Restricted Tumor Antigens Research Articles

Co-stimulatory signaling determines tumor antigen sensitivity and persistence of CAR T cells targeting PSCA+ metastatic prostate cancer

ABSTRACTAdvancing chimeric antigen receptor (CAR)-engineered adoptive T cells for the treatment of solid cancers is a major focus in the field of immunotherapy, given impressive recent clinical responses in hematological malignancies. Prostate cancer may be amenable to T cell-based immunotherapy since several tumor antigens, including prostate stem-cell antigen (PSCA), are widely over-expressed in metastatic disease. While antigen selectivity of CARs for solid cancers is crucial, it is problematic due to the absence of truly restricted tumor antigen expression and potential safety concerns with “on-target off-tumor” activity. Here, we show that the intracellular co-stimulatory signaling domain can determine a CAR's sensitivity for tumor antigen expression. A 4-1BB intracellular co-stimulatory signaling domain in PSCA-CARs confers improved selectivity for higher tumor antigen density, reduced T cell exhaustion phenotype, and equivalent tumor killing ability compared to PSCA-CARs containing the CD28 co-stimulatory signaling domain. PSCA-CARs exhibit robust in vivo anti-tumor activity in patient-derived bone-metastatic prostate cancer xenograft models, and 4-1BB-containing CARs show superior T cell persistence and control of disease compared with CD28-containing CARs. Our study demonstrates the importance of co-stimulation in defining an optimal CAR T cell, and also highlights the significance of clinically relevant models in developing solid cancer CAR T cell therapies.

Abstract 4981: Extracellular spacer and co-stimulatory domains define target sensitivity and persistence of CAR T cells for the treatment of PSCA+ bone metastatic prostate cancer

Abstract Advancing chimeric antigen receptor (CAR)-engineered adoptive T cells for the treatment of solid cancers is a major focus in the field of immunotherapy, given impressive recent clinical responses in hematological malignancies. Prostate cancer should be amenable to CAR-based immunotherapy given that several tumor antigens, including prostate stem-cell antigen (PSCA), are widely over-expressed in metastatic disease. The selectivity of CARs for solid cancers is crucial, however, due to the absence of truly restricted tumor antigen expression and potential safety concerns with “on-target off-tumor” activity. Here, we show that optimized co-stimulatory signaling and extracellular spacer domains are essential in defining a CAR’s selectivity towards tumor cells that over-express the target antigen. The 4-1BB co-stimulatory domain in PSCA-CARs confers enhanced tumor selectivity with dampened yet dose-responsive cytokine production, reduced T cell exhaustion phenotype, and equivalent tumor killing ability compared to PSCA-CARs containing the CD28 co-stimulatory domain. CARs containing a short extracellular spacer demonstrate the most selective killing of high PSCA-expressing tumors but are unable to produce the inflammatory cytokines important for a complete anti-tumor response. We show that longer extracellular spacers in PSCA-CARs are necessary for both tumor killing and cytokine production. PSCA-CARs exhibit robust in vivo anti-tumor activity in subcutaneous and orthotopic bone-metastatic patient-derived prostate cancer xenograft models, and 4-1BB-containing CARs show superior persistence in controlling metastatic disease compared with CD28-containing CARs. Our study demonstrates the critical impact of CAR components in defining an optimized tumor-selective CAR T cell, and also highlights the importance of clinically relevant animal models in developing effective solid cancer CAR T cell therapies. Citation Format: Kelly Kennewick, Dileshni Tilakawardane, Ethan Gerdts, John Murad, Anthony Park, Brook Jeang, Yukiko Yamaguchi, Stephen J. Forman, Saul Priceman. Extracellular spacer and co-stimulatory domains define target sensitivity and persistence of CAR T cells for the treatment of PSCA+ bone metastatic prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4981. doi:10.1158/1538-7445.AM2017-4981

T cell recognition of HLA‐A2 restricted tumor antigens is impaired by the oncogene HER2

