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Tumor antigenicity and cancer as non-self


Summary of key facts

  • Professor Paul Ehrlich is credited with introducing the concept of cancers as non-self and proposing the concept of tumor immune surveillance in 1909.

  • Evidence for tumor immunity was suggested by early animal tumor transplantation experiments carried out in nonhomogeneous mouse strains.

  • The development of congenic inbred murine strains was an essential contribution that enabled the study of human tumor immunogenicity.

  • TSTAs (tumor transplantation antigens) was the initial term to define tumor antigens. This term was later refined to be TSAs (tumor-specific antigens).

  • Similarly, tumor-associated transplantation antigens (TATAs) are now called TAAs (tumor-associated antigens).

  • The human immune system comprises B cells, T cells, and antigen-presenting cells. B cells are the cells responsible for interacting with T cells and antigen-presenting cells to produce highly specific antibodies against antigens.

  • T cells have subsets; for example, cytotoxic CD8 + T cells (CTLs) and CD4 + T cells.

  • In 1973, Steinman and Cohen identified a class of antigen-presenting cells termed dendritic cells that function to present antigen in association with MHC class II molecules to CTLs, other T cells, and B cells.

  • Tumor cells are genetically unstable and a source of altered genes and altered proteins that may become recognized by the host’s immune system as non-self TSAs.

  • Though SNVs are most common variant occurring in tumor cells, they have not proved to be a very good source of neoantigens. Indels (insertions and deletions) are the second most common type of variant occurring in tumor cells.

  • Studies indicate that TSAs originating from genomic variants occurring in noncoding regions of the tumor genome (alternative reading frames, antisense coding segments, variants altering splicing in formation of mRNA, 5′ untranslated regions, and long noncoding RNAs) are proving to be a more important source of actionable TSAs.

  • TAAs actually represent a form of wt peptide but are found in much greater abundance (overexpression) as a result of epigenetic alterations in tumor cell DNA methylation and/or alterations of chromatin structure resulting in shifts in gene expression. TAAs also include cancer germline antigens.

  • The proteasome and its chamber located in the cytosol function as site of proteolysis to generate short antigenic peptides from the altered proteins suitable for restricted MHC class I presentation. The proteasome can ligate distal peptides together (transpeptidation).

  • Peptides are imported into endoplasmic reticulum where they are noncovalently bound by the peptide-loading complex (involving multiple proteins) to the MHC binding groove.

  • The MHC heterodimer is composed of genetically polymorphic α and β chains and the peptide grove of MHC class I is typically closed at both ends, thus accepting only short peptides.

  • Neoantigen peptide binding is controlled by a series of four to five pockets into which amino acid side chains of the peptide selectively dock.

  • Bioinformatics tools, increasing computational power, and a growing number of critical data sets supporting machine learning all promise a future enabling antigen identification; prediction for optimal antigen processing, selective MHC binding, successful TCR presentation, and adaptive immune response; and, most important, the suitability as a therapeutic target.

Introduction

One of the great experiences in my career was the time spent at the famous Karolinska Institute, Stockholm, Sweden. I arrived there in 1970 during a break in my surgery training and with very little experience as a student of immunology. One of the special benefits of joining the Möller laboratory on the Karolinska campus was the proximity to the laboratory of the famous immunologist and cancer researcher Professor George Klein, who headed the Karolinska Institute’s Department of Tumor Biology. My mentors, Erna and Göran Möller, had both trained under Professor Klein, so there were lots of opportunities for cross-lab interactions. This was the very early days of tumor immunology and just the beginning of the recognition that the immune system involved two major, functionally unique, immune cells, T cells and B cells. Of course, this was long before the identification of T-cell subtypes such as tumor-directed cytotoxic T lymphocytes (CD8 + CTLs) and the defining of the unique functions of these cells in the adaptive antitumor immune response. The exciting story of immuno-oncology today is very much the history of our evolving understanding of the intricacies of the human immune system and its response to foreign “antigens.”

