421 Patients with immunotherapy response to PD-1 inhibitors have a high proportion of TILs, indicating that ICBs can promote the infiltration of neoantigen-reactive lymphocytes into tumors. 376 , 463 Blocking the PD-1 inhibitory signals induce the expansion of PD-1 + CD8 + T cells, resulting in a transient elevated cycling PD-1 + CD8 + T cells and an increasing amount of effector T lymphocytes at the tumor site. 376 , 400 , 464 , 465 , 466 , 467 In addition, ICBs can reinvigorate the exhausted neoantigen-specific T cells via overcoming the suppressive microenvironment. Persistent exposure to TSAs promotes the exhaustion of CD8 + T cells, which characteristically expressed high levels of PD-1 and CD39. 465 , 466 The intratumoral CD8 + T cells with high PD-1 expression show an intrinsically high capacity for tumor recognition. 468 Given the potent activation of CD39 + CD8 + T cells by high-affinity neoantigens, patients with hepatocellular carcinoma in high-affinity neoantigens-high group benefited more from anti-PD-1 therapy than high-affinity neoantigen-low group. 123

Combinations of neoantigen vaccine and ACT

Combinations of neoantigen vaccination and ACT have also been utilized successfully to boost clinical efficacy in tumor treatment. 245 Recent exciting findings showed that vaccination can increase the amount of neoantigen-reactive T cells in circulation, possibly by boosting better outgrowth of T lymphocytes. Alternatively, the vaccines can induce de novo T cell responses that overcome the insufficient recognition of neoepitope by T cells due to inadequate cross-presentation of a neoantigen by tumor cells. In addition, vaccines can be made to shield neoantigen-reactive T cells from immune checkpoint signaling or FasL-mediated apoptosis, allowing T cells to infiltrate the immunosuppressive tumor microenvironment (TME) and durably reduce epithelial malignancies. In order to increase the clinical efficacy of subsequent ACT therapy, vaccinations may be utilized to prime the patient’s neoantigen-reactive TILs or PBMCs before in vitro T cell culture. This could result in the induction of a known memory T cell response. 374

Vaccine is also used to enhance the efficacy of CAR-T therapy to eliminate solid tumors. A booster vaccine for CAR-T cells has been designed, in which the peptide neoantigens can be trafficked to lymph nodes and subsequently decorated the membrane of resident APCs by their albumin-binding phospholipid-polymers. Vaccine-boosting donor cells enhance CAR-T function in solid tumors through their chimeric receptor directly in vivo. This amph-ligand vaccine can significantly elicit the amplification and intratumoral infiltration of EGFRvIII-specific CAR-T cells compared to CAR-T cell delivery alone. This vaccine strategy safely expands CAR-T cells in vivo and boosts their function and anti-tumor activity in multiple models of solid tumors, showing the significant promise of neoantigen vaccine and CAR-T combinatorial therapy. 245 , 469 , 470 , 471

Neoantigen-based immunotherapies and conventional therapies

The majority of chemotherapeutic agents and radiation therapy were designed based on their direct cytotoxic effects without considering their impact on immune system. The genomic damage and altered gene transcription during these conventional therapies can promote the production of tumor-specific neoantigens, hence exhibiting potential of stimulating the anti-tumor immune response. Therefore, several FDA-approved combination therapies using conventional therapy together with immunotherapy have been developed. 7

Chemotherapy and radiotherapy can be used to increase the release of tumor-specific neoantigens, circumventing issues such as an insufficient number of neoantigens to stimulate T cell response. In a patient with metastatic NSCLC who has completed response to the combined CTLA4 blockade and radiotherapy, neoantigenic mutation in KPNA2 is upregulated by radiation. Peptides derived from mutant KPNA2 trigger neoantigen-reactive CD8 + T cells and induce IFNγ production, which may trigger antigen spread. 472 , 473 In addition, radiation can enhance the levels of existing peptide presentation by increasing the surface expression of MHC-I on tumor cells. Though expanding intracellular neoantigen pools and increasing the MHC-I-dependent presentation, radiation would promote cell killing by neoantigen-specific CD8 + T cells. 472 , 474 , 475 In a poorly immunogenic mouse model of TNBC, radiotherapy increases the expression of genes with immunogenic mutations. The neoantigen vaccines based on the immunogenic mutations induced by radiotherapy elicit CD8 + and CD4 + T cells that improved the therapeutic efficacy of radiotherapy. 476 Notably, highly subclonal neoantigens induced by radiation, which might be worsened by DNA-damage response (DDR) inhibitors, would interfere with the production of T lymphocytes against clonal tumor neoantigens. Additional investigations on the formation of subclonal neoantigens, as well as a thorough investigation of combined radiation, DDR inhibitors, and neoantigen-based therapies are needed to address these concerns. 472

During the chemotherapy and targeted therapy, the tumor cells often occur new mutations, including reversion mutation, contributing to drug resistance. Many reversions are predicted to encode tumor-specific neoantigens, offering a potential strategy for combating resistance with CAR-T cell therapies, immune checkpoint inhibitors or anti-cancer vaccines. Reversion mutations in breast cancer-related genes are just one example that occurs during clinical platinum and PARP inhibitor resistance. 477 The amount and functional activity of neoantigen-specific T lymphocytes can also be increased by administering a tumor vaccination followed by pretreatment with cyclophosphamide (CTX) and other drugs. 177 Together, these studies show proof-of-principle that conventional treatments can enhance tumor control when used in conjunction with immune therapies based on neoantigens.