The HER2 oncogene is frequently over-expressed in human cancers and a promising target for immune therapy. Previous studies have shown that over-expression of mouse or rat HER2 leads to markedly reduced levels of major histocompatibility complex (MHC) class I and molecules of the antigen processing and presentation machinery (APM), thus resulting in a phenotype promoting tumor escape from the immune system. Our study focuses on analyzing the effect of HER2 on MHC class I antigen presentation and sensitivity to tumor-antigen specific cytotoxic T lymphocytes (CTLs) in HLA-A2.1(+) melanoma cell lines. We demonstrate significant inverse correlations both between the expression of HER2 and total MHC class I surface expression as well as between HER2 and HLA-A2. A significant reduction of HLA-A2 levels was found when melanoma and carcinoma cell lines were transfected with a human HER2 gene. A signaling-competent HER2 molecule was crucial for the observed HLA-A2 down-regulation, as transfectants expressing high levels of HER2 mutated in the tyrosine signaling domain did not show altered HLA-A2 expression. Importantly, the human melanoma cell line EST049 demonstrated reduced HER2 and melanoma antigen-specific recognition by CTLs upon HER2 transfection. In addition, high expression of HER2 prevented both IFN-γ mediated HLA-A2 up-regulation and improved recognition by HLA-A2-restricted CTLs in treated cells. Moreover, key APM molecules were down-regulated by HER2. These findings implicate that HER2 over-expressing tumors may be more prone to escape from HLA-A2 restricted CTLs suggesting that immunotherapy approaches inducing an integrated humoral, cellular and innate immune response would be most effective.

T-Cell Receptor Gene–Modified T Cells with Shared Renal Cell Carcinoma Specificity for Adoptive T-Cell Therapy

Adoptive therapy with genetically engineered T cells carrying redirected antigen specificity is a new option for the treatment of cancer. This approach is not yet available for metastatic renal cell carcinoma (RCC), due to the scarcity of therapeutically useful reagents. We analyzed tumor-infiltrating lymphocytes (TIL) from RCC to identify T-cell specificities with shared tumor-specific recognition to develop T-cell receptor (TCR)-engineered T lymphocytes for adoptive therapy of RCC. We established a T-cell clone from TIL that recognized a human leukocyte antigen (HLA)-A2-restricted tumor antigen. The TCR alpha- and beta-chain genes were isolated, modified by codon optimization and murinization, and retrovirally transduced into peripheral blood lymphocytes (PBL). A TCR-expressing indicator line (B3Z-TCR53) was established to screen for antigen prevalence in RCC, other malignancies, and normal cell counterparts. TCR53-engineered PBL recapitulated the specificity of the TIL and showed tumor-specific HLA-A2-restricted effector activities (IFN-gamma, tumor necrosis factor-alpha, interleukin-2, macrophage inflammatory protein-1beta, cytotoxicity). PBL-TCR53 of healthy donors and RCC patients exhibited similar transduction efficiency, expansion, and polyfunctional profile. Using B3Z-TCR53 cells, 130 tumor and normal cells were screened and shared TCR53 peptide: MHC expression was found in >60% of RCC and 25% of tumor lines of other histology, whereas normal tissue cells were not recognized. To date, TCR53 is the only TCR with shared HLA-A2-restricted recognition of RCC. It fulfills the criteria for utilization in TCR gene therapy and advances T cell-based immunotherapy to patients with RCC and other malignancies expressing the TCR ligand.

Abstract B35: Dynamics of MHC class II tumor antigen presentation is critical for effector antitumor CD4+ T-cell generation and tumor rejection

Abstract Absence of CD4+ T cell help leads to defects in CD8+ T cell memory responses.Although this phenomenon is well characterized in infectious immunity, little is known about the generation of anti-tumor CD4+ T cell responses in vivo, or even how relevant is T cell help for tumor elimination. CD4+ T cells appear differ from CD8+ T cells in their requirements for differentiation, the dynamics of encounter with antigen being critical in this process. We analyzed the relevance of MHC class II antigen presentation for tumor rejection, developing a B16EaRFP line to study class II restricted tumor antigen presentation and the cognate CD4+ T cell help in vivo. We found that, in a tumor heavily infiltrated by dendritic cells (DC), a progressive decline in different cell subpopulations is observed in draining lymph nodes (DLNs), suggesting an interruption of local cell traffic. Artificial increase in precursor frequency of tumor specific CD8+ T cells by adoptive transfer of naïve OT-I cells in C57Bl/6 mice injected with B16OVA tumor was able to delay tumor growth, but was not sufficient to block it completely. While tumor antigen presentation in class II molecules was detected in the DLN, and could be increased by delivery of necrotic or apoptotic tumor cells, tumor specific CD4+ T cells showed proliferation arrest in the DLN. Tumor growth was blocked only when dendritic cells (DCs) were activated by intra-tumoral LPS injection, reestablishing cell traffic to the LN, resulting in expansion of INF-γ producing anti-tumor CD4+ T cells. Our results suggest that CD4+ T cells are a major target of tumor mediated antigen presentation interruption, and that CD4+ T cell help is necessary for rejection of an established tumor. These observations have major implications for the design of cancer immunotherapy strategies. Citation Information: Cancer Res 2009;69(23 Suppl):B35.