The primacy concept underpinning the development of our newest weapon against cancer—immunotherapy—is the understanding that when normal cells undergo genetic transformation to the cancer phenotype and acquire their unique capacity for uncontrolled division, the invasion of normal tissues, and the spread to distant sites, they have also acquired non-self-antigens. Tumor cells are, by their very nature, quite genetically unstable and have been shown to express between 50 and 1000 missense mutations. Theoretically, this array of genetic alterations should result in numerous antigenic differences from self and be adequate to trigger a reaction within the host’s immune system to destroy these abnormal cancer cells in their earliest state of development. A process often referred to as immunosurveillance.

The initial introduction of the hypothesis of immune surveillance is most often credited to the German Professor Paul Ehrlich. As early as 1909, Professor Ehrlich reasoned that without the host’s immune system monitoring the development of abnormal cancerous cells, “so konnte man vermuten, dass das Karzinom in einer geradezu ungeheurlichen Frequenz aufteten wurde,” which translates as “one could therefore suspect that the carcinoma would occur with an almost unbelievable frequency.” , However, the state of our knowledge at that time regarding the intricacies of the immune system, as well as the limitations of available scientific tools, made it difficult to validate his hypothesis. Thus, over the years the concept of “host immune surveillance” remained controversial and in fact, does so even today. (See also Chapter 6 , Tumor Immune Surveillance.)

Early experiments involved the simple grafting of fresh pieces of animal tumor tissue into mice or rats in an effort to immunize the animal to a subsequent tumor challenge. Beginning in the early years of the 20th century, scientists repeatedly observed what appeared to be immunologic differences between tumor tissues and normal tissues. Much of this early work involved the use of experimental tumors occurring spontaneously as well as those induced by chemicals such as methylcholanthrene and tumors produced by DNA and RNA oncogenic viruses. Despite somewhat variable findings, there was general recognition that tumor antigens, to be defined as such, needed to be present only in the tumor cells and not present in normal cells. Further, immunity to this tumor antigen brings with it the ability to destroy the tumor cell.

Lumsden is most often given credit for being the first to use an early attempt at developing an inbred mouse strain in his experiments to study the immune response to tumor tissue. He used an “inbred” strain of mice obtained from Lashoploeb’s laboratory in Buffalo that were more than 32 generations. For his experiments, Lumsden used a mouse tumor that had developed spontaneously. Unfortunately, of 170 mice tested, only 3 proved resistant to the tumor.

In 1935, Besredka and Gross reported a series of mouse experiments in which a small amount of a tumor cell suspension was injected intradermally. This resulted in tumor growth which soon regressed and appeared to make the mice resistant to subsequent inoculation of the tumor. Gross later reported a similar set of experiments but using the inbred mouse strain C3H developed by over 20 years of brother–sister mating. Gross used a methylcholanthrene tumor that had been induced originally in C3H mice. In these experiments, the immunized group was resistant to tumor challenge but, interestingly, Gross noted that this resistance to tumor challenge could be overcome by using a larger challenge dose of tumor cells. ,

Sjögren is most often credited with proposing that the antigens unique to tumor cells be designated as tumor-specific transplantation antigens (TSTAs). , This was based on the observation that tumors in mice induced by a small DNA virus contained a common immune response-inducing antigen. TSTAs were capable of being identified on the surface of tumor cells by antibodies they induced in the host. Though they were capable of killing tumor cells in a complement-dependent fashion in vitro, it soon became apparent that such antibodies directed at the surface TSTAs of carcinomas and sarcomas did not protect the host animal from tumor challenge.

The majority of these reported efforts to demonstrate tumor immunogenicity or the existence of TSTAs, dating from the turn of the century and through the 1940s and early 1950s, were carried out in genetically nonhomogeneous mouse strains. Therefore in almost all instances they were most often the result of normal tissue transplantation antigens also present on tumor cells rather than any unique tumor antigen. These observations by the early “giants of immunology”—R. T. Prehn, G. Klein, H. O. Sjögren, K. E. Hellström, and others—demonstrated to varying degrees evidence that cancers possessed non-self unique antigens. Much of this early work to demonstrate TSTAs, however, was rewritten once immunologists, perhaps influenced by Drosophila geneticists of the time, recognized the necessity of developing truly inbred strains of mice to study mammalian genetics and immunology.

