Tumor Antigen-specific T-cell Responses Research Articles

Enhancement of Tumor Antigen-specific T-cell Responses by Immune Checkpoint Blockade in Human Papillomavirus-related Head and Neck Squamous Cell Carcinoma.

Human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) is clinically and immunologically distinct from HPV-negative HNSCC. Herein, we investigated the presence of tumor antigens HPV E6/E7 and wild-type p53-specific T-cell responses, and the impact of immune checkpoint blockade in patients with HPV-positive HNSCC. Peripheral blood mononuclear cells (PBMCs) from patients with HPV-positive HNSCC were stimulated with HPV E6/E7 or wild-type p53-derived peptide mixture and evaluated using the interferon-γ enzyme-linked immunosorbent spot assay. Flow cytometry was performed to analyze the proportion of T-cell subsets and T cells expressing immune checkpoint molecules. HPV E6/E7-specific T cells were detected in 22 (95.7%) of 23 patients, whereas wild-type p53-specific T cells were detected in 3 (15.0%) of 20 patients. Seven (43.8%) of 16 patients exhibited wild-type p53-specific T-cell responses, as determined using whole proteins instead of peptides. Immune checkpoint blockade enhanced wild-type p53-specific T-cell responses in 9 (45.0%) of 20 patients. Flow cytometric analysis of PBMCs revealed that responders exhibiting enhanced wild-type p53-specific T-cell responses following immune checkpoint blockade had a significantly higher proportion of Ki-67+CD4+ T cells, Ki-67+CD8+ T cells, regulatory T cells, PD-1+CD4+ T cells, and TIM-3+CD4+ T cells than non-responders. Our findings indicate that tumor antigen-specific T cells are present in the peripheral blood of patients with HPV-positive HNSCC. Blockade of checkpoint pathways can enhance T-cell responses in certain patients, probably via activated T cells, Tregs, and/or exhausted CD4+ T cells.

Tumor-targeted therapy with BRAF-inhibitor recruits activated dendritic cells to promote tumor immunity in melanoma

BackgroundTumor-targeted therapy causes impressive tumor regression, but the emergence of resistance limits long-term survival benefits in patients. Little information is available on the role of the myeloid cell network, especially...

Lentinula Edodes Mycelia extract regulates the function of antigen-presenting cells to activate immune cells and prevent tumor-induced deterioration of immune function

Immune cell activation is essential for cancer rejection; however, the tumor microenvironment leads to deterioration of immune function, which enables cancer cells to survive and proliferate. We previously reported that oral ingestion of Lentinula Edodes Mycelia (L.E.M.) extract enhances the tumor antigen-specific T-cell response and exerts an antitumor effect in a tumor-bearing mouse model. In this study, we focused on antigen-presenting cells (APCs) located upstream of the immune system, induced a T-cell response, then examined the impact of L.E.M. extract on the APCs. L.E.M. extract enhanced the expression of MHC-I, MHC-II, CD86, CD80, and CD40 in bone marrow-derived dendritic cells (DCs) and strongly induced the production of IL-12. L.E.M.-stimulated DCs enhanced IFN-γ production from CD8+ T cells and induced their differentiation into effector cells. Furthermore, L.E.M. extract promoted IL-12 production and suppressed the production of IL-10 and TGF-β by transforming bone marrow-derived macrophages into M1-like macrophages. Furthermore, in a B16F10 melanoma inoculation model, DCs in the spleen were decreased and their activation was suppressed by the presence of cancer; however, ingestion of L.E.M. extract prevented this functional deterioration of DCs. In the spleen of cancer-bearing mice, the number of CD11b− F4/80+ macrophages in a hypoactivated state was also increased, whereas L.E.M. extract suppressed the increase of such macrophages. These findings suggest that L.E.M. extract may exhibit an antitumor immune response by regulating the function of APCs to induce cytotoxic T lymphocytes, as well as by suppressing the decline in antigen-presenting cell activity caused by the presence of cancer.

OncoVEXmGM-CSFexpands tumor antigen-specific CD8+ T-cell response in preclinical models

BackgroundCheckpoint inhibitors targeting cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) have demonstrated clinical efficacy in advanced melanoma, but only a...

Role of myeloid-derived suppressor cells in tumor recurrence.