Challenges and opportunities for clinical application

Despite the success in hematological malignancies and solid tumors mentioned previously, neoantigen-based immunotherapies have only shown objective efficacy in a small number of well-documented patient responses. Consequently, considerable improvements are required to improve clinical results, including increasing the accuracy of neoantigen prediction, overcoming immune evasion, and optimizing the streamlining of the production process. This section focuses on the barriers that must be surmounted to enable potent immune response specifically based on tumor-specific neoantigens and the possible solutions for offering a safe and effective therapy for solid tumors.

Limited accuracy of neoantigen prediction

The widespread application of personalized immunotherapies has been constrained by the limited discovery of targetable cancer neoantigens due to the heterogeneity of mutational burdens and significantly distinct neoantigen presentation among various tumor types. 81 Only 10% of non-synonymous tumor cell mutations can produce mutant peptides with high MHC affinity, and only 1% of the MHC-binding peptides are recognized by patient T cells. Theoretically, the higher the TMB, the greater the number of neoantigen-specific T cells in the tumor can be detected, resulting in a greater immunotherapy response rate. Nevertheless, low TMB can produce neoantigen-reactive lymphocytes in hematological malignancies and certain epithelial cancers, such as gastrointestinal cancers. 10 , 240 , 478 The insufficient neoantigen density in malignancies with low TMB, such as AML and pediatric brain cancers, requires greater powerful strategies for the accurate identification of immunogenic neoepitopes that can be detected by CD8 + T cells. 479 , 480 High-throughput technologies enable systematic assessment of suitable neoantigens for immunotherapies, overcoming the limited neoantigens caused by low TMB. 333 For example, a proteogenomic method that integrates NGS and MS data supports the development of highly target-specific, autologous, personalized neoantigen immunotherapy, especially for tumors with low TMB. 479 , 481 , 482

The prediction of neoantigens is also constrained by genetic heterogeneity, particularly the diverse somatic mutations in distinct cancer types, in different individuals and even within tumor subclone cells. A major cause of genetic heterogeneity in cancer is genomic instability, which is dynamically altered in distinct tumors and different stages. For example, TNBC patients have a higher load of frameshift and mutation-associated neoantigens (MANA) and a higher response rate to immunotherapy compared with patients with other invasive breast cancer subtypes. Furthermore, BRCA-1-mutated TNBC has an even higher mutational load. 53 , 483 , 484 , 485 Therefore, identification and prediction of neoantigens should be conducted uniquely for individuals with specific cancer. 245 Additional problems may develop depending on how the tumor sample from a patient is obtained for neoantigen identification. Recent technologies enable the investigation of the genomes and transcriptomes of single tumor samples taken at specified time points; however, this does not disclose heterogeneous mutations occurring in different lesions across a patient. The diversity of neoantigen-specific T cells present in a patient may not be fully captured by a single excised lesion due to restricted T lymphocyte infiltration, which constrains the TCR repertoire that may be established for therapy. 245 Furthermore, mutational heterogeneity within tumors contributes an additional degree of intricacy for neoantigen prediction. The genome of tumor cells undergoes extensive generation, cloning, alteration and loss of mutations. Thus, tumor clone cells that do not respond to the neoantigen-specific T cells may exist, which may outgrow other clones due to a selection advantage, thereby restricting clinical benefit. 40

Escape from immunological surveillance

A significant barrier to eliminating cancer is immune evasion, particularly for anti-cancer immunotherapies. Tumors can evade neoantigen-based immunotherapies through a number of mechanisms, including the loss of neoantigens, modification of antigen peptide presentation, and immunosuppressive TME.

Loss of neoantigens

The loss of tumor-specific neoantigens may be a significant immune escape strategy for tumors, especially if many neoantigens are by-products of tumorigenesis and do not have a critical function in tumor cell survival. The depletion of neoantigens can also present as a refractory mechanism to anti-tumor immunity, limiting the application of individualized neoantigen-specific immunotherapy. Neoantigen depletion can be induced by multiple pathways, such as copy number loss, transcriptional repression, epigenetic silencing and post-translational mechanisms. In a cohort of early-stage NSCLC tumors, 48.9% (43/88) showed evidence of neoantigen loss due to subclonal copy number events. The mutant genes encoding non-expressed neoantigens have enriched hypermethylation at their promoter regions compared with the wild-type parental genes in other purity/ploidy matched samples. 486 In addition, tumors can alter the presentation of neoantigens via modulating protein turnover. Mutant proteins are more likely to misfold and degrade quickly through the proteasome, resulting in elevated antigen presentation. Molecular chaperone HSP90, however, can be used by tumors to stabilize altered proteins, preventing them from entering the antigen presentation pathway. 487 , 488 Neoantigens that exclusively existed in specific tumor cell subpopulations can also be lost as a result of the CD8 + T cell-mediated eradication of the entire subclonal cell population. Many of the deleted mutations are identified by patients' T cells and neoantigen-encoding genes are unlikely to be produced in tumors with extensive immune cell infiltration, suggesting that neoantigen-expressing tumor subclones may be preferentially removed by the immune system. 487 , 489 Furthermore, neoantigen loss through the deletion of chromosomal regions or the elimination of tumor subclones can lead to acquired resistance to immunotherapies such as ICBs. 489 Therefore, to compensate for the loss of targetable neoantigens during immunotherapy, personalized neoantigen-specific immunotherapy should target multiple neoantigens, therefore expanding the scope of neoantigen reactivity. 487

