Cancer-associated Antigen Research Articles

Abstract 4110: Identification of PD1 B cell mimotopes with functional PD1-PDL1 blocking capacity: New strategy for cancer immunotherapy

Abstract Immune checkpoint (ICP) blockade has become a major focus in cancer immunotherapy, and the application of monoclonal antibodies (mAbs) as ICP inhibitors shows impressive results in the therapy against cancers like melanoma, renal cell carcinoma or non-small cell lung cancer. Active immunization with B cell epitopes/mimotopes of ICP inhibitors rather than application of the corresponding mAbs for passive immunotherapy may, however, provide advantages such as induction of antibodies by the patient’s own immune system and overcoming the costly treatment of the mAbs. Applying a platform for bacterial surface-display of overlapping peptides spanning the extracellular domains of human PD1 (hPD1) and mouse PD1 (mPD1), as well as in vitro solid phase-based ELISA assay and cellular-based assays involving human Jurkat T cells expressing human/mouse PD1 and/or PDL1, B cell epitopes/mimotopes of Nivolumab and an anti-mPD1 mAb (with a blocking capacity) were identified. The mimotopes were not only shown to inhibit the binding of the corresponding mAbs but also to revert the PD1-PDL1 blocking inhibitory capacity of the respective mAbs. More importantly, to proof the antitumor activity of antibodies against PD1 mimotopes in vivo, rabbits were immunized with the mimotope of anti-mPD1 mAb to generate mimotope-specific IgGs. The specific IgGs not only inhibited the interaction of mPD1 and mPDL1 in vitro, but also significantly reduced tumor growth in a syngeneic model of mice grafted with cells expressing the breast cancer-associated tumor antigen Her-2/neu. Our results show systematic identification and characterization of mimotopes of anti-PD1 ICP inhibitor B cell mimotopes conferring in vivo anti-tumor activity, and also indicate the potential of such mimotopes for active immunization in cancer therapy. Based on the results, enhancement of our second generation of anti-Her-2/neu vaccine (HerVAXX; Tobias et al, 2017*) by combining with mimotopes of ICP inhibitors is also proposed, which may potentially pave the way to a paradigm change in active immunotherapy against Her-2/neu overexpressing cancers using an effective multi-level vaccine. The study is granted by Imugene Limited, Australia, and Medical University of Vienna. * Tobias J. et al. BMC Cancer, 2017, 17:118. Citation Format: Joshua Tobias, Claire Battin, Annika De Sousa Linhares, Baier Karin, Katharina AMbroye, Mirjana Drinic, Erika Garner-Spitzer, Christoph Zielinski, Michael Kundi, Peter Steinberger, Ursula Wiedermann. Identification of PD1 B cell mimotopes with functional PD1-PDL1 blocking capacity: New strategy for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4110.

Abstract LB-187: Hsp90 inhibitor SNX-2112 enhances neoantigen presentation on the surface of tumor cells

Abstract Introduction: SNX-5422 is the orally active prodrug of SNX-2112, a potent, highly selective inhibitor of heat-shock protein 90 (Hsp90) with promising anti-tumor activity in clinical trials. SNX-5422 enhanced anti-PD-1 activity in MC38 murine model. SNX-2112 affects multiple tumor pathways, including the interferon pathway and upregulates genes associated with antigen expression, and T-cell recognition, and it interferes with tumor microenvironment immunoediting associated genes. Methods: Cells were cultured in media supplemented with 10% fetal bovine serum at 37°C, 5% CO2, and 95% humidity. The 50% effective concentration of SNX-2112 on cell viability of SkMel28, MDAMB231, SkMel2 was determined using Cell Titer-Glo® Luminescent Cell Viability Assay. Cells were treated with SNX-2112 or other anti-tumor agents, e.g., vemurafenib, to determine surface expression of MHC class I complexes by flow cytometry using MHC class I monoclonal antibody PE conjugate (Enzo-Cat# ALX-805-711R-C100) and, in parallel, induction of apoptosis by Annexin V APC. Results: At the lowest dose of SNX-2112 required to inhibit activity of model Hsp90 clients (MAPK), a 3 to 5-fold increase in MHC class I presentation was observed in all cells lines. Treatment of MDAMB231 with SNX-2112 caused a 3-fold increase in surface expression of MHC class I complexes, loss of Hsp90 client protein activity, and increased protein ubiquitination, without change in protein levels of immunoproteasome subunits LMP2 and LMP7. Increased surface expression of MHC class I complexes, loss of Hsp90 client function, increased protein ubiquitination without affecting immunoproteasome subunits were also found in both melanoma cell lines. This suggests that upregulation of cell surface MHC class I complexes is driven by increased Hsp90 client protein turnover. SNX-2112 treatment at concentrations sufficient to increase MHC class I surface expression also resulted in a small increase in apoptosis. Interestingly, treatment of SkMel28 with vemurafenib robustly induced apoptosis without any effect on surface expression of MHC class I complexes. Analysis of surface-expressed peptides by mass spectrometry revealed a change in peptide profile after treatment with SNX-2112. Conclusion: SNX-2112 increased surface expression of multiple MHC class I complexes. Results suggest that SNX-2112 can drive the selective surface presentation of Hsp90 client proteins on tumor cell surface and could improve recognition by T cells specific for various cancer-associated antigens derived from mutated genes, making it potential useful in immunotherapy of cancer. This effect of SNX-2112 is independent of the induction of apoptosis as agents like vemurafenib did not elicit the same effect. Ongoing studies are focused on identifying Hsp90 client-derived peptides in surface complexes and their role in anti-tumor immunity. Citation Format: Megan Yap, John W. Walker, Kristi Baker, Richard Fahlman, Everardus Orlemans, Paul Lapointe. Hsp90 inhibitor SNX-2112 enhances neoantigen presentation on the surface of tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-187.