In the early 1970s after returning from the Karolinska Institute, I was also privileged to be mentored by Donald C. Shreffler, an immunogeneticist and professor in the Department of Human Genetics at the University of Michigan. Shreffler was a recognized pioneer in researching the genes, including their functional roles, that comprised the murine major histocompatibility and the human leukocyte antigen (HLA) system. Don and his lab were especially recognized for developing numerous, very precious inbred congenic and recombinant H2 complex murine strains as well as strain-specific antisera to the proteins of the H2 locus. It was during these years that the time invested in developing truly isogenic mouse strains began to bear fruit and define the critically dominant genes governing tissue transplantation termed the major histocompatibility complex (MHC) and the processes of antigen recognition. Understanding the role of MHC in antigen presentation was a crucial step in beginning to define and characterize true tumor-specific antigens (TSAs).

During this time, immunology evolved to the point at which it was evident that there existed two principal immunologically competent cell types, the antibody-producing B lymphocytes and the thymus-derived lymphocytes termed T cells. , As a result, experiments demonstrated that T cells could be sensitized to TSTA and alone, in either in vitro or in vivo experimental models, could be shown to destroy targeted tumor cells by a process known as cell-mediated killing . Thus, there was beginning to be ample evidence to support the presence of TSTAs in animal models, but it remained to demonstrate their presence in human tumors. Because tumor transplantation experiments were limited to animals, the analogous antigenic proteins in humans were more appropriately termed tumor-specific antigens . TSAs are defined as being specific to the tumor and, as a result, are not proteins or oligopeptides normally present in the nontumor tissues of the body.

TSAs can result from somatically mutated genes and structural genomic variants occurring during tumor evolution and, as such, produce structurally novel tumor proteins. They can also arise secondary to exposure of the cell’s DNA to viral genomes and to carcinogens. A second class of tumor antigens have been classified as being tumor-associated transplantation antigens (TATAs). TATAs, later termed simply tumor-associated antigens (TAAs), are normal cellular proteins that, when overexpressed by growing tumors, become antigenic in terms of immune recognition and no longer elicit complete immunologic tolerance in the patient. Several examples of the presence of human TAAs can be found in breast cancer, ovarian cancer, and prostate cancer, as well as proteins normally present in skin melanocytes at low levels. Melanoma has been the most intensely studied human tumor in terms of TSAs and TAAs, perhaps because it is an example of a human malignancy that can undergo spontaneous remission (see Tumor-associated antigens ).

Identification of human tumor antigens

I have often thought that it was the surgeons’ feverish efforts to successfully replace diseased human organs with transplanted heterologous tissues that, in many ways, drove a new generation of young scientists to enter the field of immunology. Renal transplantation presented the human model for developing the needed methods of immune suppression and thus the need to understand the mechanisms underlying the complex processes of tissue rejection. I still recall my excitement as a young surgeon seeing the newly transplanted kidney produce urine even before completing the implantation of the ureter into the bladder and, as a result, markedly changing the life of the recipient. Organ transplantation and the challenges of immunosuppression really opened up the field of immunology and attracted many young scientists to study immunology.

As a result, advances in the field of immunology, driven by organ transplantation, had a tremendous effect on the study of the immune system’s interaction with a progressing human cancer. Today, the central efforts in this new era of cancer immunotherapy are directed at generating an enhanced adaptive immune response, the end result of which is a focused destruction of the cancer cells by cytolytic T lymphocytes (CTLs), while avoiding significant unwanted immune-based toxicities. This adaptive response, whether CTL-based, antibody-directed immune checkpoint inhibition, vaccines, oncolytic viruses, or some combination of these, requires the recognition of unique TSAs/TAAs and a process for their presentation that stimulates an adaptive immune response against the cancer.

A brief reflection on important historic events regarding TSAs and TAAs

It is important perhaps to pause a moment and reflect on the tools of the field and our historical understanding of the immune response to a foreign antigen, whether of an infectious origin, an evolving tumor, or a transplanted organ. As noted earlier, the demonstration of the presence of the T lymphocyte as a critical cellular component of the immune system was a landmark step forward in the mid-1960s. I remember very clearly the ability to use “antitheta” antibodies to separate mouse T cells from B lymphocytes for use in our in vitro antigen presentation experiments, as well as the challenge purifying a rabbit anti-B cell antibody during my days in the Möller lab.