Molecular cloning and characterization of MHC class I- and II-restricted tumor antigens recognized by T cells.

T cells play a central role in cancer immunosurveillance, autoimmune, and infectious diseases. Identification of MHC class I- and II-restricted T cell peptides is a critical step for the development of effective vaccines against cancer and infectious diseases. This unit describes a cDNA expression system and a genetic targeting expression system for the cloning of genes encoding for MHC class I- and II-restricted antigens recognized by antigen-specific CD8(+) and CD4(+) T cells.

Oxidation of Ovarian Epithelial Cancer Cells by Hypochlorous Acid Enhances Immunogenicity and Stimulates T Cells that Recognize Autologous Primary Tumor

Hypochlorous acid, a product of neutrophil myeloperoxidase, is a powerful enhancer of antigen processing and presentation. In this study, we examine whether ovarian epithelial cells (SK-OV-3) exposed to hypochlorous acid can stimulate T cells from patients with ovarian epithelial cancer that recognize common tumor antigens as well as autologous tumor. T cells from human leukocyte antigen (HLA)-A2(+) and HLA-A2(-) patients or healthy controls were stimulated with autologous dendritic cells cocultured with the generic ovarian tumor line SK-OV-3, previously exposed to hypochlorous acid. Hypochlorous acid-treated SK-OV-3 cells drove expansion of CD8(+) T cells from HLA-A2(+) individuals, which recognized the HLA-A2-restricted tumor antigen epitopes of HER-2/neu (E75 and GP2) and MUC1 (M1.1 and M1.2). Up to 4.1% of the T cells were positive for the HER-2/neu KIFGSLAFL epitope using pentamer staining. Dendritic cells loaded with oxidized SK-OV-3 cells and further matured with CD40 agonistic antibody or monophosphoryl lipid A additionally induced CD4(+) class II-restricted responses. Critically, T cells stimulated with mature oxidized SK-OV-3 (but not a control oxidized melanoma cell line) directly recognized autologous tumor cells isolated from patient ascites. Immunization with mature dendritic cells loaded with a generic oxidized tumor cell line stimulates a polyclonal antitumor response that recognizes autologous tumor. These findings suggest a new immunotherapeutic strategy to extend remission in ovarian cancer.

Cd8+ T-Cells Specifically Cytotoxic to Renal Cell Carcinoma (RCC) Cells Can Be Isolated from Patients with Regressing Metastatic Kidney Cancer after Allogeneic Nonmyeloablative Hematopoietic Cell Transplantation (NMHCT).

Graft-vs-tumor (GVT) effects following NMHCT induce disease regression in a subset of patients (pts) with advanced metastatic kidney cancer. At present, little is known about the antigens serving as tumor targets in responding pts. In an effort to characterize GVT effectors and their tumor antigens, we generated RCC cell lines to use as targets in cytotoxicity assays in three pts (one non-responder and two responders) undergoing a cyclophosphamide/fludarabine-based NMHCT from HLA matched siblings. Peripheral blood lymphocytes (PBL) were collected from pts at multiple time-points after transplantation and were expanded in-vitro with either irradiated patient (pre-transplant) PBL/EBV-LCL or autologous tumor cells. RCC pt #1 developed grade II skin GVHD on day 22 but did not have an objective tumor response and died from progressive tumor on day 203. In a Cr51 release assay, minor histocompatibility antigen specific (mHa) T-cell lines generated using pre-transplant pt PBL/EBV-LCL stimulators lysed 98% of pt CD40-ligand stimulated B cells (CD40L-B) but did not kill pt RCC cells. RCC pt #2 developed grade II skin GVHD on Day 51 and was noted to have regression of lung metastasis on day 183. Using PBL obtained during tumor regression, mHa- reactive T-cell lines were generated (pre-transplant pt PBL/EBV-LCL used as stimulators) that lysed 76% and 12% of pt EBV-LCL and autologous RCC tumor cells at a 20:1 E:T ratio. Following limiting dilution cloning, 6 CD8+ T cell clones were expanded that killed pt EBV-LCL (but not donor) including one MHC class-1 restricted T cell clone that lysed both pt EBV-LCL and pt tumor cells. RCC pt #3 developed grade III skin GVHD on day 120, had regression of lung metastasis on day 160, and survives more than 4 years after transplantation. PBL collected from this pt 40 months after transplant contained CD8+ T-cell populations that secreted IFN-g (0.9% by intracellular cytokine staining) when co-cultured with pt RCC cells but not after co-culture with pt CD40L-B cells. CD3+/CD8+ CTL were expanded from these PBL using pt RCC cells as stimulators; in vitro, these CTL killed the pt's RCC cells but did not kill pt fibroblasts or pt EBV-LCL (Figure A). Following co-culture with tumor, intracellular IFN-g staining combined with TCR Vb antibodies revealed 3 tumor reactive CD8+ T-cell populations (TCR Vb7+, TCRVb5.1+, and TCRVb non-staining); 25.2% of these CTL were TCRVb7+. IFN-g secretion by TCR Vb7+ T cells was blocked when tumor cells were pre-incubated with mAbs to CD8, pan MHC class I and HLA-A11 (Figure B) consistent with recognition of an HLA-11 restricted tumor antigen. Following limiting dilution cloning, several CD8+ T-cell clones (both TCR Vb7+ and TCR Vb non-staining) with RCC-specific cytotoxicity were identified. We conclude that donor T-cells with both broad alloreactivity and tumor specificity play a role in mediating GVT effects in RCC pts having disease regression following NMHCT. [Display omitted]