Abstract 3948: The sequential administration of vinca alkaloids and intermittent cyclophosphamide primes antigen-presenting and NK cells and significantly improves in vivo efficacy of anti-PD-L1 in triple-negative breast cancer models

Abstract Checkpoint inhibitors (CIs) have shown an unprecedented clinical activity in a large variety of cancer types, but only in a fraction of patients. To increase the number of responders, preclinical models are studied to define effective combinatorial regimens and the best window of therapeutic opportunities. We recently described a synergy between anti-PD-1 and -PD-L1 CIs and several types and dosages of chemotherapy in immunocompetent models of triple negative breast cancer (TNBC), likely due to unique effects of chemotherapeutics over circulating and tumor-infiltrating, suppressive, antigen-presenting and effector immune cell populations (Orecchioni et al, Br J Cancer 2018). To find a high-order drug combination, we have further studied by multiparametric flow cytometry and single-cell transcriptomic a panel of different chemotherapy drugs (including microtubular poisons, alkylating agents, topoisomerase inhibitors and antimetabolites) used in vivo at different dosages and schedule. Our aim was to define a combination, dose and schedule able to promote a) dendritic cell maturation, b) release and c) uptake of cancer-associated antigens from targeted neoplastic cells, d) cross-priming between antigen-presenting cells (APCs) and T cells, e) NK cell activation, and f) inhibition of suppressor immune cell populations. Vinca alkaloids vinblastine and vinorelbine (V), at low-medium dosages, were the most effective in a), b), c) and d). After V administration, Tregs were reduced, and circulating and tumor-infiltrating APCs showed a “maturation to activation” program with increased expression of several antigens including CD40, CD80, and CD86. The alkylating agent cyclophosphamide (CTX) targeted Tregs, mobilized APC progenitors, activated and increased the number of circulating and intratumoral NK cells. These effects were significantly increased when CTX was administered in an intermittent fashion (every 6 days) at low-medium doses. As the APC-NK cell axis has been recently found to be pivotal in CI-mediated, anti-tumor cell immunity, our data suggested a preclinical trial in immunocompetent orthotopic models of metastatic (post-mastectomy) BALB mice injected with 4T1 or EMT-6 TNBC cells. Among a large panel of different combinations and dosages, the sequential administration of V, intermittent CTX and anti-PD-L1 was the only able to completely abrogate TNBC local and metastatic growth, and this effect was not observed in T and NK cell-depleted mice. Taken together with recent data from randomized, combinatorial studies in TNBC patients (Schmid et al, NEJM 2018), our findings suggest that CIs might be included in future combinatorial regimens aimed at improving the percentage of patients receiving a clinical benefit and prolonging the duration of this benefit. Citation Format: Stefania Orecchioni, Loredana Vecchi, Paolo Falvo, Lucilla Luzi, Patrizia Mancuso, Chiara Camisaschi, Francesco Bertolini. The sequential administration of vinca alkaloids and intermittent cyclophosphamide primes antigen-presenting and NK cells and significantly improves in vivo efficacy of anti-PD-L1 in triple-negative breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3948.

