Cancer Germline Antigens Research Articles

Exploring TSGA10 Function: A Crosstalk or Controlling Mechanism in the Signaling Pathway of Carcinogenesis?

Cancer-specific antigens have been a significant area of focus in cancer treatment since their discovery in the mid-twentieth century. Cancer germline antigens are a class of antigens specifically overexpressed in germline tissues and cancer cells. Among these, TSGA10 (testis-specific gene antigen 10) is of great interest because of its crucial impact on cancer progression. Early studies explored TSGA10 expression in a variety of cancer types. More recent studies revealed that TSGA10 can suppress tumor progression by blocking cancer cell metabolism, angiogenesis, and metastasis. An open question regarding the TSGA10 is why cancer cells must express a protein that prevents their progression. To answer this question, we conducted a comprehensive review to engage the TSGA10 in the context of the current understanding of "malignant transformation". This review demonstrated that TSGA10 expression level in cancer cells depends on the cancer stage across malignant transformation. In addition, we evaluated how TSGA10 expression can prevent the "cancer hallmarks". Given this information, TSGA10 can be of great interest in developing effective targeted anti-cancer therapies.

Abstract PO4-06-08: ZP4 AS A NOVEL TARGET FOR CAR T CELL THERAPY IN TRIPLE NEGATIVE BREAST CANCER

Abstract Triple negative breast cancer (TNBC) has the worst prognosis of BC subtypes, largely due to lack of approved targeted therapies. Chimeric antigen receptor (CAR) T cells have demonstrated great success in certain hematological malignancies; however, in the treatment of solid cancers, CAR T cells are faced with a variety of obstacles that limit their efficacy, including on-target/off-tumor toxicity. To minimize these toxicities, the ideal CAR T cell target must be overexpressed by the tumor and absent from vital organs. Cancer/germline (CG) antigens satisfy this need, as their expression is restricted to male or female germ cells. Using a bioinformatic pipeline to identify novel CG antigens expressed in TNBC and other solid cancers, we have identified zona pellucida glycoprotein 4 (ZP4) as a candidate CG antigen target for CAR T cell therapy. This protein is a component of the human zona pellucida matrix, an extracellular structure that surrounds the oocyte and functions in folliculogenesis, species-specific fertilization, and early development. We have identified ZP4 expression in approximately 10% of TNBC cases from available RNAseq and proteomic databases and have confirmed ZP4 expression in several TNBC PDX models. The overall purpose of this study is to redirect T cells against ZP4 by engineering and validating a second generation ZP4-specific CAR. We have generated a library of monoclonal antibody (mAb) candidates against the extracellular portion of human ZP4. Antibody clones were screened, and their specificity validated via ELISA and flow cytometry, and the three strongest binders (z108, z128, and z164) were selected for further investigation. By IHC, we confirmed that the ZP4 mAb clones stained the area surrounding normal oocytes and did not show binding to any of the 23 normal tissues screened. The variable regions of the antibody heavy and light chains were sequenced using 5’RACE and used to assemble second generation CAR constructs composed of the variable heavy and light chain fragments separated by a linker, a CH2/CH3 hinge domain, a CD28 transmembrane domain, a CD28 co-stimulatory domain, and the CD3z signaling domain. Activated T cells transduced with retroviral supernatant express the ZP4 CAR constructs at high levels. While all ZP4 CAR T cells demonstrate antigen-specific cytotoxic killing of ZP4+ target tumor cells in short-term in vitro assays, z128 ZP4 CAR T cells demonstrate superior persistence and efficacy in long-term repeat tumor challenge in vitro assays and orthotopic in vivo mouse models. In summary, ZP4 is a promising target for TNBC and we have successfully engineered ZP4 specific CAR T cells. ZP4 CAR T cells effectively target and kill ZP4+ cells in in vitro cytotoxicity assays but perform differently in repeat tumor challenge assays and in vivo. Future studies will elucidate the precise differences between our three candidate CAR constructs and further test the effector function, trafficking, proliferation, and persistence of z128 ZP4 CAR T cells in vivo using ZP4+ PDX models. This work has exciting potential to bring a novel CAR T cell therapy to patients with TNBC, without concerns for on-target off-tumor toxicity. Citation Format: Lauren Somes, Jonathan Lei, Paul Shafer, Xinpei Yi, Diego Chamorro, Emily Madaras, Michael Lewis, Bing Zhang, Valentina Hoyos. ZP4 AS A NOVEL TARGET FOR CAR T CELL THERAPY IN TRIPLE NEGATIVE BREAST CANCER [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-06-08.

T cell receptor therapeutics: immunological targeting of the intracellular cancer proteome.