Complementary role of CD4+ T cells and secondary lymphoid tissues for cross-presentation of tumor antigen to CD8+ T cells.

MHC class I–restricted tumor antigens can be presented to CD8+ T cells by two distinct pathways: via direct and indirect presentation. The relative contribution of these two pathways toward the initial activation of tumor antigen–specific CD8+ T cells and their subsequent tumor rejection is still vigorously debated. Using a tumor model able to dissect the relative contributions of direct and indirect presentation, we show unequivocally the inefficiency of direct presentation and the essential requirement of indirect presentation for the priming of naive tumor antigen–specific T cells leading to tumor rejection. Moreover, we characterize the essential environment under which indirect presentation occurs, and find efficient cross-priming of tumor-specific CD8+ T cells in the complete absence of secondary lymphoid tissues. The independence of this process from local lymph nodes is compromised, however, in the absence of CD4+ T cell help. Therefore, our paper demonstrates that effective immune protection against tumors requires the cross-priming of CD8+ T cells under conditions that require either CD4+ T cell help, or draining lymph nodes.

MHC Class II–Restricted Tumor Antigens Recognized by CD4+ T Cells: New Strategies for Cancer Vaccine Design

The adoptive transfer of tumor-infiltrating lymphocytes (TIL) can mediate tumor regression in patients with melanoma. This finding has led to the identification and characterization of tumor-associated antigens recognized by CD8+ TIL. Several clinical trials based on the genes recognized by these CD8+ T cells have been attempted, but with only limited success. Meanwhile, increasing evidence has demonstrated that CD4+ T cells play important roles in generating and maintaining antitumor immune responses in animal models. These data suggest that it may be necessary to engage both CD4+ and CD8+ T cells for more effective antitumor immunotherapy. In this report, we review emerging molecular approaches in cloning major histocompatibility complex (MHC) class II restricted tumor antigens recognized by CD4+ T cells as well as approaches to identify new MHC class II-restricted epitopes from known tumor antigens recognized by CD8+ cytotoxic T lymphocytes and/or antibodies. Progress made in this field has shed light on the roles of tumor antigen-specific CD4+ T cells in humans; it has also provided new insights into the understanding of tumor genesis and the interaction between tumor and the immune system. More importantly, the discovery of MHC class II-restricted tumor antigens has provided opportunities for developing a new generation of cancer vaccines aimed at eliciting both CD4+ and CD8+ T-cell responses against tumor.

Enhanced Antigen-Specific Antitumor Immunity with Altered Peptide Ligands that Stabilize the MHC-Peptide-TCR Complex

T cell responsiveness to an epitope is affected both by its affinity for the presenting MHC molecule and the affinity of the MHC-peptide complex for TCR. One limitation of cancer immunotherapy is that natural tumor antigens elicit relatively weak T cell responses, in part because high-affinity T cells are rendered tolerant to these antigens. We report here that amino acid substitutions in a natural MHC class I–restricted tumor antigen that increase the stability of the MHC-peptide-TCR complex are significantly more potent as tumor vaccines. The improved immunity results from enhanced in vivo expansion of T cells specific for the natural tumor epitope. These results indicate peptides that stabilize the MHC-peptide-TCR complex may provide superior antitumor immunity through enhanced stimulation of specific T cells.