Abstract LB-205: A lipoplex-based mRNA nanovaccine for cancer immunotherapy

Abstract Intracellular delivery of messenger RNA (mRNA)-based cancer vaccines have shown great potential to activate antitumor immune response for cancer prevention and therapy. In vivo delivery efficiency of mRNA has been greatly improved by various nucleotide chemical modifications and nanovector formulations, but the efficacy of mRNA vaccine remains unsatisfied. Here, we report a lipoplex-based mRNA nanovaccine that can markedly improve mRNA delivery in antigen presenting cells, activate T cell response, and induce robust antitumor immunity. The nanovaccine formulation contains three components: cationic polymer/lipid(poly(amidoamine)/alkylene oxide), DSPE-PEG2000, and epitope-encoding mRNA. With appropriate ratio, the optimized nanovaccine show effective mRNA delivery in dendritic cells, and strongly active antigen-specific immune response in vitro and in vivo. The mRNA@lipoplex nanovaccine enhanced mRNA uptake by dendritic cells, and significantly increased T cell activation reflected by IL-2 and IFN-γsecretion in an in vitro antigen presentation assay. Besides enhancing cross-presentation, nanoparticle itself can promote the expression of inflammatory cytokines such as IL-12 via stimulating NF-κB signal pathway in dendritic cells. In a tumor prevention setting, mRNA@lipoplex nanovaccine produced strong CD8+ T cell activation and great efficacy in preventing tumor development. Furthermore, treatment of established B16 and B16-OVA melanoma and MC38-OVA colon tumors with nanovaccine containing mRNA encoding cancer-associated antigen resulted in great therapeutic efficacy and prolonged survival. Re-challenge of tumor-free mice with MC38-OVA cells led to complete tumor rejection, suggesting long-term antitumor memory. In conclusion, our work presents a new means to deliver mRNA cancer vaccine, which in principle may be applied for immunotherapy for a wide range of tumor types. Citation Format: Zhang Hongxia, You Xinru, Wang Xiaojuan, Wang Zining, Xu Feifei, Wu Jun, Xia Xiaojun. A lipoplex-based mRNA nanovaccine for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-205.

Detection of the cancer-associated T antigen using an Arachis hypogaea agglutinin biosensor

An impedimetric biosensor was developed for the selective detection of the cancer-associated T antigen, using the lectin from Arachis hypogaea (peanut agglutinin, PNA) as the recognition element. The increase in the biosensor's impedance after sample incubation was indicative of lectin recognition and complex formation between PNA and glycoproteins containing T antigen. When using asialofetuin as model glycoprotein, a minimum amount of 100 ng of glycoprotein could be detected, generating an increase in impedance of 7.2%. Albumin did not cause interference in the detection of T-carrying glycoproteins up to a concentration of 0.01 mg ml−1.The biosensor was used to evaluate the T-antigen expression in serum samples and was able to discriminate between control samples (of individuals without cancer) and case samples from patients with diverse types of carcinomas (skin, colon, breast, prostate, stomach, kidney, lung, liver and rectum) in which an increase in the expression of T antigen is well-known. The same samples were analyzed with a Vicia villosa agglutinin biosensor that has specificity for the cancer-associated Tn antigen, to compare the expression of both antigens in the diverse carcinomas. The results were different for both biosensors, confirming that the use of different lectins allows to monitor different antigen expression. Furthermore, combining different lectins, glycosylation profiles for each carcinoma type can be obtained.This work demonstrates the feasibility of employing PNA to selectively recognize the T epitope in glycoproteins and the proposed biosensor could be used for high-throughput, label-free profiling of the cancer-associated T antigen in serum samples.

Preliminary evaluation of the fully automated diagnostics testing for BRAF mutations to companion diagnostics and treatment decisions in melanoma patients

Circulating tumor DNA (ctDNA) is known to harbor tumor-specific genetic or epigenetic alterations. In the present study, the correlation of ctDNA with tumor response to neoadjuvant chemotherapy (NAC) was evaluated in primary breast cancer patients.Plasma samples were obtained from 87 primary breast cancer patients (stage II-III) before and after NAC, as well as 1 year after surgery. Methylated ctDNA (met-ctDNA) was determined by one-step methylation-specific PCR (OS-MSP) for the promoter region of RASSF1A.The positivity (23.0%, 20/87) of met-ctDNA before NAC was significantly (P < .05) higher than that of carcinoembryonic antigen (CEA) (8.6%) and cancer-associated antigen (CA) 15-3 (7.4%). In the patients with positive met-ctDNA before NAC, met-ctDNA significantly decreased after NAC in those with disease that responded to therapy (P = .006), but not in patients whose disease did not respond to therapy. Met-ctDNA after NAC was found to be significantly (P = .008) correlated to the extent of residual tumor burden. Of the 7 patients who showed an increase in met-ctDNA at 1 year after surgery, 3 developed recurrence.Met-ctDNA is a more sensitive marker than CEA and CA15-3, and it might be useful in monitoring the clinical tumor response to NAC. In addition, the potential use of met-ctDNA as a tumor marker for monitoring postoperative recurrence has been suggested.

