Unique Tumor-specific Antigens Research Articles

Fratricide-Resistant Anti-CD2 Chimeric Antigen Receptor T-Cells with Endogenous CD2 Knockout Are Highly Effective Against T-Cell Neoplasms

T cell neoplasms, such as peripheral T cell lymphomas (PTCL) and acute T cell lymphoblastic leukemia (T-ALL), have some of the worst prognoses of all hematological malignancies. Poor outcomes are driven by a limited number of effective treatments, particularly in relapsed and refractory settings. While chimeric antigen receptor T cell (CAR-T) immunotherapy has been successful in other hematological diseases, its clinical use against T cell neoplasms is limited due to a lack of unique tumor-specific antigens. Additionally, CAR specificity against pan-T cell antigens causes CAR-T fratricide and insufficient manufacturability for therapeutic dosing. Early studies of CAR-T against CD5- and CD7-expressing T cell neoplasms have shown responses, however, 1) most patients ultimately relapse, and 2) in some patients, antigen negative relapse has occurred. To circumvent these obstacles, we hypothesized that targeting CD2, a pan-T cell antigen, using anti-CD2 CAR-T cells that are engineered to lack CD2, would permit effective CAR-T manufacturing against T cell neoplasms. In a retrospective analysis of clinical flow cytometry diagnostic samples, we found CD2 expression in 25 of 33 (75.8%) and 37 of 51 (72.5%) pediatric PTCL and T-ALL cases, respectively. Similarly, we found 82 of 89 (92.1%) adult PTCL cases with CD2 immunohistochemical expression, which exceeded CD7 expression (38 of 90, 42.2%) that is currently being evaluated in early phase clinical trials. We optimized early CD2 knockout (KO) during CAR-T cell manufacturing from human donor T cells (KO efficiency: 81.2% ± 2.4, n=9 donors). We next engineered a best-in-class, 4-1BB-costimulated, second-generation CD2KO anti-CD2 CAR-T product (CART2) via lentiviral transduction ( PANEL A) using seven distinct single-chain variable fragments (transduction efficiency: 60.2% ± 5.5, n=7 donors) based on cellular expansion and stressed in vivo efficacy against CD2+ T-ALL cells (Jurkat). There were no significant differences in population doublings during CART2 expansion as compared to both CD2 wild-type (CD2 WT) untransduced T cells (Mock UTD) and CD2KO untransduced T cells (CD2KO UTD) (6.20 ± 0.42 vs. 6.12 ± 0.62 vs. 6.67 ± 0.68, respectively, Day 15, n=3 donors), while CD2 WT CART2 cells failed to expand due to fratricide. CART2 cells had no significant differences in T cell memory phenotypes or T cell exhaustion markers, including PD1 and LAG3 expression, at the end of the expansion. Expanded CART2 cells eradicated primary patient-derived CD2+ Sezary cells and CD2+ T-ALL leukemic blasts as measured by in vitro cytotoxicity assays. Cytotoxicity correlated with CD8+ CART2 cells expressing IL-2 (20.4% vs. 0.2% vs. 0.35%), TNFα (28.5% vs. 0.27% vs. 0.2%), and IFNγ (11.27% vs. 0.18% vs. 0.14%) relative to Mock UTD and CD2KO UTD controls stimulated with CD2+ Jurkat cells (n=2 donors). Importantly, we also found both CD2KO UTD and CART2 cells were capable of TNFα and IFNγ release upon interaction with cytomegalovirus and influenza peptides, suggesting that T cells with CD2 perturbations are still able to mediate effector T cell responses. We further demonstrated that CART2 cells were highly effective in controlling systemic tumor burden of T-ALL patient-derived xenograft mouse (PDX) models and significantly improved median overall survival as compared to Mock UTD and CD2KO UTD controls (138 days vs. 30 days vs. 30 days, p=0.0007, Mantel-Cox) ( PANEL B). Long-term tumor control was proportional to CART2 peripheral blood persistence at Day 28 (CD2KO UTD: 186.6 ± 83.7, n=5 vs. CART2: 936.3 ± 241.7, n=5) and Day 100 (CD2KO UTD: N/A vs. CART2: 514.0 ± 441, n=3). We also found that rescue infusion of CART2 could eliminate relapsed T-ALL in PDX mice previously in a remission after initial treatment with anti-CD5 CAR-T cells, with long-term persistence of CD2-negative T cells thereafter. In conclusion, our data demonstrate that CART2 are manufacturable and highly efficacious against T-cell neoplasms. To our knowledge, this is the first report of prolonged efficacy using a CD2-targeted cellular monotherapy in human leukemia preclinical models. This product could be delivered alone or in combination with additional CARs for dual targeting, such as anti-CD5 or anti-CD7 CARs, to reduce the potential of antigen-negative escape. Further research is ongoing to study the effect of CD2 deletion in anti-CD19 CAR T cells and the possible implication for clinical translation.