The T cell receptor (TCR) complex is a naturally occurring antigen sensor that detects, amplifies and coordinates cellular immune responses to epitopes derived from cell surface and intracellular proteins. Thus, TCRs enable the targeting of proteins selectively expressed by cancer cells, including neoantigens, cancer germline antigens and viral oncoproteins. As such, TCRs have provided the basis for an emerging class of oncology therapeutics. Herein, we review the current cancer treatment landscape using TCRs and TCR-like molecules. This includes adoptive cell transfer of T cells expressing endogenous or engineered TCRs, TCR bispecific engagers and antibodies specific for human leukocyte antigen (HLA)-bound peptides (TCR mimics). We discuss the unique complexities associated with the clinical development of these therapeutics, such as HLA restriction, TCR retrieval, potency assessment and the potential for cross-reactivity. In addition, we highlight emerging clinical data that establish the antitumour potential of TCR-based therapies, including tumour-infiltrating lymphocytes, for the treatment of diverse human malignancies. Finally, we explore the future of TCR therapeutics, including emerging genome editing methods to safely enhance potency and strategies to streamline patient identification.

Prognosis and Characterization of Microenvironment in Cervical Cancer Influenced by Fatty Acid Metabolism-Related Genes.

Increasing evidence suggests that diverse activation patterns of metabolic signalling pathways may lead to molecular diversity of cervical cancer (CC). But rare research focuses on the alternation of fatty acid metabolism (FAM) in CC. Therefore, we constructed and compared models based on the expression of FAM-related genes from the Cancer Genome Atlas by different machine learning algorithms. The most reliable model was built with 14 significant genes by LASSO-Cox regression, and the CC cohort was divided into low-/high-risk groups by the median of risk score. Then, a feasible nomogram was established and validated by C-index, calibration curve, net benefit, and decision curve analysis. Furthermore, the hub genes among differential expression genes were identified and the post-transcriptional and translational regulation networks were characterized. Moreover, the somatic mutation and copy number variation landscapes were depicted. Importantly, the specific mutation drivers and signatures of the FAM phenotypes were excavated. As a result, the high-risk samples were featured by activated de novo fatty acid synthesis, epithelial to mesenchymal transition, angiogenesis, and chronic inflammation response, which might be caused by mutations of oncogenic driver genes in RTK/RAS, PI3K, and NOTCH signalling pathways. Besides the hyperactivity of cytidine deaminase and deficiency of mismatch repair, the mutations of POLE might be partially responsible for the mutations in the high-risk group. Next, the antigenome including the neoantigen and cancer germline antigens was estimated. The decreasing expression of a series of cancer germline antigens was identified to be related to reduction of CD8 T cell infiltration in the high-risk group. Then, the comprehensive evaluation of connotations between the tumour microenvironment and FAM phenotypes demonstrated that the increasing risk score was related to the suppressive immune microenvironment. Finally, the prediction of therapy targets revealed that the patients with high risk might be sensitive to the RAF inhibitor AZ628. Our findings provide a novel insight for personalized treatment in CC.

Co-Delivery of the Human NY-ESO-1 Tumor-Associated Antigen and Alpha-GalactosylCeramide by Filamentous Bacteriophages Strongly Enhances the Expansion of Tumor-Specific CD8+ T Cells.

Tumor-associated antigens (TAAs) represent attractive targets in the development of anti-cancer vaccines. The filamentous bacteriophage is a safe and versatile delivery nanosystem, and recombinant bacteriophages expressing TAA-derived peptides at a high density on the viral coat proteins improve TAA immunogenicity, triggering effective in vivo anti-tumor responses. To enhance the efficacy of the bacteriophage as an anti-tumor vaccine, we designed and generated phage particles expressing a CD8+ peptide derived from the human cancer germline antigen NY-ESO-1 decorated with the immunologically active lipid alpha-GalactosylCeramide (α-GalCer), a potent activator of invariant natural killer T (iNKT) cells. The immune response to phage expressing the human TAA NY-ESO-1 and delivering α-GalCer, namely fdNY-ESO-1/α-GalCer, was analyzed either in vitro or in vivo, using an HLA-A2 transgenic mouse model (HHK). By using NY-ESO-1-specific TCR-engineered T cells and iNKT hybridoma cells, we observed the efficacy of the fdNY-ESO-1/α-GalCer co-delivery strategy at inducing activation of both the cell subsets. Moreover, in vivo administration of fdNY-ESO-1 decorated with α-GalCer lipid in the absence of adjuvants strongly enhances the expansion of NY-ESO-1-specific CD8+ T cells in HHK mice. In conclusion, the filamentous bacteriophage delivering TAA-derived peptides and the α-GalCer lipid may represent a novel and promising anti-tumor vaccination strategy.