Derivation of mimetic γδ T cells endowed with cancer recognition receptors from reprogrammed γδ T cell.

Using induced pluripotent stem cells (iPSCs) to derive chimeric antigen receptor-modified T (CAR-T) cells has great industrial potential. A previous study used αβ T cell-derived CAR-modified iPSCs to produce CAR-T cells. However, these αβ T cells are restricted to autologous use and only recognize single cancer antigen. To make CAR-T alternative for allogeneic use, we reprogrammed γδ T cell into iPSCs (γδ T-iPSCs) to circumvent the risk of graft-versus-host disease. To target multiple cancer-associated antigens, we used an “NK cell-promoting” protocol to differentiate γδ T-iPSCs and to induce expression of natural killer receptors (NKRs). Through such two-step strategy, mimetic γδ T cells endowed with an array of NKRs and thus designated as “γδ natural killer T (γδ NKT) cells” were derived. With no/low-level expression of inhibitory killer cell immunoglobulin-like receptors (KIRs) and immune checkpoint receptors, γδ NKT cells may provide a potent “off-the-shelf” cytotoxic cell source to recognize multiple ubiquitous antigens in a broad spectrum of cancers.

OVA66 promotes tumour angiogenesis and progression through enhancing autocrine VEGF-VEGFR2 signalling.

BackgroundActivation of autocrine VEGF-VEGFR2 signalling in tumour cells activates cell proliferation, survival, and angiogenesis, all of which are crucial for tumour progression. Ovarian cancer-associated antigen 66 (OVA66) is now known to be overexpressed in multiple tumours and plays a role in tumour development, but the underlying mechanisms has not been fully investigated.MethodsWe employed ovarian and cervical cancer cells and mouse models to detect the role of OVA66 in angiogenesis, growth, and metastasis of cancer cells. Immunofluorescence and western blot were used to determine the function of OVA66 in regulating autocrine VEGF-VEGFR2 signalling. Immunohistochemistry and bioinformatics analysis were used to detect the correlation of OVA66 and VEGF expression.FindingsOVA66 overexpression in the cancer cell lines promoted VEGF secretion, tumour growth and angiogenesis in vitro and in vivo. Conversely, shRNA-mediated OVA66 knockdown had the opposite effects. Mechanistically, OVA66 overexpression was found to boost an autocrine VEGF–VEGFR2 positive-feedback signalling loop in the tumour cells, leading to amplified effect of VEGF on tumour angiogenesis and proliferation and increased migration in vitro and in vivo, respectively. Finally, we identified a significant positive correlation between the expression levels of OVA66 and VEGF in ovarian and cervical cancer specimens, and found that OVA66 was significantly associated with advanced ovarian cancer.InterpretationWe identify a novel function for OVA66 in regulating an autocrine VEGF–VEGFR2 feed-forward signalling loop that promotes tumour progression and angiogenesis.FundThis work was supported by the National Natural Science Foundation of China (81602262); and Excellent Youth Scholar Program of Tongji University (2015KJ062).

Abstract B049: Empty MHC class I molecules for improved detection of antigen-specific T-cells

