No abstract

The transfer of T cell receptor (TCR) genes allows to endow T cells with a new antigen specificity. For clinical applications of TCR-redirected T cells, efficient functional expression of the transgenic TCR is a key prerequisite. Here, we compared the influence of the transgene cassette on the expression and function of the murine TCR P14 (recognizing a LCMV gp33 epitope) and the human TCR WT-1 (recognizing an epitope of the tumor-associated antigen WT-1). We constructed different vectors, in which TCRalpha- and beta-chain genes were either (a) linked by an internal ribosomal entry site (IRES), (b) combined by a 2A peptide, or (c) introduced into two individual retroviral constructs. While in a TCR-deficient T cell line TCR P14 was expressed equally well by all constructs, we found that IRES- but not 2A-employing TCR expression is hampered in a TCR-bearing cell line and in primary murine T cells where the transgenic TCR has to compete with endogenous TCR chains. Similarly, 2A-linked TCR WT-1 genes yielded highest expression and function as measured by tetramer binding and peptide-specific IFN-gamma secretion. Differences in expression were independent of copy number integration as shown by real-time PCR. Thus, linking TCRalpha- and beta-chain genes by a 2A peptide is superior to an IRES for TCR expression and T cell function.

T cell receptor (TCR) gene transfer is a convenient method to produce antigen-specific T cells for adoptive therapy. However, the expression of two TCR in T cells could impair their function or cause unwanted effects by mixed TCR heterodimers. With five different TCR and four different T cells, either mouse or human, we show that some TCR are strong -in terms of cell surface expression -and replace weak TCR on the cell surface, resulting in exchange of antigen specificity. Two strong TCR are co-expressed. A mouse TCR replaces human TCR on human T cells. Even though it is still poorly understood why some TCRa/b combinations are preferentially expressed on T cells, our data suggest that, in the future, designer T cells with exclusive tumor reactivity can be generated by T cell engineering.

The apoptosis inhibitor protein survivin is overexpressed in many tumors, making it a candidate target molecule for various forms of immunotherapy. To explore survivin as a target antigen for adoptive T cell therapy using lymphocytes expressing survivin-specific transgenic T cell receptors (Tg-TCRs), we isolated HLA-A2-allorestricted survivin-specific T cells with high functional avidity. Lymphocytes expressing Tg-TCRs were derived from these T cells and specifically recognized HLA-A2 + survivin + tumor cells. Surprisingly, HLA-A2 + but not HLA-A2 -lymphocytes expressing Tg-TCRs underwent extensive apoptosis over time. This demise was caused by HLA-A2-restricted fratricide that occurred due to survivin expression in lymphocytes, which created ligands for Tg-TCR recognition. Therefore, survivin-specific TCR gene therapy would be limited to application in HLA-A2-mismatched stem cell transplantation. We also noted that lymphocytes that expressed survivin-specific Tg-TCRs killed T cell clones of various specificities derived from HLA-A2 + but not HLA-A2 -donors. These results raise a general question regarding the development of cancer vaccines that target proteins that are also expressed in activated lymphocytes, since induction of high-avidity T cells that expand in lymph nodes following vaccination or later accumulate at tumor sites might limit themselves by self-MHC-restricted fratricide while at the same time inadvertently eliminating neighboring T cells of other specificities.

The ease of sequencing the cancer genome, identifying all somatic mutations and grafting mutation-specific T cell receptor (TCR) genes into T cells for adoptive transfer allow, for the first time, a truly tumor-specific and effective therapy. Mutation-specific TCR gene therapy might achieve optimal efficacy with least possible toxicity. Recent clinical data confirm the long-standing evidence from experimental cancer models that antigens encoded by the tumor-specific somatic mutations are potentially the best targets for adoptive T cell therapy. Open questions are, how many somatic mutations create suitable epitopes, whether only individual-specific or also recurrent somatic mutations qualify as suitable epitopes and how neoantigen-specific TCRs are most efficiently obtained. Tumor heterogeneity needs to be considered; therefore, it will be important to identify immunogenic driver mutations that occurred early, are essential for cancer cell survival and present in all cancer cells.

