Simplified Screening Approach Identifies Mutated Proteins Expressed in Patient Tumors

Expressed genes from a patient’s tumor can be sequenced to identify candidate mutant T cell epitopes. Relevant epitopes that may bind to the MHC molecules of the patient could be predicted using peptide prediction algorithms. If peptides derived from mutant proteins are able to form new MHC-restricted target structures, the candidate peptides could be used in one of at least three ways. If the T cell populations generated are specific for the patient’s tumor, they could be expanded and adoptively transferre

Expressed genes from a patient’s tumor can be sequenced to identify candidate mutant T cell epitopes. Relevant epitopes that may bind to the MHC molecules of the patient could be predicted using peptide prediction algorithms. If peptides derived from mutant proteins are able to form new MHC-restricted target structures, the candidate peptides could be used in one of at least three ways. If the T cell populations generated are specific for the patient’s tumor, they could be expanded and adoptively transferred if they are of human origin. Alternatively, mouse T cells can be used to identify suitable T cell receptors (TCRs) for gene-engineering approaches.

Adoptive cell therapy using tumor-infiltrating lymphocytes (TILs) is a very effective treatment for patients with metastatic melanoma. In phase 2 clinical trials, up to 70 percent of patients with melanoma who received autologous TILs had considerable regressions of metastatic lesions. Recently, in another trial, 40 percent of patients treated had complete regressions of all measurable lesions lasting more than five years after treatment. Identifying antigens associated with TIL-mediated tumor regression has been a difficult task due to the diversity of these large lymphocyte populations and the complexity of current screening approaches.

Steven Rosenberg, M.D., Ph.D., in CCR’s Surgery Branch, along with Paul Robbins, Ph.D., a Staff Scientist in his lab, and their colleagues set out to investigate the possible correlation between the ability of TILs to be clinically effective and their ability to recognize potent antigens that likely include mutated gene products. They developed a new screening method that used whole-exome sequencing of tumor-cell DNA followed by the use of MHC binding algorithms to identify mutated antigens recognized by TILs.

The researchers focused on identifying T cell epitopes presented in the context of HLA-A class I gene products, which were previously found to be expressed at higher levels in melanomas than HLA-B and HLA-C products, in three individuals with metastatic melanoma who had regression of bulky metastatic lesions after adoptive transfer of autologous TILs.

The researchers began their studies by isolating DNA from cells generated from a metastatic lesion of an individual with melanoma and subjecting it to whole-exome sequencing. They synthesized select mutated candidate peptides identified from the melanoma cells that were predicted to bind with high affinity to HLA-A*0201 and evaluated them for their ability to sensitize a HLA-A*0201-positive cell line for recognition by autologous TILs. Their screening assay measured the release of IFN-γ from TILs. They found that several of the peptides in the predicted high-affinity HLA-A*0201 binders, stimulated release of amounts of IFN-γ from TILs that were comparable to those released after stimulation by autologous tumor cells.

The screening method was further evaluated by identifying mutated antigens recognized by TILs from two other individuals with melanoma that expressed different HLA-A gene products. The researchers then evaluated the expression of genes encoding mutated candidate epitopes by amplifying partial cDNA transcripts from tumor cell RNA. The amplified transcripts were also subjected to Sanger sequencing to validate the results from whole-exome sequencing. Their peptide prediction results combined with gene expression analysis demonstrated that TILs from two individuals with melanoma recognized mutated peptides with at least the second-highest predicted binding affinity for the corresponding HLA-A gene products among proteins expressed by autologous tumor cells. Taken together, these results indicate that TILs may generally recognize mutated epitopes that bind with relatively high affinities to MHC class I molecules.  

To evaluate the contribution of T cells recognizing mutated targets to in vitro and in vivo antitumor responses, enzyme-linked immunosorbent spot assays measuring IFN-γ were used on infused TILs and samples of peripheral blood mononuclear cells obtained before and after therapy. Overall, results indicated that therapeutic TILs contained dominant populations of T cells reactive with mutated epitopes that persisted in the peripheral blood for at least one month after adoptive transfer. This finding suggests that T cells present in these TILs that recognize mutated epitopes may have played a part in the tumor regressions observed in these patients.

Taken together, whole-exome sequencing of tumor cell DNA, followed by the use of MHC binding algorithms, has enabled the identification of eight mutated epitopes recognized by four of the five melanoma TILs evaluated by the research team. This simplified approach to identify mutated antigens recognized by T cells makes the arduous task of generating and screening cDNA libraries unnecessary. Immunogenic peptides identified using this approach could be used to isolate or generate tumor reactive T cells in vitro from patient peripheral blood or cultured tumors that are then administered to patients following further expansion. Whole-exome sequencing may also facilitate the identification of mutated antigens that are targetable by adoptive transfer in a variety of tumor types.

Summary Posted: 06/2013

Reference

Robbins PF, Lu YC, El-Gamil M, Li YF, Gross C, Gartner J, Lin JC, Teer JK, Cliften P, Tycksen E, Samuels Y, Rosenberg SA. Mining exomic sequencing data to identify mutated antigens recognized by adoptively transferred tumor-reactive T cells. Nat Med. 2013 May 5 PubMed Link