Abstract Major histocompatibility complex (MHC) class I multimers have been widely used to identify antigen specific T-cells for immune monitoring, epitope discovery, and T-cell isolation. A bottleneck to many peptide-MHC driven applications for T-cell interrogation is the peptide ligand dependent stability of the MHC class I proteins, which thus compels high-affinity peptide dependent in-vitro folding of each MHC protein and the use of a peptide-exchange technology to investigate antigens of interest. To overcome this challenge, we demonstrate the use of empty peptide-receptive MHC class I molecule for detection of antigen specific T-cells. This strategy is based on an HLA-A*02:01 variant which is stabilized by a disulfide bond to link the alpha-1 and alpha-2 helices close to the F pocket. Determined by the crystal structure, peptide-loaded disulfide-stabilized HLA-A*02:01 show complete structural overlap to wild-type HLA-A*02:01. Following peptide loading, we used such disulfide-stabilized HLA-A*02:01 molecules to form fluorescence labeled tetramers and applied them for detections of T-cell responses against common viruses in healthy donor peripheral blood mononuclear cells. In all tested samples, disulfide-stabilized HLA-A*02:01 tetramers detected T-cell with same specificity as wild-type MHC tetramers and they consistently provide a better staining index for antigen-specific T-cell detection. Importantly, disulfide-stabilized MHC class I molecules can be loaded with peptide in the multimerized form without impacting the T-cell staining capacity. We demonstrate the value of empty loadable tetramers, converted to antigen-specific tetramers by a single-step peptide addition, for identification of T-cells specific to several neo- and cancer-associated antigens among tumor-infiltrating lymphocytes in melanoma.To evaluate if the disulfide linkage has an impact on TCR recognition of peptide-MHC complexes, we determined and compared TCR fingerprints of T-cell clones specific to a given peptide-MHC complex using both the wild-type and the disulfide-stabilized HLA-A*02:01 multimers. No differences were observed in the TCR interaction profile between the disulfide optimized and the wild-type MHC class I. In conclusion, disulfide-stabilized empty HLA class I proteins are a potentially powerful tool for interrogating T-cell recognition—offering a fast and flexible transformation from an empty peptide receptive state to a set of personalized reagents generated to match individual tumor characteristics for T-cell monitoring or selection. Citation Format: Tripti Tamhane, Sunil Kumar Saini, Raghavendra Anjanappa, Ankur Saikia, Sofie Ramskov, Marco Donia, Inge Marie Stenfoft Svane, Søren Nyboe Jakobsen, Maria Garcia-Alai, Martin Zacharias, Rob Meijers, Sebastian Springer, Sine Reker Hadrup. Empty MHC class I molecules for improved detection of antigen-specific T-cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B049.

Abstract B166: Mass spectrometric characterization of MHC peptides on therapeutic EBV transformed primary lymphoblastoid cells from HLA A*11 patients

Abstract EBV transformed lymphoblastoid cell lines (LCL) derived from patients with Epstein Barr virus (EBV)-related cancer have been used as "feeder cells" after terminally irradiation in multiple cell therapy protocols targeting EBV-related cancers. These cells are subsequently co-cultured with patient peripheral blood mononuclear cells (PBMC) and are sufficient expansion of autologous cell product, given therapeutically back to patients. LCL are known to present both host genome derived cancer-associated antigens relevant for cancer, alongside EBV antigens. PBMC that are co-cultured with LCL have been used in multiple small studies across multiple EBV related cancers. This study was performed in the context of LCL used as feeder cells in the context of EBV-related nasopharyngeal cancer that was treated with autologous cell product after co-culture with LCL as part of a phase II clinical trial in Singapore. The aims of this study were, firstly, to perform unbiased identification of MHC peptides using mass spectrometry; secondly, to map the peptides to respective HLA alleles using publicly available HLA peptide binding prediction algorithms; and thirdly, to look specifically at HLA A*11 allele specific EBV-derived peptides. 6 patients with HLA A*11 were identified and LCL from these patients were grown in cell culture for 1-3 months each, until 5E8 cells were available. Cells were treated in suspension with acidification in order to enable acid elution of MHC peptides. Mass spectrometric analysis was performed for unbiased evaluation of MHC peptides. Based on the consensus peptide sequences derived from 8-mer and 9-mer peptides, HLA A*11:01 appeared to be a dominant allele with at least 20% of peptides having basic end residues that are typical for binding to HLA allele. Between 3,000 to 5,000 distinct peptide sequences across lengths characteristic for both class I and II MHC alleles were identified for each sample, including multiple peptides derived host genome DNA, and some from EBV proteins including LMP1, BARF1, EBNA6, and TTK. There was some overlap of peptides seen across the samples. For example, 2 patient samples, with 3,636 and 4,033 distinct peptides identified respectively, had 1,025 overlapping peptides. 2 patient samples with HLA A*11:01 and HLA A*11:02 respectively, had 1 overlapping EBV protein derived set of peptides that demonstrated differences in trimming of peptides derived from the same epitope across these 2 HLA classes. Only 1 EBV protein was found to be consistently represented in the MHC peptidome across the patient samples, with the same epitope represented across all the samples within the scope of this study. This small study demonstrates that mass spectrometric analysis of large numbers of cells is able to directly deconvolute the MHC peptide, and this is relevant in the context of adoptive cell therapies that utilize "feeder cells" that are required to reliably present clinically relevant antigens to generate autologous cell product. Furthermore, in the absence of reliable peptide prediction algorithms for HLA alleles that are more prevalent in Asia, such as HLA A*11:02, this approach is useful for direct characterization of peptides that bind. Citation Format: Amit Jain. Mass spectrometric characterization of MHC peptides on therapeutic EBV transformed primary lymphoblastoid cells from HLA A*11 patients [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B166.