186P Synergistic antitumor effect of gemcitabine combined with Nyeso1-vaccine treatment evaluated in mouse PDAC organoids

Pancreatic adenocarcinoma (PDAC) is projected to significantly increase and become the second cause of cancer-related deaths before 2030. Immunotherapy has revolutionized the treatment of cancer, however, it has failed to show benefits in PDAC treatment. Recent efforts to synergize chemotherapy and immunotherapy have shown promising results in clinical trials. With this rationale, we tested the anti-tumor effect of the combination of the chemotherapeutic agent gemcitabine with a chitosan-based nanoparticle vaccine that contains the immunogenic cancer germline antigen Ny-eso1.

IMA402, an Off-the-Shelf, Next-Generation TCR Bispecific (TCER®) for Efficiently Targeting an HLA-Presented Peptide from the Pan-Cancer Antigen PRAME

Preferentially expressed antigen in melanoma (PRAME) is an intracellular protein that has been shown to have attractive target attributes that can be leveraged by T cell receptor (TCR)-based immunotherapy. PRAME is cancer-specific, homogenously expressed and presented at high target density. In contrast to most other cancer germline antigens, PRAME is expressed across a multitude of solid tumors as well as hematological malignancies including acute myeloid leukemia and diffuse large B-cell lymphoma. We have developed IMA402, a bispecific T cell engaging receptor (TCER®) molecule targeting an HLA-A*02-presented peptide derived from PRAME. TCER® molecules are fusion proteins consisting of a high-affinity TCR for peptide-HLA targeting and a humanized T cell-recruiting antibody coupled to an effector function-silenced IgG1 Fc domain that aims to extend half-life together with providing antibody-like stability and manufacturability characteristics. For IMA402, a TCR recognizing the selected PRAME pHLA molecule with high avidity and specificity was affinity maturated via yeast surface display resulting in more than 1,000-fold increased binding affinity. During maturation we applied an off-target toxicity screening against normal tissue peptides selected from the our immunopeptidome database to retain specificity and deselect cross-reactive candidate TCRs. The affinity maturated TCR was engineered into the bispecific TCER® scaffold and produced in Chinese hamster ovary cells. In preclinical assays, IMA402 required only picomolar concentrations to induce lysis of various tumor cells with PRAME target peptide levels in the physiological range as found in patients. In different in vivo xenograft mouse models, IMA402 induced consistent tumor regression including complete remissions. In these models, pharmacokinetic profiling determined a terminal half-life of several days. In vivo analyses of various T cell recruiting domains with different affinities confirmed the superiority of the low affinity T cell recruiting domain used in IMA402 when compared to analogous TCER® molecules with higher-affinity T cell recruiter domains. For the safety assessment, we measured TCER® reactivity against more than 20 different human normal tissue cell types. We could demonstrate that IMA402 did not induce killing of normal tissue cells at concentrations of at least 1,000-fold higher than required for tumor cell lysis, suggesting a broad therapeutic window. In patients, the design of our next-generation TCER® molecule harbouring a high-affinity TCR, a low-affinity T cell recruiter and a Fc domain aims at optimizing biodistribution and activation of T cells at the tumor site instead of the periphery. This might reduce occurrence of immune-related toxicities, such as cytokine release syndrome and allow reaching relevant doses in the tumor tissue. Taken together, our preclinical studies demonstrate that our novel, next-generation bispecific T cell engager IMA402 could be a promising therapeutic option for PRAME-positive patients. The phase 1 clinical trial designed as basket trial will start recruitment in 2023.

A phase I trial of T-cell receptor gene therapy targeting KK-LC-1 for gastric, breast, cervical, lung and other KK-LC-1 positive epithelial cancers.

