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 thesame. 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.
Cross-reactivity of TCRs: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. Another observation that suggests significant polyspecificity of T cells was the identification of diverse peptide ligands using synthetic combinatorial peptide libraries. Using these libraries, it was estimated that between two thousand and two million stimulatory peptides contribute to each T cell activation.
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. 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.
In cancer:Early clinical trials, targeting antigens shared by melanoma and differentiating melanocytes, with substitutions in the MHC anchor residues, such as Melan-A/MART-126–35 A27L, showed little efficacy (WT EAAGIGILTV, A27L ELAGIGILTV, the substituted amino acid is bolded). Later trials were more promising. In a prime/boost study, melanoma patients were first vaccinated with a plasmid encoding Melan-A/ MART-126–35 A27L, followed by a boost with peptides.
Another trial with the MART-1 mimotope compared the responses to peptide encapsulated in noninfectious virus-like particles with and without LAG-3Ig designed to engage the LAG-3 checkpoint protein. One patient of 12 had a partial response in this trial. Examined differences between the Melan-A/MART-126–35 wild type and mimotope peptides. Using two different T cell clones, they showed that the peptides bind to the MHC and TCR molecules with different binding properties and stimulated different responses in vitro.
In autoimmunity:In autoimmunity, delivery of high affinity signals into T cells using mimotopes can improve therapeutic efficacy through a number of mechanisms. Induction of stronger antigen-specific responses by Tregs, modulation of functions and phenotypes of Tpaths, and deleting Tpaths all assist identifying and targeting self antigen-reactive T cells in an efficient manner.
An important aspect of the latter two strategies that aim to kill or modify response of Tpaths is that targeting T cells specific for a single epitope may not be sufficient to stop autoimmune responses; antigen spreading of therapy effects to non-mimotope-specific Tpaths may also be necessary to halt autoimmunity. Genetic risks of a number of autoimmune diseases are detected within the HLA loci. Specific HLA alleles and haplotypes provide susceptibility or protection from the disease, such as susceptibility with DQ2 and DQ8 in celiac disease, DR3-DQ2/DR4-DQ8 in type 1 diabetes, DQB1*0602 in narcolepsy, and DR15- DQ6 in multiple sclerosis.
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. In addition, many other receptor-ligand interactions take place after TCRpMHC binding resulting in a stronger avidity. Krogsgaard and colleagues showed the threshold affinity is 10 μM—stronger binding does not further increase the avidity of the interaction. The overall function of mimotopes in therapies of cancer and autoimmunity is to increase the biding of this interaction.
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. Unlike in tumor immunity, in autoimmunity some mimotopes have ultimately been found to bind with lower affinity than the wild type
Author: Jill E Slansky, Maki Nakayama