The progress of study on the targeted immunotherapy based on XAGE-1b antigen in prostate cancer

Prostate cancer is one of the most common malignancies in men, which threats to men’s healthseriously. Its development is a complex process of multi-gene interaction and interaction, the immune response plays an important role in the whole process. With the development of immunology and tumor microenvironmentinprostate cancer, the targeted immunotherapy isconsidered the most promising treatment for prostate cancer. XAGE-1b antigen is a prostate cancer-associated antigen with strong immunogenicity. A large number of studies have shown that it is closely related to the occurrence and progress of prostate cancer, many researchers believe that XAGE-1b antigen isa potential target forimmunotherapy in advanced prostate cancer. Inthis review, we perform a review of the research progress on the targeted immunotherapy based on XAGE-1b antigen in prostate cancer. Key words: XAGE-1b; Prostate Cancer; Cancer-testis antigen; Targeted immunotherapy

Affinity-enhanced T-cell receptors for adoptive T-cell therapy targeting MAGE-A10: strategy for selection of an optimal candidate

ABSTRACTCirculating T-cells that have passed thymic selection generally bear T-cell receptors (TCRs) with sub-optimal affinity for cancer-associated antigens, resulting in a limited ability to detect and eliminate tumor cells. Engineering TCRs to increase their affinity for cancer targets is a promising strategy for generating T-cells with enhanced potency for adoptive immunotherapy in cancer patients. However, this manipulation also risks generating cross-reactivity to antigens expressed by normal tissue, with potentially serious consequences. Testing in animal models might not detect such cross-reactivity due to species differences in the antigenic repertoire. To mitigate the risk of off-target toxicities in future clinical trials, we therefore developed an extensive in vitro testing strategy. This approach involved systematic substitution at each position of the antigenic peptide sequence using all natural amino acids to generate a profile of peptide specificity (“X-scan”). The likelihood of off-target reactivity was investigated by searching the human proteome for sequences matching this profile, and testing against a panel of primary cell lines. Starting from a diverse panel of parental TCRs, we engineered several affinity-enhanced TCRs specific for the cancer-testis antigen MAGE-A10. Two of these TCRs had affinities and specificities which appeared to be equally optimal when tested in conventional biochemical and cellular assays. The X-scan method, however, permitted us to select the most specific and potent candidate for further pre-clinical and clinical testing.

Antibody-initiated beneficial and harmful immune responses

A critical function of the immune system is to maintain tolerance to self by corrective immune responses throughout life, including preventing or correcting changes that may interfere with organ function and architectural integrity. These changes have two broad categories, namely (1) exogenous antigen-induced mishaps (e.g., due to bacterial, viral or fungal infections) and (2) endogenous antigen-caused ailments initiated by modified self-antigens derived from damaged organs following exposure to smoke, certain drugs, chemicals, infectious agents, radiation, etc., resulting in autoimmune diseases or cancer. In some cases, cells of the immune system are unable to respond with a corrective antibody response. For example, presentation of a modified self-antigen can initiate a pathogenic IgG immune response, thereby causing an autoimmune disease. Furthermore, if cancer-associated antigens are not appropriately presented to the cells of the immune system, there is failure to mount a specific pathogenic lytic IgG autoantibody response for recognition and elimination of cancer-associated antigens, and as a consequence, the cancer continues to proliferate.The third vaccination technique that we have developed and designated a modified vaccination technique (MVT) is able to correct these immunological mishaps. The premise of the MVT is that it can prevent both exogenous (infectious and contagious diseases) and endogenous (autoimmune diseases and cancer) antigen-caused diseases, as well as terminate established diseases. Therefore, by exploiting the immune system's natural abilities to make corrective responses, it has both prophylactic and therapeutic actions, with minimal side effects.