TPS2678 Background: T cell receptor (TCR)-T cell therapy is an emerging cancer treatment strategy. Thus far, demonstration of clinical activity has been limited to a subset of solid tumors including melanoma, synovial cell sarcoma and HPV-associated cancer. It is estimated that metastatic epithelial cancers are responsible for approximately 90% of cancer deaths in the United States. The estimated 600,000 cancer deaths each year is driven largely by lung adeno- and squamous cell carcinoma and invasive breast cancer, which account for approximately 30% of all cancer-related deaths. Kita-Kyushu Lung Cancer Antigen 1 (KK-LC-1) is a cancer germline antigen with expression restricted to germ cells in adults and certain epithelial cancers including lung, breast, gastric and cervical. We identified a KK-LC-1 TCR from the tumor-infiltrating lymphocytes (TIL) of a patient with cervical cancer who had a complete tumor response to TIL therapy. The KK-LC-1 TCR was the most dominate clonotype in the infused TIL product and persisted in the peripheral blood following infusion, suggesting that it may have contributed to cancer regression in this patient. Methods: We are conducting a phase I cell dose escalation trial to test the safety and efficacy of KK-LC-1 TCR-T cell therapy in patients with metastatic KK-LC-1 positive epithelial cancer. Patients receive a lymphocyte depleting conditioning regimen followed by a one-time infusion of genetically engineered T cells expressing the KK-LC-1 TCR (KK-LC-1 TCR-T cells) and high-dose systemic aldesleukin. KK-LC-1 positivity is determined by RNAscope assay measuring the percentage of cancer cells expressing CT83 (gene encoding KK-LC-1) with a percentage of 25 or greater considered positive. Main inclusion criteria include HLA-A*01:01 allele, prior first-line therapy, ECOG of 0 or 1 and adequate organ and hematologic function. Main exclusion criteria include active major medical illness of the cardiovascular, respiratory or immune system, primary or secondary immunodeficiency and autoimmune disease. Participants will be entered in sequential dose levels and receive escalating doses of cells beginning at 1x108 and ending with 6x1010. Dose-limiting toxicities will be assessed during the first 30 days of cell infusion. The primary objective is to determine the maximally tolerated dose of KK-LC-1 TCR-T cells. Exploratory objectives include assessing the safety and efficacy of KK-LC-1 TCR-T cells and to conduct immunologic studies to understand and improve the administered treatment. Clinical trial information: NCT05035407.

Cancer germline antigen gene MAGEB2 promotes cell invasion and correlates with immune microenvironment and immunotherapeutic efficiency in laryngeal cancer

By multiple transcriptome datasets (TCGA, GSE59102, GSE25727, GSE27020 and GSE65858) and multi-omics (RNA-seq, SNP, CNV, DNA methylation) in-depth analysis, we found that cancer germline antigen (CGA) family/genes MAGEB2 is involved in the imitation, progression and prognosis in LC as well as correlated positively with lymphatic metastasis and negatively with DNA methylation. Then, in vitro experiment verified that MAGEB2 expression renders significant alteration in LC tissues and cells via immunohistochemical (IHC), qRT-PCR and western blot (WB), and up-regulation of MAGEB2 expression could facilitate the proliferation, migration and invasion of LC cells and vice versa. Subsequently, MAGEB2 knockdown suppressed tumor growth and lung metastasis in vivo animal experiment, while MAGEB2 overexpression promoted tumor growth and lung metastasis. Lastly, MAGEB2 is significantly associated with immune cell infiltration (CD8+ T cells particularly, IHC staining confirmed that as the protein expression of MAGEB2 increased, the protein level of CD8 (representing tumor-infiltrating CD8 + T cells) decreased in vitro), immunomodulators (knockdown or overexpression of MAGEB2 on LC cell lines can significantly affect the chemokine/cytokine secretion in vitro), and immunogenicity(TMB) in LC, which hints that MAGEB2 is tightly correlated with immune characteristics and might guide more effective immunotherapy strategies for LC patients.

Emerging Strategies in TCR-Engineered T Cells.

Immunotherapy of cancer has made tremendous progress in recent years, as demonstrated by the remarkable clinical responses obtained from adoptive cell transfer (ACT) of patient-derived tumor infiltrating lymphocytes, chimeric antigen receptor (CAR)-modified T cells (CAR-T) and T cell receptor (TCR)-engineered T cells (TCR-T). TCR-T uses specific TCRS optimized for tumor engagement and can recognize epitopes derived from both cell-surface and intracellular targets, including tumor-associated antigens, cancer germline antigens, viral oncoproteins, and tumor-specific neoantigens (neoAgs) that are largely sequestered in the cytoplasm and nucleus of tumor cells. Moreover, as TCRS are naturally developed for sensitive antigen detection, they are able to recognize epitopes at far lower concentrations than required for CAR-T activation. Therefore, TCR-T holds great promise for the treatment of human cancers. In this focused review, we summarize basic, translational, and clinical insights into the challenges and opportunities of TCR-T. We review emerging strategies used in current ACT, point out limitations, and propose possible solutions. We highlight the importance of targeting tumor-specific neoAgs and outline a strategy of combining neoAg vaccines, checkpoint blockade therapy, and adoptive transfer of neoAg-specific TCR-T to produce a truly tumor-specific therapy, which is able to penetrate into solid tumors and resist the immunosuppressive tumor microenvironment. We believe such a combination approach should lead to a significant improvement in cancer immunotherapies, especially for solid tumors, and may provide a general strategy for the eradication of multiple cancers.