Eradication of Large Solid Tumors by Gene Therapy with a T-Cell Receptor Targeting a Single Cancer-Specific Point Mutation.

Cancers usually contain multiple unique tumor-specific antigens produced by single amino acid substitutions (AAS) and encoded by somatic nonsynonymous 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. 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. When the neoepitope was expressed at high levels and by all cancer cells, their direct recognition sufficed to destroy intratumor 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. 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. Clin Cancer Res; 22(11); 2734-43. ©2015 AACRSee related commentary by Liu, p. 2602.

Tumor antigen discovery through translation of the cancer genome

Cancer cells harbor unique mutations that theoretically create corresponding unique tumor-specific antigens. This class of mutated antigens represents an attractive target for cancer immunotherapy, but their identification has been cumbersome. By combining cancer genome sequencing with computational analysis of MHC binding, it is possible to predict and rank all of the possible mutated tumor antigens. This form of antigen screen is being combined with high throughput methods to measure the immune response to each candidate mutated antigen. Using these techniques, it is possible to systematically test each mutated tumor antigens for an associated immune response. Only a small fraction of the putative mutated antigens tested in this manner have been found to elicit an immune response, yet these responses appear to be both robust and durable. It is becoming increasingly clear that these mutated tumor antigens are an important target in the antitumor response. Studies incorporating this approach promise to improve our understanding of the inherent immunogenicity of individual cancers, potentially providing an explanation for the varying clinical responses to novel immunotherapeutic agents.

A point mutation in the NFYC gene generates an antigenic peptide recognized by autologous cytolytic T lymphocytes on a human squamous cell lung carcinoma

We have identified an antigen recognized by cytolytic T lymphocytes (CTL) on the autologous tumor cells of a nonsmall cell lung cancer patient. The antigenic peptide, presented by HLA-B*5201 molecules, was encoded by a mutated sequence in the gene coding for the C subunit of transcription factor NF-Y. The mutation was present in the tumor sample from which the cell line was derived, and appeared to be unique to the tumor of this patient. In a lymph node draining the tumor, precursors of CTL recognizing the autologous tumor cells were detected at a frequency of about 1/30,000 of the CD8 cells, and 85% of them recognized the mutated NF-YC peptide, suggesting that the patient mounted a T cell response against this antigen. These results strengthened the notion that unique tumor-specific antigens are highly represented not only in melanoma but also in other types of tumors, like nonsmall cell lung cancer.

Vaccination with syngeneic, lymphoma-derived immunoglobulin idiotype combined with granulocyte/macrophage colony-stimulating factor primes mice for a protective T-cell response.