Genomics Role in Cancer Immunosurveillance: Impact on Immunotherapy Response

Context: One of the most important achievements in cancer research is the development of cancer immunotherapy. However, only a subset of patients respond to immunotherapy modalities, and few patients respond for a durable time. Here, we review the possible genomic mechanisms of response and resistance to these therapies, which can lead to the selection of responders, who may benefit most from immunotherapy. Evidence Acquisition: We searched PubMed, Scopus, and Web of Science Core Collection with the following keywords: “Immunotherapy, Resistance, Response, Programmed cell death 1 receptor, CTLA-4, Cancer immunity, Tumor Genomics, and Somatic Mutations”. Results: T cells that specifically recognize cancer-associated antigens, are responsible for the immune system response against cancer. Nonsynonymous mutations, which are transcribed and translated into polypeptides, may generate new epitopes (neoepitopes), which can lead to their presentation on major histocompatibility (MHC) class I molecules and subsequently recognized by the adaptive immune system. Despite the unprecedented durable responses, the majority of patients treated with cancer immunotherapies do not respond to the therapy (primary resistance), and some patients relapse after an initial response (acquired resistance). Resistance to immunotherapy can be a result of tumor cell intrinsic or extrinsic factors. There is correlation between tumor mutation burden (TMB) and response to immunotherapy. In addition, mismatch repair deficient tumors harbor considerably more somatic mutations compared to mismatch repair proficient tumors and respond better to anti-programmed cell death protein 1 (anti-PD1) therapy. Mutations in other DNA repair genes may also affect immunotherapy response. Conclusions: Neoantigen specific T cells constitute a major “active component” for the success of cancer immunotherapies. The genetic damage that confers tumorigenic growth, can also be targeted by the immune machinery to inhibit cancer development and progression. With further validation of experiments, genomics-based approaches can allow to select patients most likely to achieve durable responses to immunotherapy modalities.

Attaining threshold antibody cytotoxicity for selective tumor cell destruction: an opinion article.

We propose a novel immunotherapeutic paradigm that justifies application of several antibodies to various membrane-associated antigens to achieve a critical threshold density of immune complexes on the surface of cancer cells sufficient for triggering downstream cytolytic pathways. Indeed, some cancer-associated antigens (such as cancer/testis antigens) were found to be expressed on many cancer (but not normal) cells, with their baseline membrane expression levels being originally quite low for some of them, or even further down-regulated due to immune-driven cell selection. To achieve the mandatory threshold density of membrane-associated immune complexes on malignant cells, the concept stipulates combined application of antibodies specific for a cancer-associated antigen along with antibodies against an antigen expressed not only on tumor, but also on normal cells. In the proposed scenario it is of vital importance that the latter antibodies should be applied in suboptimal dosage to exclude the destruction of normal cells devoid of a cancer-associated antigen. Malignant cells often co-express antigens not present concurrently on normal cells at high levels. In such cases, suboptimal dosages of antibodies specific for those antigens could also be applied to achieve cumulative effect leading to selective destruction of tumour cells. Hence, the described immunotherapeutic technology could be used metaphorically speaking as a kind of ‘immunological knife’, which is capable of highly selective destruction of cancer cells without destroying normal cells.

Multicellular Human Gastric-Cancer Spheroids Mimic the Glycosylation Phenotype of Gastric Carcinomas.

Cellular glycosylation plays a pivotal role in several molecular mechanisms controlling cell–cell recognition, communication, and adhesion. Thus, aberrant glycosylation has a major impact on the acquisition of malignant features in the tumor progression of patients. To mimic these in vivo features, an innovative high-throughput 3D spheroid culture methodology has been developed for gastric cancer cells. The assessment of cancer cell spheroids’ physical characteristics, such as size, morphology and solidity, as well as the impact of glycosylation inhibitors on spheroid formation was performed applying automated image analysis. A detailed evaluation of key glycans and glycoproteins displayed by the gastric cancer spheroids and their counterpart cells cultured under conventional 2D conditions was performed. Our results show that, by applying 3D cell culture approaches, the model cell lines represented the differentiation features observed in the original tumors and the cellular glycocalix underwent striking changes, displaying increased expression of cancer-associated glycan antigens and mucin MUC1, ultimately better simulating the glycosylation phenotype of the gastric tumor.

The Potential of CAR T Cell Therapy in Pancreatic Cancer.

Pancreatic cancer has a dismal prognosis and effective treatment options are limited. It is projected to be the second most common cause of cancer related mortality in the United States by 2030 and there is urgent unmet need for novel systemic treatment options. Immunotherapy with antibodies targeting PD-1, PD-L1, CTLA-4 has not shown clinical activity in unselected pancreatic cancer, emphasizing the need for combination immunotherapy approaches or other therapeutic strategies. As such, chimeric antigen receptor (CAR) T cell therapy represents an emerging therapeutic option for pancreatic cancer. This modality utilizes genetically engineered T cells that are redirected to specific cancer-associated antigens to elicit potent cytotoxic activity. This review summarizes the available preclinical data and highlights early phase clinical trials using CAR T cell approaches in pancreatic cancer, a disease state that is gaining attention as a conduit for cell therapy. Future directions in application of CAR T cell therapy are also considered including its ability to be directed against novel epitopes and combined with other therapeutic regimens.