Cancer Vaccine in Solid Tumors: Where We Stand

AbstractCancer immunotherapy has achieved landmark progress in the field of medical oncology in the era of personalized medicine. In the recent past, our knowledge has expanded regarding how tumor cells escape from the immune system, introducing immunosuppressive microenvironments, and developing tolerance. Therapeutic cancer vaccine leads to activation of immune memory that is long-lasting, safe, and effective; hence, it is becoming an attractive method of immunotherapy. Various cancer vaccine trials in the past have taught us the types of target selection, magnitude of immune response, and implementation of appropriate technologies for the development of new successful cancer vaccines. Tumor-associated antigens, cancer germline antigens, oncogenic viral antigens, and tumor-specific antigens, also known as neoantigens, are potential targets for designing therapeutic cancer vaccines. Cancer vaccine could be cell based, viral vector based, peptide based, and nucleic acid based (DNA/RNA). Several preclinical and clinical studies have demonstrated the mechanism of action, safety, efficacy, and toxicities of various types of cancer vaccines. In this article, we review the types of various tumor antigens and types of cancer vaccines tested in clinical trials and discuss the application and importance of this approach toward precision medicine in the field of immuno-oncology.

PLGA Nanoparticles Co-encapsulating NY-ESO-1 Peptides and IMM60 Induce Robust CD8 and CD4 T Cell and B Cell Responses.

Tumor-specific neoantigens can be highly immunogenic, but their identification for each patient and the production of personalized cancer vaccines can be time-consuming and prohibitively expensive. In contrast, tumor-associated antigens are widely expressed and suitable as an off the shelf immunotherapy. Here, we developed a PLGA-based nanoparticle vaccine that contains both the immunogenic cancer germline antigen NY-ESO-1 and an α-GalCer analog IMM60, as a novel iNKT cell agonist and dendritic cell transactivator. Three peptide sequences (85–111, 117–143, and 157–165) derived from immunodominant regions of NY-ESO-1 were selected. These peptides have a wide HLA coverage and were efficiently processed and presented by dendritic cells via various HLA subtypes. Co-delivery of IMM60 enhanced CD4 and CD8 T cell responses and antibody levels against NY-ESO-1 in vivo. Moreover, the nanoparticles have negligible systemic toxicity in high doses, and they could be produced according to GMP guidelines. Together, we demonstrated the feasibility of producing a PLGA-based nanovaccine containing immunogenic peptides and an iNKT cell agonist, that is activating DCs to induce antigen-specific T cell responses.

The Tumor Associated Antigen PRAME Exhibits Dualistic Functions That Are Targetable in Diffuse Large B-Cell Lymphoma