It soon became clear that the TSAs were recognized by the T cells often to be found in the tumor itself and in the tumor-draining lymph nodes. The recognition that it was T cells interacting with antigenic determinants on the tumor cells and the resultant tumor cell destruction marked the beginning of characterizing the subclasses of T lymphocytes, including CD8 + cytolytic T cells (CTLs) and eventually CD4 + T cells.

In 1973, Ralph Steinman and Zanvil Cohn reported on the identification of a novel “glass and plastic” adherent cell found in the skin and peripheral lymphoid tissues termed dendritic cells (DCs). These cells soon became recognized as a critical cellular component of the immune system response to foreign antigen. Today, a number of morphologically similar cells have been defined as DCs with a diversity of functions. The classic DC has the ability to be an immunostimulatory cell in response to specific stimuli and in this higher state of differentiation to express high levels of peptide-bound MHC class II molecules that can interact directly with T cells. DCs are capable of recognizing foreign antigen materials—bacteria, viruses, toxins, and TSAs (tumor-derived peptides and lipids)—and through their own proteosome converting them to small peptide fragments to be presented in association with MHC class II molecules for adaptive immune response presentation. The classic DC, we now know, is also capable of interacting with and transmitting information to B cells, as well as other immune system cells such as CD4 + T cells and natural killer (NK) cells (for an in-depth review, see Cabeza-Cabrerizo et al. ). Another important advance was the development by Kohler and Milstein in 1975 of the ability to generate immortalized B cells and to create hybridoma cell lines that would produce an abundance of specific monoclonal antibodies. These murine monoclonal antibodies were used early on to identify TSAs on mouse tumors and eventually on some human cancers. The evolution of this discovery has today become a central feature of immuno-oncology.

In 1989, Lurquin and colleagues reported a series of experiments demonstrating that a peptide derived from a normal self-protein that had become mutated in cancer cells could be recognized by CTLs. This observation has been expanded experimentally over the years to define the process of antigen presentation and T-cell receptor recognition. Recognition of the antigenic peptide begins in the cytosol of the tumor cell where genetically altered self-proteins occur as part of the tumor cell’s cancerous progression. These altered proteins are cleaved into small peptides that are presented at the surface of the tumor cell, complexed in the binding cleft of the MHC class I molecules where they can be presented and recognized by the T-cell receptors (TCRs).

These MHC class I–associated peptides, often referred to as MAPs, are key to the process of CD8 + T-cell maturation and their ability to functionally discriminate between normal cells (self) and those cells that are infected or cancerous (non-self). Thus, the vast majority of TSAs are products of normal cellular proteins that have been genetically altered during tumor initiation and progression and subsequently processed to be non-self-MAPs. This intracellular process of the presentation of MAPs on the cell surface can occur in virtually all cells of the body and is often referred to as the immunopeptidome processing mechanism. , It should be remembered that MHC class I alleles are highly polymorphic and, therefore, each allotype has a unique MAP peptide-binding motif. It is important to note that in contrast to MAPs, the process of antigen presentation by MHC class II determinants is the special activity of the classical antigen-presenting cells: the macrophages, dendritic cells, and B cells.

Two caveats appear worth noting. First, though much of the search for TSAs has naturally focused on the genetic alterations occurring in the exome of the gene, it is increasingly apparent that a much more common occurrence is MAPs derived from the unmutated peptides of cancer-specific epigenetic and splicing alterations. , The existence of MAPs derived from noncoding regions of the genome greatly increases the number of TSAs potentially recognizable by CD8 + T cells from roughly 2% to perhaps as great as 75%. Secondly, cancer cells have proven to be rather poor antigen-presenting cells and therefore the response to TSAs is significantly dependent on cross-presentation by DCs. If this DC presentation does not occur in sufficient robustness, then many potential TSAs are not immunologically active. ,