Human tumor antigens: implications for cancer vaccine development.

The adoptive transfer of tumor-infiltrating lymphocytes along with interleukin 2 into autologous patients resulted in the objective regression of tumor in about 30% of patients with melanoma, indicating that these T cells play a role in tumor rejection. To understand the molecular basis of the T cell-cancer cell interaction we and others started to search for tumor antigens expressed on cancer cells recognized by T cells. This led to the identification of several major histocompatibility complex (MHC) class I restricted tumor antigens. These tumor antigens have been classified into several categories: tissue-specific differentiation antigens, tumor-specific shared antigens, and tumor-specific unique antigens. Because CD4+ T cells play a central role in orchestrating the host immune response against cancer, infectious diseases, and autoimmune diseases, a novel genetic approach has recently been developed to identify these MHC class II restricted tumor antigens. The identification of both MHC class I and II restricted tumor antigens provides new opportunities for the development of therapeutic strategies against cancer. This review summarizes the current status of tumor antigens and their potential applications to cancer treatment.

In situ analysis of tumor-specific CTL effector and memory responses elicited by tumor vaccination.

In this study tumor-specific CTL effector and memory responses were analyzed in normal mice, in tumor bearing mice and in animals treated by active specific immunotherapy (ASI) with an autologous virus modified tumor vaccine. In the well defined DBA/2 mouse lymphoma model ESb, the tumor specific CTL response requires interactions of four different cell types and recognition of MHC class I and class II restricted tumor antigens either on host antigen presenting cells (APCs) or on the tumor cells. The most effective in situ activation of syngeneic tumor specific CTL can be generated within no more than 9 days by primary immunization in the ear pinna and restimulation in the peritoneal cavity. Here we describe modulatory effects of either cytokines or of virus infection of tumor stimulator cells during restimulation in the peritoneal cavity on the CTL memory response. The in situ peritoneal effector cell (PEC) response was augmented when Newcastle Disease Virus (NDV) was used to infect tumor cells which expressed the correct tumor associated antigen of the memory response but not when a third party tumor cell was infected and admixed. A response could also be augmented by co-administration of interferons-alpha, -beta or IL-2 and by pre-treatment with low dose cyclophosphamide. Therapeutic vaccination effects could be achieved in mice inoculated s.c. with this aggressive and metastatic tumor variant if the vaccine was given very early, i.e. 1-2 days after priming and when the vaccine was applied post-operatively, i.e. in situations of low tumor burden. A long-term CTL memory response could be demonstrated even two months after post-operative active specific immunotherapy with ESb-NDV vaccine.

Identification of a novel major histocompatibility complex class II-restricted tumor antigen resulting from a chromosomal rearrangement recognized by CD4(+) T cells.

CD4(+) T cells play an important role in antitumor immune responses and autoimmune and infectious diseases. Although many major histocompatibility complex (MHC) class I-restricted tumor antigens have been identified in the last few years, little is known about MHC class II- restricted human tumor antigens recognized by CD4(+) T cells. Here, we describe the identification of a novel melanoma antigen recognized by an human histocompatibility leukocyte antigen (HLA)-DR1-restricted CD4(+) tumor-infiltrating lymphocyte (TIL)1363 using a genetic cloning approach. DNA sequencing analysis indicated that this was a fusion gene generated by a low density lipid receptor (LDLR) gene in the 5' end fused to a GDP-L-fucose:beta-D-galactoside 2-alpha-L-fucosyltransferase (FUT) in an antisense orientation in the 3' end. The fusion gene encoded the first five ligand binding repeats of LDLR in the NH2 terminus followed by a new polypeptide translated in frame with LDLR from the FUT gene in an antisense direction. Southern blot analysis showed that chromosomal DNA rearrangements occurred in the 1363mel cell line. Northern blot analysis detected two fusion RNA transcripts present only in the autologous 1363mel, but not in other cell lines or normal tissues tested. Two minimal peptides were identified from the COOH terminus of the fusion protein. This represents the first demonstration that a fusion protein resulting from a chromosomal rearrangement in tumor cells serves as an immune target recognized by CD4(+) T cells.

MHC class II restricted tumor antigens and the role of CD4 + T cells in cancer immunotherapy

Recent studies in human and murine systems suggest a central role for CD4 + T cells in initiating, effecting and maintaining anti-tumor immunity. Characterization of the tumor-associated antigens recognized by CD4 + T cells may prove critical to the success of cancer immunotherapy strategies.