Disrupted presentation of neoantigen peptides

Tumors may evolve mutations that change not just neoantigen expression but also HLA heterozygosity and MHC stability in response to anti-tumor immune pressure. These changes impede neoantigen processing and presentation, hence inhibiting T cell recognition and tumor killing. 316 , 490 The tumors may be able to avoid recognition by adoptively transferred T lymphocytes if there are mutations in key antigen presentation genes like β2M or a lack of HLA allele heterozygosity. 316 , 380 For example, all seven lung metastases from a colorectal cancer patient regressed after receiving an infusion of TILs containing four unique T cell clonotypes that target KRAS-G12D. However, an evaluation nine months after treatment found that one of these lesions had advanced progress. Further analysis of this excised lesion discovered that the chromosome 6 haplotype responsible for the HLA-C*08:02 MHC-I molecule had been deleted, contributing to the tumor immune evasion. 387 A second verified mechanism for epitope loss has been found as the downregulation of MHC molecules in tumor cells owing to aberrant transcription, translation or protein stability events. 491 , 492 In multiple myeloma cell lines, higher levels of splicing factor expression are associated with lower levels of MHC-II activity, while spliceosome inhibition improved MHC-II activity, suggesting that abnormal alternative splicing is responsible for the loss of MHC-II. 123 Moreover, autophagy-dependent degradation causes the lower expression of MHC-I in pancreatic ductal adenocarcinomas that are resistant to ICB treatment. Inhibiting autophagy improves antigen presentation and anti-tumor T cell responses, slowing tumor growth in syngeneic host mice. 493 Due to the decreased neoantigen presentation, these mechanisms together may help to partially explain why higher neoantigen loads in some cancers were not linked to better prognostic outcomes. Based on these findings, ICB therapy for cancers may be more effective if MHC-I presentation is activated using splicing inhibitors or autophagy inhibitors. 123 , 493

The immunosuppressive TME

Loss of neoantigens and insufficient presentation are only two of the many immune evasion mechanisms possessed by tumor cells. The recognition of neoantigens and activation of T cells can also be compromised by immunosuppressive TME processes, including the suppression of immunological checkpoints, the immunosuppressive effects of various TME cells, and the release of ions or proteins from within tumor cells following necrosis. 245 The immunosuppressive checkpoint ligand molecules like PD-L1 and CTLA-4, which can restrict T cell growth and function biologically, are often upregulated in tumor cells during immunotherapeutic treatment. 245 , 433 , 491 ICBs are combined with neoantigen vaccines to prevent immune escape. 245

Inducible neoantigen expression, combinatorial CARs, and leveraging epitope spreading are compensatory strategies that may be used to address the immune escape of tumor cells. 494 The MHC-I immunopeptidomes can be extended by splicing-derived neoepitopes that result from defective interactions of splicing complex with RNA, improper degradation of the accessory splicing factors, or aberrant splicing factor PTMs. The considerable number of highly immunogenic neoantigens created by pharmacologically altered splicing enhances tumor immunogenicity and improves the immune response to ICB treatment in mouse model. 495 These findings open the exciting possibility of employing immunotherapy, which was until now only effective in diseases with greater mutation burdens like melanoma, to treat cancers that are resistant to current treatment.

Insufficient production of neoantigen-specific T cells

The immunotherapies, including vaccination, adoptive transfer of tumor-reactive TILs and TCR-T cell therapy, as well as ICBs, all rely on neoantigen-specific T cells. Thus, streamlining the sufficient production of tumor-reactive T cells from cancer patients or healthy donors should accelerate the application of neoantigen-based therapies, including broadening the TCR repertoire, enhancing the neoantigen presentation and the proper expansion of T cells.

Limited neoantigen-reactive T cell repertoire

TILs include a large number of neoantigen-reactive T cells, making them a valuable source of T lymphocytes for ACTs. 380 However, the implementation of TILs treatment will be hampered by the scarcity of fresh tumor samples and cold tumors with low TILs. 389 , 421 First, the acquisition of TILs requires invasive surgery to remove a resectable lesion, which enables only some patients to be suitable. Second, in cold tumors, the suppressive TME may reduce the efficacy and quantity of TIL-derived neoantigen-specific T cells. 379 , 389 , 421 Therefore, the majority of cancer patients are ineligible for TIL therapy due to insufficient TCR repertoire. 379 Efforts are currently being made to develop efficient strategies for the isolation and rapid expansion of neoantigen-specific T cells, which could benefit neoantigen-based ACTs. Utilizing particular cell-surface markers such as CD39, immunomagnetic cell sorting can efficiently detect and separate potent tumor-specific TILs with self-renewing ability from solid tumors, enabling the long-term effectiveness of ACTs. 407