Synthesis of H (type 4) trisaccharide, key structural fragment of globo-H and fucosyl-GM1 cancer-associated antigens

A convenient synthesis of Fucα1-2Galβ1-3GalNAcβ-OCH2CH2CH2NH2 (trisaccharide H type 4) is described. This glycan is the terminal part of glycosphingolipids globo-H and fucosyl-GM1 known as cancer-associated carbohydrate antigens.

Abstract 4616: Cancer-associated MUC1 epitope-recognizing antibody as a novel immunohistochemical marker for breast carcinoma

Abstract Background: MUC1 has been used in clinical practice as a serum tumor marker (CA15-3) for monitoring recurrence and response to the treatment of breast cancers. However, it is not so practical to use the conventional anti-MUC1 antibody (C-Ab) for the differential diagnoses among malignant and benign tumors, because of its limited specificity to cancer-associated sugar chain structures. Recently, a novel epitope-defined (ED) antibody, which specifically recognizes MUC1 with cancer-associated carbohydrate antigens including Tn and sialyl-T antigens, was developed by our collaborators. In the present work, we focused on this Tn antigen-recognizing ED antibody (Tn-ED Ab) for MUC1, and examined the potential utility of this novel antibody as an immunohistochemical diagnostic marker of the breast.Design: Tissue microarray (TMA) consisting of invasive lesions and their non-neoplastic counterparts were constructed from 169 lesions of 167 individuals with invasive breast carcinomas (including 2 bilateral cases) resected and diagnosed at Hokkaido University Hospital from 2000 to 2003. We compared MUC1 immunoreactivity of Tn-ED Ab (clone SN-102) to commercially available C-Ab (clone Ma552) using TMA to evaluate its diagnostic performance and to analyze the association with clinicopathologic factors. Results: In invasive cancer parts, the immunoreactive positivity of Tn-ED Ab was observed predominantly in the cytoplasm (146/169) rather than in the cell membrane (5/169). The positive reaction of C-Ab was found in the cytoplasm and the membrane as well (156/169 and 93/169). Tn-ED Ab showed a lower expression rate in non-neoplastic parts and a high specificity in invasive cancer parts. Sensitivity/specificity in invasive parts was 86%/92% for Tn-ED Ab and 92%/47% for C-Ab. Moreover, the invasive cancer group with Tn-ED-positivity was significantly correlated with their molecular subtypes, low Ki-67 indices, and high ages, compared to those with Tn-ED-negativity (p=0.008, p=0.010 and p=0.027, respectively). Conclusion: Compared to C-Ab, the newly-developed Tn-ED Ab for MUC 1 showed a higher specificity for invasive breast cancer with a low reactivity to non-neoplastic components. Future study plans are ongoing to show that this cancer-specific Tn-ED MUC1 Ab could be useful for differentiating malignant from benign intraductal breast lesions as well. Citation Format: Ai Shimizu, Kanako Hatanaka, Yutaka Hatanaka, Kentaro Naruchi, Masaharu Sato, Hiroshi Kase, Tomoko Mitsuhashi, Hiroko Yamashita, Yoshihiro Matsuno. Cancer-associated MUC1 epitope-recognizing antibody as a novel immunohistochemical marker for breast carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4616.

Synthesis, Liposomal Formulation, and Immunological Evaluation of a Minimalistic Carbohydrate-α-GalCer Vaccine Candidate.

Fully synthetic glycan-based vaccines hold great potential as preventive and therapeutic vaccines against infectious diseases as well as cancer. Here, we present a two-component platform based on the facile conjugation of carbohydrate antigens to α-galactosylceramide (α-GalCer) to yield fully synthetic vaccine candidates. Formulation of the cancer-associated Tn antigen glycolipid model vaccine candidate into liposomes of different sizes and subsequent immunization of mice generated specific, high-affinity antibodies against the carbohydrate antigen with characteristics of T cell-dependent immunity. Liposome formulation elicited more reproducible glycan immunity than a conventional glycoconjugate vaccine bearing the same glycan antigen did. Further evaluation of the immune response revealed that the size of the liposomes influenced the glycan antibody responses toward either a cellular (Th1) or a humoral (Th2) immune phenotype. The glycolipid vaccine platform affords strong and robust antiglycan antibody responses in vivo without the need for an external adjuvant.