Peptide mimotopes alter T cell function in cancer and autoimmunity

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The relative risk of developing cancer increases with autoimmunity, and autoimmunity increases the risk of cancer, although both disease categories involve diverse cell types and mechanisms. One similarity in both conditions, however, is that T cells interact with self antigens.

Specifically, the antigen receptor on T cells (TCRs) interacts directly with peptides derived from self proteins presented in the groove of MHC molecules. One case study involving a melanoma patient suggests that T cells and antigens in both diseases could be the same. The patient’s melanoma was treated with expanded autologous tumor infiltrating lymphocytes (TILs) resulting in durable and complete remission of the cancer.

This patient also developed autoimmunity resembling Vogt-Koyanagi-Harada disease (VKH). VKH is an autoimmune disease targeting organs that contain melanocytes, such as eye, ear, skin, and meninges. Most T cells that effectively react to self antigens and attack self tissues, so called antitumor T cells in cancer or pathogenic T cells (Tpaths) in autoimmunity, are culled in the thymus during T cell development.

Regulatory T cells (Tregs) promote tolerance to self antigens and stop the function of the other T cells to avoid autoimmunity. Interventions to activate T cells against self/tumor antigens for immunotherapies of cancer or to impair effector T cells against self antigens in autoimmunity therapies require innovative methods.

To achieve such goals, here we discuss the use of mimotopes, mimics of epitopes, which substitute amino acids in the antigenic peptide with either natural or artificial amino acids. Mimotopes have also been tested in HIV and other infectious diseases, but are beyond our scope and have recently been reviewed.

For T cell immunity to be successful in modulating immune responses, TCRs must efficiently bind to more than one peptide-MHC (pMHC) molecule. Each αβ T cell expresses one TCR generated by somatic recombination of gene fragments and random addition/subtraction of nucleotides at the gene fragment junctions.

This process results in vast TCR diversity making it possible for TCRs to interact with pMHC, and contributes to antigen binding specificity of the T cell. However, there are not enough T cells in our bodies for one T cell clone to interact with one peptide [eg, assuming 20 amino acids and 10 amino acid long peptides, there are 2010 (∼1 × 1013) possible peptides, and there is the possibility of even more because peptides come in different lengths, may receive post-translational modifications, and there are multiple MHC-restricting molecules).

In addition, there is no mechanism for a single cognate antigen to find a single T cell clone during an infection. There are a number of hints that suggest TCRs interact with multiple peptide antigens. First, T cells are exposed to at least two pMHC molecules: one during thymic development and another as they function in the periphery.

Substitutions in the MHC anchor amino acids

MHC class I (MHCI) and MHC class II (MHCII) molecules bound to peptide are a classic example of how form reflects function. The peptide binding grooves of the MHC molecules face away from the antigen presenting cell, so that the peptide can interact with the TCR (reviewed in and by many others).

The groove is bordered by two alpha helices and the floor of the groove is made of a beta-pleated sheet. The peptide lies in the groove in an extended state allowing the amino acid side chains to point in optimal directions for interactions with TCR.

In MHCI molecules, depending on the allele and the polymorphisms, the floor of the groove has a series of 6 pockets, A through F, that interact with the peptide. The amino terminus of the peptide binds directly to the A pocket in a side-chain-independent manner. The residue of the last amino acid is buried in the F pocket locking the peptide into the groove, elucidating one position of the MHCI allele’s peptide binding motif.

Since the ends of the peptide are “attached” and the ends of the MHCI grooves are closed, the peptide can bulge in the middle allowing for conformational changes upon TCR binding. The size, shape, and electronic charge of the pockets determine which peptides interact with that MHC allele and with what binding affinity.

Many mimotopes for human antigens are designed and tested for presentation by the MHCI HLA-A*0201 allele (HLA-A2) because, relative to other HLA alleles, HLA-A2 is frequently identified in many human populations and there are many reagents (such as antibodies, antigen databases, atomic structures, and transgenic animals) that facilitate its study.

Peptide binding to MHCII molecules differ from MHCI in that the TCR binding surface is made of two proteins, alpha and beta, and the peptide extends beyond the open ends of the groove. Hydrogen bonds between the peptide backbone and the MHCII molecules are generally conserved and sequence-independent.

Substitutions in secondary MHC anchor amino acids

One strategy to improve binding of peptides to MHC molecules is to make substitutions in secondary anchor amino acids. These amino acids are unique from the dominant consensus anchor amino acids discussed above, but also point into the MHC groove and contribute to stabilizing the pMHC interaction.

One testable assumption made with these substitutions is that the interaction between the peptide and MHC molecule may change, but with the right substitution the surface that interacts with the TCR may not change and a similar repertoire of T cells may respond to the mimotope as the wild type peptide. The majority of studies of peptides with altered secondary anchor residues are in MHCI-restricted peptides, although some mimotope substitutions, synthesized or natural, in secondary anchor residues for MHCII-restricted antigens have been characterized.

Peptides that bind to MHCII are more heterogeneous in length and more degenerate in MHC binding specificity than those that bind to MHCI. In addition, substitutions in peptides that bind weakly to MHCII molecules, might change the register of the peptide in the MHCII molecule since the ends of the peptide binding groove are open.

Amino acid substitutions that improve T cell responses

Research using surface plasmon resonance to study binding affinity and kinetics of the monomeric TCR-pMHC interaction suggest that the physiologic affinity range is 100 to 1 micromolar (μM). However, a recent study by Zhang et al., which examined Hepatitis C-specific T cells using an in situ TCR affinity and sequence assay, found 1000-fold range in affinity in specific CD8+ T cells.

Since TCRs are the only antigen-specific molecules on the surface of T cells, a simple assumption is that the affinity of the TCR for pMHC correlates with the strength of the T cell response, although there are a number of noted exceptions. The monomeric affinity between TCR and pMHC molecule is weak relative to other receptor–ligand interactions and T cells have on average 105 TCR molecules on their surfaces.

Future mimotopes

The concept that adding antigen-specific T cell responses to immunotherapies has reemerged as patients are relapsing after checkpoint therapies for cancer and antigens are being discovered in autoimmunities. Methods to improve mimotopes that enhance binding of peptide to MHC or pMHC to T cell repertoire are evolving, and technologies that predict epitopes and mimotopes recognized by particular TCRs are being developed.

Chemically modified antigens or antigens with unnatural amino acids, such as d-amino acids, may help to implement subtle changes in antigens; antigen changes that improve binding but do not change the antigen surface. In the search for the perfect antitumor mimotope for the GP70 antigen expressed by the CT26 tumor, we identified many that were suboptimal and did not protect from tumor growth.

Most of these mimotopes raised T cell repertoires that were not cross-reactive with the wild type antigen. However, we determined that we could improve these suboptimal mimotopes by boosting with the wild type antigen. The first immunization with the mimotope would stimulate a small fraction of the cross-reactive T cells with higher affinity than the wild type antigen did, then the booster immunization with the wild type antigen did not have to be as strong to expand the T cells differentiating into cytotoxic T lymphocytes as these T cells have a much lower threshold for stimulation. For this reason, the order of the vaccination could not be reversed.

Author: Jill E Slanskya, Maki Nakayama