The easily accessible peripheral blood could be a suitable source for generating large amounts of neoantigen-reactive T cells for ACTs. 379 Both circulating neoantigen-specific T cells and peripheral blood lymphocytes modified to express neoantigen-specific TCRs recognize and kill autologous malignancies. 467 , 496 , 497 There are several clinical trials utilizing neoantigen-reactive T cells generated from peripheral blood to treat patients with epithelial cancer (NCT02959905, NCT05020119, and NCT04596033). 498 Nonetheless, these T cells are significantly scarce and need to be precisely isolated by identified neoantigens or stimulated in vitro to proliferate to detectable levels. 374 Cancer patient-derived PBMCs can be stimulated in vitro with mutant peptides to enrich neoantigen-reactive T lymphocytes. However, it should be highlighted that the substantial in vitro expansion can both further differentiate T cells and expand falsely positive neoantigen-reactive T cells. 499 , 500 Moreover, the tumor-reactive TCR repertoire of PBMC-derived T cells can be expanded by transducing TCRs specific for tumor neoantigens of each patient. 374 In addition, numerous neoantigen-reactive TCRs can be simultaneously transduced into the PBMC of a patient to boost the immune response. Based on these advantages, minimally cultured T cells with individualized TCRs will have better cytolytic capacity than exhausted and senescent TILs. Therefore, future studies improving neoantigen-reactive T lymphocytes generated from peripheral blood will benefit for their clinical applications.

The efficacy of ACTs can be restricted when tumor-reactive T cells are weakly persistent and terminally differentiated effector cells. 501 , 502 , 503 Naive and very early memory T cells with stem cell-like properties can be produced using induced pluripotent stem cell (iPSC) technology. 504 The functionally regenerated CTLs produced from iPSC that are specific for the EWS/FLI1 fusion gene-derived neoantigen elicit an anti-tumor immune response in both cultured EWS/FLI1 + sarcoma cells and Ewing sarcoma xenograft mouse model. 505 iPSC-derived immature T cell lineages can now be further differentiated into neoantigen-specific T cells based on the unique three-dimensional thymic organ culture system that faithfully recapitulates the disease in vitro. The ability of iPSC-derived thymic emigrants to express heterodimeric αβ co-receptor of T cells can be maintained by imitating the thymus in vitro. 506 Another benefit is that iPSCs can be established from a single neoantigen-specific CTL clone and re-differentiated into a large number of CTLs. 505 In the near future, iPSCs derived from a single CTL clone will be employed to generate a sufficient number of neoantigen-specific TCR-T cells that preserve a naive-like condition and contain the TCRs in its endogenous state, significantly boosting neoantigen-based immunotherapies.

Neoepitope presentation

Presently, mature DCs or EBV-transformed B cell lines that are pulsed with peptide or transfected with TMGs for APCs are used to present antigens to T cells. 374 Neoantigen-expressing mRNA-transfected DCs are also employed to prime the autologous naive CD8 + T lymphocytes in healthy donors, who are not exposed to the immunosuppressive milieu of tumor hosts. 101 However, TCRs triggered by APCs pulsed with mRNA-encoding or synthetic peptides might not recognize tumor cells that present antigens endogenously. 507 Directly detecting the neoantigens presented by tumor cells may be the most efficient method to establish neoantigen-reactive T cells, ensuring they can recognize the neoepitope in vivo. Tumor single-cell suspension, PDXs in highly immunodeficient mice and three-dimensional patient-derived tumor organoid cultures can be used for the recognition. Notably, PDXs and tumor organoids exhibit more typical features in neoantigen processing and presentation in contrast to the synthetic peptides or TMGs presented by DCs or B cells. PDXs maintain the endogenous expression, natural processing, and presentation of neoepitopes. 508 When little tumor biopsies are available, PDX tumors can be employed to steadily extend the authentic peptidomes, creating possibilities for identifying neoantigens and sufficiently presenting neoepitopes to T cells. 509 The tumor organoid cultures that extend the in vitro capacity of patient tumor cells are another alternative for autologous neoepitope presentation. A unique preclinical therapeutic model was created using tumor organoid-T cell co-culture systems to precisely measure each patient’s sensitivity to various immunotherapies. This approach was able to assess the effectiveness of cellular immunotherapies in vitro while also preserving the heterogeneity and microenvironment of tumors. In addition, co-culturing autologous PBMCs with tumor organoids produced personalized tumor-reactive CD8 + T lymphocytes that significantly reduced the growth of tumors. 405 , 510 Although further improvements are required to conserve TME, including myeloid and stromal components, the organoid model may offer a better in vitro opportunity for neoantigen presentation and recognition by T cells.