The establishment of primary tumor cells in distant organs, termed metastasis, is the principal cause of cancer mortality and is a crucial therapeutic target in oncology. Thus, it is critical to establish a better understanding of metastatic progression for the future development of improved therapeutic approaches. Indeed, such development requires insight into the timing of tumor cell dissemination and seeding of distant organs resulting in occult lesions. Following dissemination of tumor cells from the primary tumor, they can reside in niches in distant organs for years or decades, following which they can emerge as an overt metastasis. This timeline of metastatic dormancy is regulated by interactions between the tumor, its microenvironment, angiogenesis, and tumor antigen-specific T-cell responses. An improved understanding of the mechanisms and interactions responsible for immune evasion and tumor cell release from dormancy would help identify and aid in the development of novel targeted therapeutics. One such mediator of dormancy is myeloid derived suppressor cells (MDSC), whose number in the peripheral blood (PB) or infiltrating tumors has been associated with cancer stage, grade, patient survival, and metastasis in a broad range of tumor pathologies. Thus, extensive studies have revealed a role for MDSCs in tumor escape from adoptive and innate immune responses, facilitating tumor progression and metastasis; however, few studies have considered their role in dormancy. We have posited that MDSCs may regulate disseminated tumor cells resulting in resurgence of senescent tumor cells. In this review, we discuss clinical studies that address mechanisms of tumor recurrence including from dormancy, the role of MDSCs in their escape from dormancy during recurrence, the development of occult metastases, and the potential for MDSC inhibition as an approach to prolong the survival of patients with advanced malignancies. We stress that assessing the impact of therapies on MDSCs versus other cellular targets is challenging within the multimodality interventions required clinically.

Vanadyl sulfate-enhanced oncolytic virus immunotherapy mediates the antitumor immune response by upregulating the secretion of pro-inflammatory cytokines and chemokines.

Oncolytic viruses (OVs) arepromisinganticancer treatmentsthat specifically replicate in and kill cancer cells and haveprofoundimmunostimulatory effects.We previously reported the potentialofvanadium-based compounds such as vanadyl sulfate(VS) as immunostimulatory enhancers ofOVimmunotherapy.Thesecompounds,in conjunction with RNA-basedOVssuch as oncolytic vesicular stomatitis virus (VSVΔ51),improve viral spread and oncolysis,leadingto long-term antitumor immunity and prolonged survival in resistant tumor models. This effectis associated with a virus-induced antiviral type I IFN response shifting towards a type II IFN response in the presence of vanadium.Here, we investigated the systemic impact of VS+VSVΔ51 combination therapy tounderstand theimmunological mechanism of action leading to improved antitumor responses.VS+VSVΔ51 combination therapy significantly increased the levels of IFN-γ and IL-6, and improved tumor antigen-specific T-cell responses.Supported by immunological profiling and as a proof of concept for the design of more effective therapeutic regimens, we found that local delivery of IL-12 using VSVΔ51 in combination with VS further improved therapeuticoutcomes in a syngeneic CT26WT colon cancer model.

A first-in-human phase 1 trial of nx-1607, a first-in-class oral CBL-B inhibitor, in patients with advanced solid tumor malignancies.

TPS2691 Background: Casitas B-lineage lymphoma proto-oncogene B (CBL-B) is an E3 ubiquitin ligase expressed in multiple immune cell lineages, which in contrast to cell surface immune checkpoints, acts as a regulator of both T and NK cell activation. Inhibition of CBL-B enhances T-cell response, increases response to suboptimal priming, and restores response in exhausted T cells. Thus CBL-B is a promising immune-oncology target and may overcome challenges seen with other T-cell directed therapies. NX-1607 is an oral small molecule inhibitor of CBL-B that has demonstrated anti-tumor activity and long-term survival in murine models as both a single agent and in combination with programmed cell death protein-1 (PD-1) antibodies. Further, NX-1607 elicits dose-dependent increases in cytokine secretion and proliferation in T-cell receptor-stimulated primary human T cells with enhanced tumor antigen-specific T-cell and NK cell anti-tumor responses. Thus, NX-1607 may be effective as a single agent or it may significantly enhance efficacy of other anti-tumor agents. Methods: NX-1607-101 is a first-in-human, multicenter, open-label, Phase 1 dose escalation and expansion trial evaluating NX-1607 in a variety of indications including platinum-resistant epithelial ovarian cancer (EOC), gastric cancer, squamous cell carcinoma of the head and neck (HNSCC), metastatic melanoma, non-small cell lung cancer (NSCLC), metastatic castration-resistant prostate cancer (mCRPC), malignant pleural mesothelioma (MPM), triple-negative breast cancer (TNBC), locally advanced or metastatic urothelial cancer, cervical cancer, microsatellite stable colorectal cancer (MSS CRC), and diffuse large B-cell lymphoma with Richter transformation (DLBCL-RT). The main objective is to establish the safety and tolerability of NX-1607, characterize PK/PD, and determine the recommended Phase 1b dose. NX-1607 will be given orally once daily at doses ranging from 5 to 100 mg in up to 6 dose levels. Dose escalation will proceed using an accelerated modified Fibonacci dose escalation design that transitions to a standard 3 + 3 design based on protocol-specific criteria. Up to 8 expansion cohorts in Phase 1b will be composed of patients with subsets of advanced cancers. Key eligibility criteria include patients with metastatic or unresectable disease that have progressed after prior therapy and for whom standard therapy with proven clinical benefit does not exist, is no longer effective, or is not appropriate. Prior treatment with immune checkpoint inhibitors or CAR-T cells with washout is allowed, but a history of active autoimmune disease is not. Up to 336 patients (60 in Phase 1a, 276 in Phase 1b using a Simon 2-stage design) will be enrolled at approximately 20 sites in the UK and US and treated until disease progression or unacceptable toxicity. Dose escalation is ongoing. Clinical trial information: NCT05107674.