Identifying T-cell receptors (TCRs) that bind tumor-associated antigens (TAAs) with optimal affinity is a key bottleneck in the development of adoptive T-cell therapy of cancer. TAAs are unmutated self proteins, and T cells bearing high-affinity TCRs specific for such antigens are commonly deleted in the thymus. To identify optimal-affinity TCRs, we generated antigen-negative humanized mice with a diverse human TCR repertoire restricted to the human leukocyte antigen (HLA) A*02:01 (ref. 3). These mice were immunized with human TAAs, for which they are not tolerant, allowing induction of CD8⁺ T cells with optimal-affinity TCRs. We isolate TCRs specific for the cancer/testis (CT) antigen MAGE-A1 (ref. 4) and show that two of them have an anti-tumor effect in vivo. By comparison, human-derived TCRs have lower affinity and do not mediate substantial therapeutic effects. We also identify optimal-affinity TCRs specific for the CT antigen NY-ESO. Our humanized mouse model provides a useful tool for the generation of optimal-affinity TCRs for T-cell therapy.

To prepare Translational relevanceAdoptive T cell therapy with neoantigen-specific T cell receptor (TCR)-engineered T cells is considered as a promising novel immunotherapy strategy. It takes four steps to prepare; (1) prediction of neoantigen epitopes, (2) neoantigen peptides synthesis, (3) identification of neoantigen-specific TCR and (4) production of virus vector to express TCR. Among them, the most challenging part is identification of neoantigen-specific TCRs. Our protocol required only two weeks from stimulation of T cells with peptides to the identification of neoantigen-specific TCRs. We conducted a pilot study to validate our time-efficient protocol in solid cancers with relatively lower mutational loads such as ovarian cancer. We successfully induced neoantigen-specific T cells against three neoantigens and established corresponding TCR-engineered T cells. One case of neoantigen-specific TCR-engineered T cells showed cross-reactivity against the corresponding wild-type peptide. These results give an important insight into the clinical application of adoptive T cell therapy with neoantigen-specific TCR-engineered T cells.Research. Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on May 2, 2018; DOI: 10.1158/1078-0432.CCR-18-0142 4 ABSTRACTPurpose: Current evolution of cancer immunotherapies, such as immune checkpoint blockade, has implicated neoantigens as major targets of anti-cancer cytotoxic T cells. Adoptive T cell therapy with neoantigen-specific T cell receptor (TCR)-engineered T cells would be an attractive therapeutic option for advanced cancers where the host antitumor immune function is strongly inhibited. We previously developed a rapid and efficient pipeline for production of neoantigen-specific TCR-engineered T cells using peripheral blood from an HLA-matched healthy donor. Our protocol required only two weeks from stimulation of T cells with neoantigen-loaded dendritic cells to the identification of neoantigen-specific TCRs. We conducted the pilot study to validate our protocol. Experimental Design: We used tumors from 7 ovarian cancer patients to validate our protocol.Results: We chose 14 candidate neoantigens from 7 ovarian tumors (1-3 candidates for each patient), and then successfully induced 3 neoantigen-specific T cells from one healthy donor and identified their TCR sequences. Moreover, we validated functional activity of the three identified TCRs by generating TCR-engineered T cells which recognized the corresponding neoantigens and showed cytotoxic activity in an antigendose-dependent manner. However, one case of neoantigen-specific TCR-engineered T cells showed cross-reactivity against the corresponding wild-type peptide. Conclusion/discussions: This pilot study demonstrated the feasibility of our efficient process from identification of neoantigen to production of the neoantigen-targeting cytotoxic TCR-engineered T cells for ovarian cancer and revealed the importance of careful validati...

Purpose Cancers usually contain multiple unique tumor-specific antigens produced by single amino acid substitutions (AAS) and encoded by somatic non-synonymous single nucleotide substitutions. We determined whether adoptively transferred T cells can reject large, well-established solid tumors when engineered to express a single type of T cell receptor (TCR) that is specific for a single AAS. Experimental Design By exome and RNA sequencing of an UV-induced tumor, we identified an AAS in p68 (mp68), a co-activator of p53. This AAS seemed to be an ideal tumor-specific neoepitope because it is encoded by a trunk mutation in the primary autochthonous cancer and binds with highest affinity to the MHC. A high-avidity mp68-specific TCR was used to genetically engineer T cells as well as to generate TCR-transgenic mice for adoptive therapy. Results When the neoepitope was expressed at high levels and by all cancer cells, their direct recognition sufficed to destroy intra-tumor vessels and eradicate large, long-established solid tumors. When the neoepitope was targeted as autochthonous antigen, T cells caused cancer regression followed by escape of antigen-negative variants. Escape could be thwarted by expressing the antigen at increased levels in all cancer cells or by combining T cell therapy with local irradiation. Therapeutic efficacies of TCR-transduced and TCR-transgenic T cells were similar. Conclusions Gene therapy with a single TCR targeting a single AAS can eradicate large established cancer but a uniform expression and/or sufficient levels of the targeted neoepitope or additional therapy are required to overcome tumor escape.