Improved expansion approaches for tumor-reactive T cells

A significant mechanism of immunotherapy resistance is the death of tumor-reactive T lymphocytes. Since the finding that the expression of Fas-ligand (FasL) in melanoma cells caused TIL mortality, the role of the Fas/Fas-ligand pathway in establishing tumoral immune resistance has been debated for several decades. 511 T cells separated from cancer patients express more Fas than healthy donors. Moreover, most cancer cells and intratumoral vascular endothelial cells express high levels of Fas ligand (FasL), which is related to a lack of CD8 + infiltration. 374 , 512 Fas-FasL interactions in the TME during ACT may lead to T cell death. Therapies to control the Fas-mediated apoptosis and differentiation may be helpful to generate the appropriate cell products for efficient ACT. The mutant Fas that failed to bind FADD are used as a dominant negative receptor (DNR) to prevent FasL-mediated apoptosis in Fas-competent T cells. 380 The Fas DNR can be transduced together with a neoantigen-reactive TCR or CAR into T cells, which can be further enriched using a magnetic bead of the introduced TCR or Fas. These Fas DNR-engineered TCR-T or CAR-T cells showed improved persistent anti-tumor activity against established solid and hematologic malignancies. 380 Other strategies involve the systemic administration of Fas-Fc or anti-FasL to neutralize FasL. These FasL-neutralizing methods may reduce the death of TILs, enhance the tumoral infiltration of CD8 + T cells, and improve the persistence and activity of T cells at the tumor site. 513 Notably, when administered in concert with ACT, these cell-extrinsic reagents may impair the capacity of T cells to use Fas/FasL signaling to cause cytolysis in tumor cells.

Determination and monitoring of neoantigen-specific T cell responses

Reliable immune monitoring will be essential to assess whether neoantigen-based immunotherapy achieves its expected immunologic effects and to expand the immunologically effective candidates to larger and suitable patient subsets. 514 The tumor-reactive T cell responses are crucial for anti-tumor efficacy of various therapies, including cancer vaccines, ACTs, bsAbs and ICBs. The quantity and quality of tumor-reactive T cells can be measured and tracked in order to anticipate how well cancer immunotherapy will work. 378 , 515 , 516 A multiparameter phenotypic and functional readout of T cell reactivity will be likely necessary in the absence of extensive evaluation using several types of T cells and APCs. 380 Numerous effective markers, including CD39, PD-1, TIM-3, OX40, 4-1BB, IFNγ, and TNFα, can be used to determine the proportion of neoantigen-reactive T cells in infusion products and their capacity to recognize autologous tumors. CCR5 and CXCL13 can also be used as T cell-intrinsic indicators for CPI sensitivity. In addition, the neoepitope-specific CD8 + T cells in the blood can be used to identify the ongoing anti-tumor immune response at the tumor location. Blood neoepitope-specific T cells analyzed on a single-cell level revealed substantial clonal T-cell expansions with different effector transcription patterns, which are also present in the corresponding malignancies, indicating the recognition and destruction of tumor cells. 401 It should be noted that the level of circulating tumor DNA (ctDNA), a proxy for tumor burden, can be utilized to dynamically detect the mutations that generate neoantigens. The ctDNA sequencing-base method can also monitor the neoantigen evolution during ICB treatment, thereby guiding personalized immunotherapy. 517

The function and specificity of T cells can also be understood through examination of cellular states, which can also serve as a predictor for how well neoantigen-directed ACT would work. A reservoir of stem-like neoantigen-reactive TILs, such as CD8 + cells expressing activation or exhaustion markers (PD-1, TIM-3, and LAG-3), expand prolifically and supply differentiated subsets promotes T cell persistence and long-term tumor control, thereby strengthening the anti-tumor response. 518 , 519 With high-throughput transcriptomic and TCR sequencing, neoantigen-specific TCR clonotypes dysfunctional characteristics were utilized to detect anti-tumor TCRs with minimal TIL material. Signatures of neoantigen-specific TCR clonotypes characterize the landscape of TILs across tumors, allowing TCR prediction based solely on TIL transcriptomic states for neoantigen-based cancer immunotherapy. 520 These signatures may provide a degree of clonality predicting clinical response to various immunotherapies by identifying anti-cancer TCRs in the blood and, more crucially, in the tumor without the need for functional screening of putative neoantigens. 378 , 516

Conclusion and perspective

In summary, neoantigens play a pivotal role in cancer immunotherapies, including cancer vaccines, ACTs, antibody-based therapies, and ICBs. This review summarizes the compelling evidence indicating the therapeutic strategies by targeting these cancer-specific neoantigens without normal tissue destruction and provides a strong rationale that supports the relevance of neoantigens in clinically successful immunotherapies. Numerous initiatives are being made to develop personalized or off-the-shelf anti-cancer medicines based on neoantigens. Nevertheless, experimental and theoretical improvements are required to address the time and economic issues for advanced personalized neoantigen-based immunotherapies, including efficient recruitment of patients, optimizing sequencing technology and neoantigen prediction algorithms, and taking off-the-shelf therapies against public neoantigens. 15 , 245 , 302 , 309

Effective patient recruitment is essential for neoantigen-based immunotherapy. First, early resection may improve clinical outcomes by giving clinicians more time to carefully design, produce and test neoantigen-based therapeutic medicines. Second, early resection makes it easier to select patients who might be eligible for the off-the-shelf therapies, including vaccines, TCRs or TCRm antibodies that target well-characterized cancer driver mutations. Third, given cancer treatment, including chemotherapy, radiation therapy and ICBs will stimulate T cells to become excessively differentiated, isolating autologous T cells or TILs as soon as a patient is diagnosed with cancer may enable the collection of the highest-quality and least differentiated T cells from patients. 409 In addition, early patient enrolment in neoantigen-based therapies enable effective infusion of superior neoantigen-based cellular products and ameliorate the severity of co-morbidities caused by advanced metastatic cancer clones.