The idiotype of the Ig expressed by a B-cell malignancy (Id) can serve as a unique tumor-specific antigen and as a model for cancer vaccine development. In murine models of Id vaccination, formulation of syngeneic Id with carrier proteins or adjuvants induces an anti-idiotypic antibody response. However, inducing a potent cell-mediated response to this weak antigen instead would be highly desirable. In the 38C13 lymphoma model, we observed that low doses of free granulocyte/macrophage colony-stimulating factor (GM-CSF) 10,000 units i.p. or locally s.c. daily for 4 days significantly enhanced protective antitumor immunity induced by s.c. Id-keyhole limpet hemocyanin (KLH) immunization. This effect was critically dependent upon effector CD4+ and CD8+ T cells and was not associated with any increased anti-idiotypic antibody production. Lymphocytes from spleens and draining lymph nodes of mice primed with Id-KLH plus GM-CSF, but not with Id-KLH alone, demonstrated significant proliferation to Id in vitro without any biased production of interferon gamma or interleukin 4 protein or mRNA. As a further demonstration of potency, 50% of mice immunized with Id-KLH plus GM-CSF on the same day as challenge with a large s.c. tumor inoculum remained tumor-free at day 80, compared with 17% for Id-KLH alone, when immunization was combined with cyclophosphamide. Taken together, these results demonstrate that GM-CSF can significantly enhance the immunogenicity of a defined self-antigen and that this effect is mediated exclusively by activating the T-cell arm of the immune response.

Identification of a gene encoding a tumor-specific antigen that causes tumor rejection.

Transplantation experiments have clearly demonstrated the existence of unique (individual) tumor-specific antigens on cancers induced by physical or chemical carcinogens. These antigens often induce a tumor-specific immune response upon immunization with a tumor which protects the host against a subsequent challenge with the same tumor, but not against a challenge with any other independently induced tumor [1]. Unique antigens were observed even when the tumors were induced with the same carcinogen in the same organ system in the same strain of mice [2]. This finding of unique tumor specificity raises questions about the mechanism by which these tumorspecific antigens are generated. The critical questions regarding such unique tumor-specific antigens are their composition, genetic origin, and possible role as target antigens for the immune system.KeywordsTumor RejectionCific AntigenAntigen Loss VariantAntibody CP28Autochthonous HostThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Identification of a unique tumor-specific antigen as a novel class I major histocompatibility molecule.

Cancers induced by physical or chemical carcinogens express tumor-specific antigens that are uniquely specific for any given tumor; therefore, there is a seemingly endless variety of these unique antigens. We have studied a UV-induced fibrosarcoma, designated 1591, to elucidate the obscure molecular nature and genetic origins of unique tumor-specific antigens. A monoclonal antibody raised against syngeneic 1591 tumor cells has unique tumor specificity. This tumor-specific monoclonal antibody precipitated from the tumor a 45-kDa molecule associated with a 12-kDa molecule having the pI of beta2-microglobulin. This and other evidence indicated that the 1591 tumor expresses a novel class I molecule. A 1591 variant selected for the absence of binding to the monoclonal antibody lacked the novel class I MHC molecule as well as reactivity with cytotoxic T lymphocytes specific for the 1591 tumor. Furthermore, tumor cells bearing the antigen are rejected while variants that have lost the antigen grow progressively. Fourteen of 14 host-selected progressor tumor variants lost reactivity with the monoclonal antibody and provided further evidence that this novel class I molecule is a transplantation antigen on the parental 1591 tumor required for immune rejection. The identification of a unique tumor-specific antigen as a novel class I major histocompatibility complex gene product allows us to search for the possible genetic mechanisms involved and to explore further the role such molecules play in tumor immunity and malignancy.

Specific sensitization of lymphocytes to tumor antigens by Co‐cultivation with peritoneal cells exposed to such antigens

Peritoneal cells from BALB/c mice were seeded into 50-mm plastic Petri dishes and exposed either to antigen extracts prepared from individual BALB/c sarcomas having unique tumor-specific antigens, to similar extracts prepared from 14- to 18-day-old mouse embryos, or to culture medium only. Lymphoid cells from BALB/c lymph nodes were than added to the peritoneal cells. Following 4-5 days of co-cultivation, the lymphoid cells were harvested and tested in a 30 h microcytotoxicity assay for reactivity against tumor cells carrying the respective sensitizing antigens as well as against control cells which consisted of either tumor cells lacking the sensitizing antigens or normal skin fibroblasts. By this approach sensitization to individually unique tumor antigens, as well as to antigens shared by mouse embryos and mouse sarcoma cells, was achieved.