Background: With the goal of translating biological discovery into clinical actionability, deciphering crosstalk in the cellular ecosystem of the tumor microenvironment (TME) has emerged as a research focus. Although comparatively little is known about the immune biology of diffuse large B-cell lymphoma (DLBCL), as reflected in clonal selection of specific somatic gene mutations in response to immune system pressure and the specific composition of the TME, PRAME has emerged as a prominent member of the cancer germline antigen/ tumor associated antigen (TAA) family of proteins. It is expressed in various types of cancers, but generally not in normal tissues, apart from male germinal cells, and triggers autologous T-cell mediated immune responses. PRAME is highlighted as a new cancer therapeutic target of T-cell or antibody-based immunotherapies with promising anti-tumor responses in early phase clinical trials and pre-clinical models for several types of cancers. In the context of developing new immunotherapies, targeting TAAs that are presented by major histocompatibility complexes on tumor cells is a promising therapeutic strategy for patients that experience treatment failure. Material and methods: We performed integrative genomic analysis of whole-transcriptome RNAseq, targeted genomic sequencing, and high-resolution copy number analysis in 347 de novo DLBCL tumors from patients uniformly treated with R-CHOP. Findings were correlated with pathological and clinical parameters, as well as TME composition. Using DLBCL-derived cell lines (7 EZH2 mutated and 5 wt) and CRISPR-Cas9 genome editing, we performed in vitro functional studies to characterize cell-intrinsic effects of PRAME knockout. Moreover, we studied EZH2 inhibition in a murine model of Ezh2 mutant lymphoma with a focus on mechanistic links between EZH2 activity and PRAME expression, as well as TME composition (cell-extrinsic effects). Results: We discovered recurrent, and highly focal deletions of 22q11.22 including the PRAME gene, which were associated with poor treatment outcome, independent of pathological and clinical risk factors. To explore PRAME-deletion-associated phenotypes and interaction with the tumor microenvironment (TME), we analyzed corresponding RNAseq, immunohistochemistry (IHC), and flow-cytometry data from our DLBCL cohort and utilized enzyme-linked immunospot (ELISPOT) assays using patient-derived peripheral blood mononuclear cells. PRAME deletions contributed to an immunologically "cold" TME, representing a somatically acquired mechanism to evade anti-tumor T-cell response (cell-extrinsic effect). Using PRAME knock out by CRISPR-Cas9 in vitro, TRAIL-mediated apoptotic signaling was impaired. In addition, PRAME down-modulation was strongly associated with somatic EZH2 Y641 mutations in DLBCL. Using proximity ligation assays and co-IP, we demonstrated that PRAME directly interacted with EZH2 as a negative regulator, establishing a link between PRAME deletions and EZH2 mutations with anti-apoptotic signaling in DLBCL (cell-intrinsic effect). An in vivo murine model of Ezh2 mutant lymphoma showed decreased T-cell infiltration in the TME and Ezh2 inhibition induced PRAME restoration as compared to vehicle controls. IHC for CD3, CD4, FOXP3, and GZMB revealed significant increases in various T-cell populations of the TME, including Tregs and cytotoxic T cells. EZH2 inhibition with EPZ-6438 abrogated these dualistic effects leading to increased immune cell infiltration in the tumor microenvironment and acceleration of apoptosis via PRAME restoration. Moreover, restoration of PRAME antigen presentation by EZH2 inhibition resulted in enhancement of PRAME binding using a T-cell receptor mimic PRAME antibody (Pr20), suggesting immunotherapeutic potential. Conclusion: Our findings highlight multiple functions of PRAME during lymphomagenesis. PRAME restoration by EZH2 inhibition provides a preclinical rationale for synergistic therapies combining epigenetic re-programming with PRAME-targeted therapies. Disclosures Dao: Eureka Therapeutics: Consultancy. Melnick:Jubilant: Consultancy; Epizyme: Consultancy; Constellation: Consultancy; Janssen: Research Funding; Daiichi Sankyo: Research Funding. Scheinberg:Lantheus: Current equity holder in private company; Eureka Therapeutics: Consultancy, Current equity holder in private company, Patents & Royalties: Eureka Therapuetics and MSKCC have filed patent on this ScFV and TCRm; Actinium: Consultancy, Current equity holder in private company; Contrafect: Current equity holder in private company; Sapience: Consultancy, Current equity holder in private company; Iovance: Current equity holder in private company; Enscyse: Current equity holder in private company; Arvenas: Current equity holder in private company; Pfizer: Consultancy, Current equity holder in private company; Sellas: Consultancy, Current equity holder in private company; Oncopep: Consultancy. Scott:Janssen: Consultancy, Research Funding; Abbvie: Consultancy; AstraZeneca: Consultancy; NIH: Consultancy, Other: Co-inventor on a patent related to the MCL35 assay filed at the National Institutes of Health, United States of America.; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoString, Research Funding; Celgene: Consultancy; Roche/Genentech: Research Funding. Steidl:Curis Inc: Consultancy; Roche: Consultancy; AbbVie: Consultancy; Seattle Genetics: Consultancy; Bayer: Consultancy; Bristol-Myers Squibb: Research Funding; Juno Therapeutics: Consultancy.

Proteogenomic Analysis Unveils the HLA Class I Presented Immunopeptidome in Melanoma and EGFR Mutant Lung Adenocarcinoma

Immune checkpoint inhibitor and adoptive lymphocyte transfer-based therapies have shown great therapeutic potential for cancers with high tumor mutation burden (TMB). Here, we employed mass spectrometry (MS)-based proteogenomic large-scale profiling to identify potential immunogenic human leukocyte antigen (HLA) Class ǀ-presented peptides in both melanoma, a “hot tumor” with high TMB, and EGFR mutant lung adenocarcinoma, a “cold tumor” with low TMB. We identified several classes of neopeptides, including mutated neoantigens and more than 1000 post-translationally modified peptides representing 58 different PTMs. We constructed a cancer germline (CG) antigen database with 285 antigens and identified 42 Class I-presented CG antigens. Finally, we developed a non-canonical peptide discovery pipeline to identify 44 lncRNA-derived peptides and validated Class I binding for select neopeptides. We provide direct evidence of HLA Class I presentation of a large number of neopeptides for potential vaccine or adoptive cell therapy in melanoma and mutant EGFR lung cancer.