The precise identification of immunogenic neoantigens and their cognate TCRs is the most crucial and rate-limiting step in the creation of personalized cancer immunotherapies. 101 Immunogenic neoantigens can be identified by both immunogenomic approach that builds virtual peptidomes using in silico algorithm based on NGS data and immunopeptidomic approach that uses MS to analyze the MHC-loaded peptides. The genomic and transcriptomic sequencing data have also been integrated with MS profiling of HLA-associated peptidomes to improve the sensitivity and specificity of neoantigens identification. Neoantigen-based therapies could, however, be quite affordable due to the less expensive high-throughput sequencing and the application of powerful deep learning algorithms. 245 , 521 A comprehensive and efficient one-stop computational workflow or a categorized benchmark of the available in silico neoantigen detection approaches is still necessary for clinical application. 522 The efficient one-stop computational methods might also enable determining the potential of neoantigens as biomarkers for survival prognosis or ICB response prediction using huge data cohorts. Most crucially, the accuracy of these epitope prediction pipelines should also be confirmed by thorough immunomonitoring in early clinical investigations to improve the development of neoantigen-based cancer therapies. In addition to these in silico approaches that predict the immunogenic neoantigens and cognate TCRs based on high-throughput sequencing data, several T cell antigen discovery strategies have recently been developed to unbiased map the immunogenic neoantigens. A variety of pMHCs libraries, including yeast display library, SABRs, BATTLES, have been established, which allow for the flexible and scalable screening of antigenic epitopes. By relying on the physiological activity of T cell killing rather than evaluating TCR-pMHC binding affinity, T-Scan enables the interrogation of a significantly larger antigen space independent of predictive algorithms. Thus, the simplicity and scalability of T cell ligand discovery techniques will be useful for studying the immunogenicity of candidate neoantigens and aiding the development of novel neoantigen-based immunotherapies.

Off-the-shelf precision immunotherapies against public neoantigens is another possible strategy to overcome the time and financial issues in individualized treatment based on personalized neoantigens. 300 An excellent public neoantigen shared across patients would be produced by a peptide having a hotspot mutation in a driver gene or a TSG that is presented by a relatively common HLA allele. Public neoantigens are more possibility of being clonally conserved across metastases and systematically reappearing among patients. 523 Numerous general therapeutic techniques are readily available to target public neoantigens, including vaccines, bsAbs, adoptive transfer of CTLs and TCR-T cells. 300 , 524 A library of TCRs that specifically target shared neoantigens in an HLA-specific manner has subsequently been developed for patients with advanced cancers. 446 , 525 , 526 , 527 , 528 , 529 , 530 As more public neoantigens and corresponding TCRs are discovered, more patients with frequent genetic alterations that drive cancers will benefit from the public neoantigen-reactive TCR library. In addition, the widespread use of cancer genome sequencing and neoantigen prediction pipelines will facilitate matching patients with therapies that target the public neoantigens of their tumors. Therefore, this off-the-shelf strategy based on public neoantigens is anticipated to shorten the time needed for neoantigen identification and extensive T cell cultures, increasing the application of neoantigen-based therapies in a significant portion of patients.

In addition to the neoantigens generated by spontaneous mutations during carcinogenesis, certain covalent molecules can be employed to induce the production of tumor-specific public neoantigens by modifying hotspot residues in highly recurrent somatic mutations. Covalent KRAS-G12C inhibitors, like ARS1620, irreversibly modify the mutant cysteine. The haptenated peptides that carry a covalently attached small molecule can be presented by MHC-I on the cell surface. The haptenated peptide:MHC complexes can serve as tumor-specific neoantigens, triggering a cytotoxic T cell response. 191 , 192 Based on this principle, the mutant tumor suppressor proteins can be targetable by a new class of molecules, which induce neoantigen generation and trigger a specific immune response by covalently modifying the hotspot residues like TP53Y220C and TP53R273C. Therefore, the range of tumor-specific neoantigens suitable for therapeutic targeting can be significantly expanded by haptens that specifically modify a mutant oncoprotein rather than functioning as pharmacologic inhibitors.

The neoantigens provide powerful targets for cancer vaccines, which can not only precisely eliminate residual tumor lesions, but also effectively target distant metastatic cells due to their systemic characteristics. Personalized neoantigen vaccines are produced in accordance with the individual tumor conditions in the following steps: collection of tumor tissues and normal samples, sequencing, and analysis of unique mutations, prediction, and validation of immunogenic neoantigens as well as design and production of vaccines. A variety of platforms, including peptides, nucleic acids, and DCs, can be used to develop vaccines based on predicted personalized or matched public neoantigens. Peptide, RNA and DNA-based neoantigen vaccines are high feasibility, generally safety and economical manufacture. However, the majority of patients have not been reliably induced by peptide-based neoantigen vaccinations to elicit substantial neoantigen-specific CD8 + T cell responses. The recent success of the COVID-19 mRNA vaccine has accelerated the development of mRNA-based vaccination for cancers. All the active components of tumor vaccines, such as neoantigens, formulations and delivery systems, have undergone ongoing improvement. Synthetic self-amplifying mRNAs (samRNAs), which contain replicase genes that encode RNA-dependent RNA polymerase (RdRp), are gaining interest due to their higher and longer-lasting expression of antigens compared to conventional mRNA. 531 The in vivo expression of vaccine neoantigens can also be enhanced by natural carriers of genetic instructions, including adenoviruses (Ads), retroviruses and adeno‐associated viruses (AAV). 532 In addition, various nanoparticle formations, such as lipid nanoparticles, exosomes, virus-like particles, caged protein nanoparticles, bacterial membrane materials-based nanocarrier, high density lipoprotein-mimicking nanodiscs, polyplexes and polymeric nanoparticles, are being developed to enhance the capacity of transport and tissue penetration, boosting the immunogenicity of personalized vaccinations. 533 , 534 , 535 , 536 , 537 , 538 , 539 Compared to the viral vectors, the nanoparticles can also efficiently co-delivery vaccines and immune adjuvants to lymphoid organs, strengthening the neoantigen presentation.