As indicated earlier in this chapter, antigenic neoepitopes as in TSAs unique to the tumor (or a class of tumors such as melanoma) must be recognized by T cells through the T-cell surface receptor (TCR). What makes this MHC class I peptide complex a valuable therapeutic tool in cancer immunotherapy is the tremendous antigenic epitope binding diversity of these T-cell receptors—a receptor-binding diversity similar to that of B cell–produced antibodies. The generation of the immunogenic neoepitope has its origin in the cytosol, where in the case of a cancer cell, altered self-proteins are cleaved by the proteasome and aminopeptidases into peptide fragments. The peptides are then translocated in the endoplasmic reticulum (ER) via a transporter associated with antigen processing and further resolved by enzymes (ER aminopeptidases) to reach a size of ∼8 to 10 amino acids. These neoepitope peptides are then loaded into the specific peptide cleft of an MHC class I molecule, and if the complex is stable, it is exported as a MAP complex to the tumor cell surface where it can be recognized by TCRs of the T cell. A more detailed description of the intracellular processes operative in creating the short neoantigen peptides for MHC class I binding is presented later in this chapter in the section Tumor antigens—Intracellular processing and MHC presentation .

The origins of targetable tumor-specific antigens—today’s science

The rapid evolution of affordable high-throughput sequencing—based assays, advances in mass spectroscopy, the National Cancer Institute’s Cancer Genome Atlas (TCGA) project, the accumulating number of critical databases, and advances in computational biology have resulted in exciting advances in the field of tumor biology. These tools of science are providing new insights into tumor-specific antigenicity and the potential for developing novel immunotherapies. This rapid progress since 2005 has led to some confusion in the field regarding the true definition of tumor antigens and their actual potential as useful targets. This section is designed to add some definition and clarity to our understanding of the various sources of tumor antigens arising in the genetically unstable cancer cell.

TSAs can be subdivided broadly into two groups: those derived from exon or nonexon mutated/altered DNA gene sequences (mTSAs) and those derived from aberrant expression of transcripts not expressed in nonmalignant tissues, including in medullary thymic epithelial cells, which provide a source of antigen tolerance. A second group of TSAs are derived from human endogenous retroelements (EREs) that comprise approximately 42% of our genome. Much of this part of our genomic DNA results from the integration of transposable elements into the genome millions of years ago (retroviruses) that, through genetic evolution, have lost their ability to be expressed. Aberrantly expressed EREs, however, can occur in the genomically unstable cancer cell via epigenetic dysregulation of the cancer genome such as H3K27me3 loss. You will see these latter TSAs frequently denoted as aeTSA. ,

Single nucleotide variant neoantigens

Tumor cells as altered self are by their very nature genetically unstable and undergo a series of genetic and epigenetic alterations during tumor growth and metastasis. Single nucleotide variants (SNVs) are the most common genetic alteration even in normal cells and, as such, have long been considered an excellent initiator of novel tumor TSAs. These neoantigens and their resultant MAPs have been considered a source of potential antitumor T-cell targets. Nonsynonymous SNVs are most commonly the result of simple DNA replication errors during abnormal tumor cell division and are sometimes positively selected. SNVs can also result from genetic alterations in the tumor cell that damage critical enzymes essential for faithful DNA damage repair. SNVs can, of course, result from ongoing exposure to external mutagens during cancer treatment.

As therapeutically promising as SNV-generated TSAs may appear, it has proved much more difficult to identify suitable targets for the development of novel immunotherapies. As pointed out by Minati and colleagues at the Université de Montreal, Canada, in their excellent review, except perhaps for highly mutated tumors such as melanoma and lung, “all studies based on whole-exome or ribonucleic acid sequencing (WES and RNAseq respectively) combined or not with mass spectrometry (MS) analysis, could only identify a very limited number of SNV-derived MAPs.” ,

It appears, therefore, that sources of neoantigens other than those originating from exome SNVs will be more critical to the design of future immunotherapies. In fact, in mass spectroscopy studies by Laumont and colleagues, some 90% of the identified TSAs in two mouse cancer cell lines and seven primary human tumors were unexpectedly found to be derived from noncoding regions of the genome. These potentially more important sources for actionable TSAs will be discussed in later sections of this chapter. ,

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