Abstract 6613: Identification of targetable HLA*A2:01 restricted peptides in osteosarcoma

Abstract Background: Osteosarcoma is the most common primary tumor of bone with 800-900 new cases diagnosed annually in the United States. The development of effective chemotherapeutic agents in the early 1970's led to a dramatic increase in osteosarcoma survival from less than 10% to over 60%. Unfortunately, little progress has been made in the past several decades despite intensified treatment and the application of targeted therapies with the 5-yr overall survival stalled at ~70%. Several phase I studies have reported occasional clinical responses to immune checkpoint inhibitors in osteosarcoma patients, although the role of immunotherapy in osteosarcoma treatment is largely unexplored. The goal of this work was to perform a deep immuno-genomic and proteomic profile of osteosarcoma tumors as the basis for developing effective immune-based therapy for osteosarcoma. Methods: Osteosarcoma tumors and cell lines were subjected to whole transcriptomes analysis. Intratumoral T cell receptor sequences were identified using MiXCR and VDJtools from the RNA-seq data. Immune signatures were scored in each tumor sample using single-sample GSEA. MHC class I bound peptides were isolated by immunoprecipitation of MHC class I complex, acid elution of bound peptides, and identification using LC-MS/MS. Healthy donor T cells were transduced with a lentiviral construct targeting PRAME and co-cultured with U2OS cells to evaluate specific PRAME peptide targeting by T cells. Results: Gene expression profiling of extracranial pediatric solid tumors identified high T cell infiltration in osteosarcoma patient tumors. High CD8+ T cell infiltration were associated with favorable outcome among osteosarcoma patients, suggesting the presence of an underlying endogenous T cell response against osteosarcoma tumors. We identified a set of tumor associated antigens, such as cancer germline antigens (CGA), which are highly expressed in osteosarcomas and display low/absent expression in normal tissues. PRAME, a CGA expressed in multiple solid tumors and hematological malignancies, was identified as one of the most frequently overexpressed CGAs in osteosarcoma. Immunoprecipitation of MHC Class I complexes followed by LC-MS/MS from osteosarcoma cells identified peptides derived from PRAME and other tumor associated antigens that are presented by HLA*A2:01 in osteosarcoma. In a proof-of-concept experiment, we demonstrate effective T cell cytotoxicity against osteosarcoma using a TCR which specifically recognizes an PRAME HLA*A2:01 peptide identified by mass spectrometry. Conclusions: Osteosarcoma tumors have prognostic high CD8+ T cell infiltration, expression of cancer testis antigens, and presentation of tumor associated peptides on MHC class I complex. These data warrant a broad evaluation of the efficacy of immunotherapy in osteosarcoma. Citation Format: David Milewski, Sivasish Sindiri, Jun S. Wei, Andrew Brohl, Young Song, Xinyu Wen, Andrew Qi, Udayan Guha, Javed Khan. Identification of targetable HLA*A2:01 restricted peptides in osteosarcoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6613.

Adoptive therapy with TCR gene-engineered T cells to treat patients with MAGE-C2-positive melanoma and head and neck cancer

Background & Aim We are scheduling a patient study, in which we will target the Cancer Germline Antigen MAGE-C2 (MC2), and use T cells with a young phenotype. MC2 is highly expressed in, amongst others, melanoma, head-and-neck squamous cell carcinoma (HNSSC), bladder, and triple-negative breast cancers but not in healthy adult tissues. Methods, Results & Conclusion We isolated MC2-specific TCRs from melanoma patients who showed clinical responses following vaccination that were accompanied by significant frequencies of anti-MC2 CD8 T cells in blood and tumor without apparent side effects. Following extensive evaluation of in vitro anti-tumor and self-reactivities, we have selected a TCR that recognizes the ALK epitope in the context of HLA-A2 for clinical development. Furthermore our preclinical studies showed that epigenetic pretreatment of tumor cells, but not normal cells, up-regulated MC2 gene expression and resulted in enhanced recognition of MC2 by the selected TCR. In parallel to the above studies, we renewed our GMP protocol to process T cells, using stimulating antibodies and cytokines, to generate T cells with a young phenotype. In a phase I/II study, we will explore the safety and anti-tumor efficacy of T cells engineered with the selected TCR in patients with MC2-positive melanoma and HNSSC. The study contains the following unique elements: - CT antigen not targeted before by T cell therapy - a new T cell processing method to generate young T cells - pretreatment of patients with epigenetic drugs to increase MC2 expression in the tumor - no chemotherapy prior to T cell infusion. All preparations have been successfully completed - including production and testing of the clinical vector batch and the clinical scale process validations. To date regulatory approvals have been obtained - including license for use of the vector and approval for the clinical trial. Patient treatment will start Q1 2020.

Cellular immune responses against cancer-germline genes in cancers.