Natural killer cell engagers in cancer immunotherapy: Next generation of immuno‐oncology treatments

Immuno-oncology is revolutionizing the treatment of cancers, by inducing the recognition and elimination of tumor cells by the immune system. Recent advances have focused on generating or unleashing tumor antigen-specific T-cell responses, leading to alternative treatment paradigms for many cancers. Despite these successes, the clinical benefit has been limited to a subset of patients and certain tumor types, highlighting the need for alternative strategies. One innovative approach is to broaden and amplify antitumoral immune responses by targeting innate immunity. Particularly, the aim has been to develop new antibody formats capable of stimulating the antitumor activity of innate immune cells, boosting not only their direct role in tumor elimination, but also their function in eliciting multicellular immune responses ultimately resulting in long-lasting tumor control by adaptive immunity. This review covers the development of a new class of synthetic molecules, natural killer cell engagers (NKCEs), which are built from fragments of monoclonal antibodies (mAbs) and are designed to harness the immune functions of NK cells in cancer. As currently shown in preclinical studies and clinical trials, NKCEs are promising candidates for the next generation of tumor immunotherapies.

CCL5 production by fibroblasts through a local renin-angiotensin system in malignant melanoma affects tumor immune responses.

To enhance the antitumor effects of anti-programmed death-1 (PD-1) antibodies, it is important to reverse cancer-induced immunosuppression. We previously reported that a localized renin-angiotensin system in the tumor microenvironment inhibited tumor immunity via macrophages. In this study, we analyzed the underlying mechanism through which fibroblasts express tumor immunity influenced by the angiotensin receptor. We used an angiotensin receptor inhibitor (ARB) to inhibit renin-angiotensin system. Furthermore, angiotensin receptors were knocked out from mice fibroblasts, which were then collected. The fibroblasts and a malignant melanoma were then transfused into a mouse model and tumor immunity response was analyzed. Fibroblasts produced CC motif chemokine ligand 5 (CCL5) on renin-angiotensin system stimulation, and this production decreased after ARB administration. In mice with transplanted malignant melanoma, ARB administration resulted in decreased CCL5 concentration in the blood, increase in tumor-infiltrating T cells, decrease in regulatory T cells, as well as an increase in tumor antigen-specific T-cell responses. The mice in which the angiotensin receptor knockout fibroblasts and malignant melanoma were transplanted showed a similar decrease in CCL5 concentration and increased tumor antigen-specific T-cell responses. Furthermore, ARB and anti-PD-1 antibody were administered in combination, which resulted in significantly better tumor growth inhibition over monotherapy. Inhibiting renin-angiotensin system restored the therapeutic efficacy of inhibited anti-PD-1 antibodies. Thus, this could be considered a valid approach to enhance the therapeutic efficacy of anti-PD-1 antibodies.

Oncolytic measles virus therapy enhances tumor antigen-specific T-cell responses in patients with multiple myeloma