Ex vivo loading of blood-isolated monocytes or hematopoietic progenitor cells with tumor neoantigens effectively improves the anti-cancer effects of neoantigen-based vaccines. Autologous DCs can be loaded the neoantigens in the forms of peptides, RNA and DNA. Compared to the time-and cost-intensive sequencing and computational analysis of patient-specific neoantigens, using autologous WTLs is a more convenient and economical method to induce neoantigen-specific immune responses. Whole tumor cells have both MANA and non-mutated TAAs, which might overcome the potential immune escape and resistance. 367 , 368 , 369 However, the higher abundance of nonimmunogenic self-antigens might limit the capacity of neoantigens to elicit immune responses. Various immunosuppressive factors are also present in WTLs, which inhibit DC maturation and T cell activation. 30 , 540 To overcome these issues, extracellular vesicles (EVs) produced by tumor cells have recently been shown to be a vaccination platform that supports DC maturation and neoantigen presentation. Tumor cell-derived EVs can deliver tumor antigen repertoires into DCs and facilitate neoantigen cross-presentation. EVs also have high immunostimulatory factors that trigger DCs to release innate immune signals. 541 , 542 , 543 In addition to tumor cell-derived EVs, DC-derived EVs can also serve as a neoantigen-presenting unit to immune cells. 544 , 545 Though modulating the tumor immune microenvironment and systemic immune responses, EV-based vaccination can turn a ‘cold’ tumor into a ‘hot’ one. Therefore, EVs may provide an option format for neoantigen-based cancer vaccines, which may potentially be given orally.

Although neoantigen-based immunotherapies have shown promising outcomes in earlier preclinical and clinical investigations, significant advancements are still required, especially for patients with epithelial malignancies. Cancer cells have evolved inherent defenses to evade immune recognition at every stage of the cancer-immunity cycle. 7 , 461 Given a complicated mechanisms of immune escape in cancers, combination therapies that simultaneously target different stages of the cancer-immunity cycle may be more effective (Fig. 6 ). The neoantigen generation and release will be boosted by the cell death following chemotherapy, radiation, targeted therapy, photodynamic therapy and oncolytic viral therapies, which further enhance the anti-tumor immunity cycle. Neoantigen presentation can be facilitated by the administration of IFN-α, GM-CSF, anti-CD40, TLR agonist and STING agonists. 546 , 547 , 548 , 549 , 550 , 551 , 552 , 553 To promote the infiltration of immune cells into tumors, the TME modification method and intratumor cytokines can be used. 554 , 555 The ICBs and IDO inhibitor will also alter the immunosuppressive TME to enhance neoantigen-based immunotherapy. 556 , 557 Nano- and EV-based drug delivery systems have been recently employed as an integrated platform for simultaneous administration of numerous drugs or therapeutic medications that work in concert to activate different stages of cancer-immunity cycle, reverse the immunosuppression and create an immunosupportive TME. 558 , 559 , 560 These combining strategies using therapeutic agents with different mechanisms of action induce a robust effective, long-lasting and tumor-specific immune response in cancer patients.

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Acknowledgements

This work was supported by grants from the National Key R&D Program of China (2020YFA0509400), Guangdong Basic and Applied Basic Research Foundation (2019B030302012), the National Natural Science Foundation of China (81821002, 82130082, 81790251, 81972665, 82173003, 82102738 and 82103168), 1·3·5 project for disciplines of excellence, West China Hospital, Sichuan University (ZYGD22007 and ZYJC21004), and the Science and Technology Foundation of Shenzhen (JCYJ20200109113810154). BioRender was used to create the figures.

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These authors contributed equally: Na Xie, Guobo Shen.

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State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, and West China School of Basic Medical Sciences and Forensic Medicine, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, 610041, China

Na Xie, Guobo Shen, Zhao Huang & Canhua Huang

Clinical Genetics Laboratory, Affiliated Hospital & Clinical Medical College of Chengdu University, Chengdu, 610081, China

Guangdong Provincial Key Laboratory of Regional Immunity and Diseases, Department of Pharmacology and International Cancer Center, Shenzhen University Health Science Center, Shenzhen, 518060, Guangdong, People’s Republic of China

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N.X. and G.S. wrote the manuscript and made the figures, W.G. and Z.H. organized the tables, C.H. and L.F. conceived, designed, and edited the manuscript. All authors have read and approved the article.