Cancer-germline genes are a class of genes that are normally expressed in testis, trophoblast and few somatic tissues but abnormally expressed in tumor tissues. Their expression signature indicates that they can induce cellular immune responses, thus being applied as targets in cancer immunotherapy. To obtain the data of cellular immune responses against cancer-germline genes in cancer. We searched PubMed/Medline with the key words cancer-germline antigen, cancer-testis antigen, CD4+ T cell, CD8+ T cell and cancer. About 40 cancer-germline genes have been shown to induce T cell specific responses in cancer patients. Melanoma, lung and breast cancer are among the mostly assessed cancer types. Several epitopes have been identified which can be used in immunotherapy of cancer. Cellular immune responses against cancer-germline genes are indicative of appropriateness of these genes as therapeutic targets.

Relationship between Cancer Stem Cell Marker CD133 and Cancer Germline Antigen Genes in NCI-H292 Lung Cancer Cells.

BackgroundPrevious studies have shown that lung cancer stem cells express CD133 and that certain cancer stem cells express cancer germline antigens (CGAs). The transcriptional regulation of CD133 is complicated and poorly understood. We investigated CD133 and CGA expression in a non-small cell lung cancer cell line.MethodsThe expression levels of CD133 and CGAs (MAGE-6, GAGE, SSX, and TRAG-3) were measured in an NCI-H292 lung cancer cell line. The methylation status of the CD133 gene promoter region was analyzed. The expression levels and promoter methylation statuses of CD133 and CGAs were confirmed by treatment with the demethylating agent 5-aza-2′-deoxycytidine (ADC).ResultsAfter treatment with ADC, CD133 expression was no longer detected. MAGE-6 and TRAG-3 were detected before ADC treatment, while GAGE and SSX were not detected. ADC treatment upregulated MAGE-6 and TRAG-3 expression, while GAGE expression was still undetected after treatment, and only weak SSX expression was observed. GAGE expression was not correlated with expression of CD133, while the levels of expression of MAGE-6, TRAG-3, and SSX were inversely correlated with CD133 expression.ConclusionThese results showed that CD133 expression can be regulated by methylation. Thus, the demethylation of the CD133 promoter may compromise the treatment of lung cancer by inactivating cancer stem cells and/or activating CGAs.

Low-grade glioma harbors few CD8 T cells, which is accompanied by decreased expression of chemo-attractants, not immunogenic antigens

In multiple tumor types, prediction of response to immune therapies relates to the presence, distribution and activation state of tumor infiltrating lymphocytes (TILs). Although such therapies are, to date, unsuccessful in gliomas, little is known on the immune contexture of TILs in these tumors. We assessed whether low and high-grade glioma (LGG and HGG, grade II and IV respectively) differ with respect to number, location and tumor reactivity of TILs; as well as expression of molecules involved in the trafficking and activation of T cells. Intra-tumoral CD8 T cells were quantified by flow cytometry (LGG: n = 12; HGG: n = 8) and immunofluorescence (LGG: n = 28; HGG: n = 28). Neoantigen load and expression of Cancer Germline Antigens (CGAs) were assessed using whole exome sequencing and RNA-seq. TIL-derived DNA was sequenced and the variable domain of the TCRβ chain was classified according to IMGT nomenclature. QPCR was used to determine expression of T cell-related genes. CD8 T cell numbers were significantly lower in LGG and, in contrast to HGG, mainly remained in close vicinity to blood vessels. This was accompanied by lower expression of chemo-attractants CXCL9, CXCL10 and adhesion molecule ICAM1. We did not observe a difference in the number of expressed neoantigens or CGAs, nor in diversity of TCR-Vβ gene usage. In summary, LGG have lower numbers of intra-tumoral CD8 T cells compared to HGG, potentially linked to decreased T cell trafficking. We have found no evidence for distinct tumor reactivity of T cells in either tumor type. The near absence of TILs in LGG suggest that, at present, checkpoint inhibitors are unlikely to have clinical efficacy in this tumor type.

Sex Differences in Cancer Immunotherapy Efficacy, Biomarkers, and Therapeutic Strategy.

Sex differences in innate and adaptive immune responses are known, and women generally mount a stronger immune response than men. Cancer immunotherapy, represented by immune checkpoint inhibitors (ICIs), has revolutionized the treatment of cancer, and sex differences in cancer immunotherapy are just starting to be revealed. Here, we summarize recent research progress concerning sex differences in cancer immunotherapy efficacy. On their own, ICIs tend to be more effective in male cancer patients compared with female patients, while ICIs combined with chemotherapy tend to be more effective in female patients than male patients. Male tumors are usually more antigenic than female tumors, and this is reflected by their increased number of tumor mutations and cancer germline antigens. The biomarker tumor mutational burden (TMB), which reflects tumor antigenicity, is more effective at predicting immunotherapy response for female lung cancer patients than for male patients. In this review, we propose different therapeutic strategies for the different sexes: For male cancer patients, the immune environment should be enhanced, whereas for female cancer patients, tumor antigenicity should be enhanced.