Oncolytic virus therapy leads to immunogenic death of virus-infected tumor cells and this has been shown in preclinical models to enhance the cytotoxic T-lymphocyte response against tumor-associated antigens (TAAs), leading to killing of uninfected tumor cells. To investigate whether oncolytic virotherapy can increase immune responses to tumor antigens in human subjects, we studied T-cell responses against a panel of known myeloma TAAs using PBMC samples obtained from ten myeloma patients before and after systemic administration of an oncolytic measles virus encoding sodium iodide symporter (MV-NIS). Despite their prior exposures to multiple immunosuppressive antimyeloma treatment regimens, T-cell responses to some of the TAAs were detectable even before measles virotherapy. Measurable baseline T-cell responses against MAGE-C1 and hTERT were present. Furthermore, MV-NIS treatment significantly (P < 0.05) increased T-cell responses against MAGE-C1 and MAGE-A3. Interestingly, one patient who achieved complete remission after MV-NIS therapy had strong baseline T-cell responses both to measles virus proteins and to eight of the ten tested TAAs. Our data demonstrate that oncolytic virotherapy can function as an antigen agnostic vaccine, increasing cytotoxic T-lymphocyte responses against TAAs in patients with multiple myeloma, providing a basis for continued exploration of this modality in combination with immune checkpoint blockade.

Abstract A87: The A2AR antagonist AZD4635 prevents adenosine-mediated immunosuppression in tumor microenvironment and enhances antitumor immunity partly by enhancing CD103+ dendritic cells

Abstract Adenosine signaling via the adenosine 2A receptor (A2AR) is emerging as an important regulatory mechanism of immune responses. Adenosine levels are increased in the tumor microenvironment by the CD39/CD73 axis and adenosine signaling through the adenosine 2A receptor (A2AR) on immune cells elicits a range of immunosuppressive effects that promote tumor growth and limit the efficacy of immune checkpoint inhibitors. We provide evidence that AZD4635, an oral A2AR antagonist, inhibits downstream signaling causing enhanced antigen presentation by CD103+ DCs and increased T-cell activation. In our in vitro assays, NECA (a stable analog of adenosine) caused potent suppression of CD3/CD28 stimulated IFNγ secretion in T cells with 1 μM NECA leading to 80% suppression of IFNγ secretion. Incubation of CD8+ T cells with AZD4635 restored IFNγ secretion back to baseline levels with an observed EC50 of 0.053μM and 0.598 μM at 0.1 and 1 μM NECA, respectively. Treatment of mice bearing CT26 colorectal carcinoma and MC38 colorectal carcinoma with AZD4635 monotherapy led to tumor growth inhibition with (TGI) of 44% and 73%, respectively, and in combination with anti-PD-1 with a TGI of 73% and 91%, respectively. Ex vivo analysis of MC38 OVA tumor-bearing mice showed increase in intratumoral CD8 T cells and CD103+DCs in treated groups compared to control. In particular, combination treatment increased the frequency of OVA antigen specific CD8+ T cells as measured by % of cells expressing the activation marker CD25 (2-fold increase) or the proliferation marker Ki67 (2-fold increase). Aligned with increased tumor antigen-specific T-cell response was a 2-fold increase of intratumoral CD103+ DCs. CD103+DCs play a critical role in tumor antigen trafficking and cross-priming to T cells, which are crucial for successful tumor rejection. In vitro, treatment of CD103+ DCs with NECA suppressed about 50% of pinocytosis (uptake of fluorescent beads) and antigen presentation (OVA peptides), which was almost completely rescued by AZD4635 treatment. We extend these studies into human DCs, and show adenosine promotes a tolerogenic phenotype that can be reversed with AZD4635 restoring antigen presentation and T-cell costimulation by DCs, leading to 50% increase in priming and expansion of antigen-specific T cells compared to adenosine-treated group. Our results support the role of adenosine signaling as an intrinsic negative regulator of CD103+ DC maturation and priming. We show that potent inhibition of A2AR with AZD4635 reduces tumor burden and enhances antitumor immunity. Our preclinical data support the evaluation of AZD4635 as a single agent and in combination with durvalumab (αPD-L1 antibody) in patients with solid malignancies (www.clinicaltrials.gov- NCT02740985, NCT03980821). Citation Format: Dinesh Chandra, Christine M. Barbon, Alexandra Borodovsky, Yanjun Wang, Minwei Ye, Laura Prickett, Kris Sachsenmeier, Wenlin Shao, Carl Barrett, Stephen Fawell, Deanna A. Mele, Alwin Schuller. The A2AR antagonist AZD4635 prevents adenosine-mediated immunosuppression in tumor microenvironment and enhances antitumor immunity partly by enhancing CD103+ dendritic cells [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A87.