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Correspondence to Canhua Huang or Li Fu .

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The authors declare that they have no potential conflicts of interest. C.H. is the editorial board member of Signal Transduction and Targeted Therapy, but he has not been involved in the process of the manuscript handling.

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Xie, N., Shen, G., Gao, W. et al. Neoantigens: promising targets for cancer therapy. Sig Transduct Target Ther 8 , 9 (2023). https://doi.org/10.1038/s41392-022-01270-x

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Received : 12 October 2022

Revised : 14 November 2022

Accepted : 27 November 2022

Published : 06 January 2023

DOI : https://doi.org/10.1038/s41392-022-01270-x

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• The New York Times - FILM VIEW; Neo-Noir's a Fashion That Fits Only a Few
• Academia - 'British neo-noir'
• BFI - Where to begin with neo-noir

neo-noir , a genre of films that use the visual style and themes of classic film noir (French: “dark film”) but add a modern sensibility. They also usually contain more graphic depictions of violence and sexuality.

(Read Martin Scorsese’s Britannica essay on film preservation.)

Classic film noir thrived in the 1940s and ’50s. The genre was characterized by dark stylized cinematography and a pessimistic mood, perhaps reflecting the uncertainty of the postwar era. Plots typically featured troubled cynical characters involved in the underworld. The earliest neo-noirs came in the decades after, typically in the 1960s and ’70s. They often used groundbreaking narrative techniques to reinterpret conventional story lines. French New Wave director Jean-Luc Godard ’s Breathless (1960) and Alphaville (1965) used improvised scripts to tell the stories of a petty crook and a secret agent, respectively. British director John Boorman employed Godard’s techniques of jump cuts and nonlinear narratives for his nihilistic gangster drama Point Blank (1967).

Neo-noir filmmakers also took advantage of relaxed censorship, specifically the end (1968) of the Hays Office Code , a self-regulatory set of standards that had been self-imposed on film production since 1930. Under the code, profanity and depictions of illegal drug use , illicit sex, and many methods of crime were taboo. To depict such activities, directors relied on oblique references or manipulated imagery and symbolism, such as cutaways, shadows, or off-screen violence. Moreover, in films made under the code, the “bad guys” and immoral behaviour were usually punished by the film’s end. After the Hays Code was supplanted by the Motion Pictures Association of America ratings system, movies reveal changing sensibilities. In both Billy Wilder ’s Double Indemnity (1944) and Lawrence Kasdan’s Body Heat (1981), a woman lures a man into killing her husband to collect on the insurance. The earlier film’s depiction of their affair, however, is markedly chaste compared to the later film. Likewise, whereas Double Indemnity ’s femme fatale “gets her due” in the end, Body Heat ’s comes away triumphant.

The neo-noir that emerged in the 1970s also placed greater emphasis on the hero’s, or antihero’s , moral ambiguity and distrust of authority, reflecting the pessimism of the post- Vietnam War and Watergate era. In Roman Polanski ’s Chinatown (1974), the private eye protagonist comes up against a bureaucratic conspiracy and is ultimately unable to solve the crime. Vigilante justice became another theme of neo-noir, such as in Clint Eastwood ’s popular Dirty Harry franchise (1971–88), about a ruthless police inspector, and Martin Scorsese ’s Taxi Driver (1976), about a disturbed, insomniac Vietnam veteran who patrols the crime-infested streets of New York City at night.

By the 1990s subverting the tropes of classic noir became a signature of neo-noir. Quentin Tarantino ’s highly stylized neo-noir films, beginning with Reservoir Dogs in 1992, are characterized by dark humour, plot twists, pop culture references, and ridiculous levels of graphic violence. The Coen brothers also subverted classic noir themes and styles. Fargo (1996) features a wintery small-town Minnesota setting and an unflappable, cheerful, pregnant cop as its protagonist. The Big Lebowski (1998) offers an absurdist take on classic noir plots, with a middle-aged slacker and his bowling buddies attempting to solve a rather obvious kidnapping. In the acclaimed but bleak thriller No Country for Old Men (2007), which tracks the wake of destruction left by a sociopathic killer, the Coen brothers offered no clear-cut heroes and villains or neat endings.

Some filmmakers further stretched the definition of film noir to include elements from other genres . Ridley Scott ’s futuristic Blade Runner (1982), regarded as one of the greatest science-fiction movies, has classic hard-boiled film noir conventions at its core. Other filmmakers expanded the film noir canon by including perspectives and social issues that were overlooked in earlier films. Black directors in the 1990s expanded the predominantly white genre by introducing issues of racism, including Carl Franklin in One False Move (1992), about three fugitives on the run from Los Angeles to small-town Arkansas. In Franklin’s stylish murder mystery Devil in a Blue Dress (1995), the protagonist is a Black private eye (played by Denzel Washington ) investigating the disappearance of a femme fatale character ( Jennifer Beals), who may be passing as white in 1940s Los Angeles. Meanwhile, in Bound (1996), Lana and Lilly Wachowski offer a woman-centred noir thriller with a queer romantic triangle, while The Matrix (1999) launched a series of spectacular science-fiction films that contain not only elements from film noir but also an allegorical transgender narrative.