Viral and tumor antigen-specific CD8 T-cell responses in Merkel cell carcinoma

Merkel cell carcinoma (MCC) is a rare and aggressive cutaneous cancer, which is immunogenic, regardless of the presence of MCPyV (80% of cases). The identification of MCC-specific epitopes recognized by CD8 T cells is crucial to expand the arsenal of immunotherapeutic treatments. Until now, most efforts focused on the identification of virus-specific epitopes, whereas immune responses directed against shared cellular tumor-specific antigens have not been evidenced. In this study, we measured T-cell responses against viral (n = 3) and tumor antigens (n = 47) from TILs derived from 21 MCC tumors. Virus-specific CD8 T-cell responses dominated MCC-specific immune responses, and we identified two new HLA-peptide complexes derived from the LT antigen, located in a region encompassing 3 previously identified epitopes. Finally, we show that MAGE-A3 antigen, frequently expressed by MCC tumors, was recognized by CD8 TILs from a virus-negative MCC tumor and thus could be a target for immunotherapy in this setting.

High-throughput Screening of Human Tumor Antigen-specific CD4 T Cells, Including Neoantigen-reactive T Cells.

Characterization of tumor antigen-specific CD4 T-cell responses in healthy donors and malignant melanoma patients using an in vitro amplified T-cell library screening procedure. A high-throughput, human leukocyte antigen (HLA)-independent approach was used to estimate at unprecedented high sensitivity level precursor frequencies of tumor antigen- and neoantigen-specific CD4 T cells in healthy donors and patients with cancer. Frequency estimation was combined with isolation and functional characterization of identified tumor-reactive CD4 T-cell clones. In healthy donors, we report frequencies of naïve tumor-associated antigen (TAA)-specific CD4 T cells comparable with those of CD4 T cells specific for infectious agents (Tetanus toxoid). Interestingly, we also identified low, but consistent numbers of memory CD4 T cells specific for several TAAs. In patients with melanoma, low frequencies of circulating TAA-specific CD4 T cells were detected that increased after peptide-based immunotherapy. Such antitumor TAA-specific CD4 T-cell responses were also detectable within the tumor-infiltrated tissues. TAA-specific CD4 T cells in patients displayed a highly polyfunctional state, with partial skewing to Type-2 polarization. Finally, we report the applicability of this approach to the detection and amplification of neoantigen-specific CD4 T cells. This simple, noninvasive, high-throughput screening of tumor- and neoantigen-specific CD4 T cells requires little biologic material, is HLA class II independent and allows the concomitant screening for a large number of tumor antigens of interest, including neoantigens. This approach will facilitate the immunomonitoring of preexisting and therapy-induced CD4 T-cell responses, and accelerate the development of CD4 T-cell-based therapies.

Abstract 3206: Host immunity following near infrared photoimmunotherapy is enhanced with PD-1 checkpoint blockade to eradicate established highly antigenic tumors

Abstract Near infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer treatment that employs a targeted monoclonal antibody-photo-absorber conjugate (APC). Following antibody localization of the APC to a tumor cell surface antigen, NIR light is used to induce highly selective cytolysis. Extensive pre-clinical evidence demonstrates that NIR-PIT is effective at inducing tumor cell lysis using a number of different antibody-APC conjugates and NIR-PIT targeting EGFR with a cetuximab-APC conjugate has shown promising results in phase 2 clinical evaluation for the treatment of recurrent head and neck squamous cell carcinoma. NIR-PIT induces rapid, necrotic cell death that yields innate immune ligands that activate dendritic cells, consistent with immunogenic cell death. Yet, NIR-PIT treatment of syngeneic tumors in wild-type mice has mostly failed to induce durable regression of established tumors, suggesting the presence of one or more mechanisms of resistance to formation of meaningful anti-tumor immunity. In this study, we hypothesized that NIR-PIT could induce anti-tumor immunity that was being restricted by the PD-1/PD-L1 signaling axis, and that PD-1 could reverse innate immune resistance to induce durable, effective anti-tumor immune responses. Using a CD44-targeting APC, we demonstrated the ability of PD-1 immune checkpoint blockade to significantly enhance antigen-specific anti-tumor immunity induced by NIR-PIT in multiple syngeneic tumor models (MC38, LLC, and MOC1 cancer cell line). In two of three models, NIR-PIT monotherapy halted tumor growth, enhanced dendritic cell tumor infiltration, and induced de novo tumor antigen-specific T-cell responses absent at baseline. The addition of PD-1 blockade appeared to reverse adaptive immune resistance, resulting in both enhanced pre-existing tumor antigen-specific T-cell responses and enhanced de novo T-cell responses induced by NIR-PIT. Enhanced immune responses appeared to correlate with shared tumor antigen expression, suggesting that antigenicity is a major determinant of response to combination NIR-PIT and PD-1 blockade. Combination treatment induced complete rejection of MC38 tumors treated with NIR-PIT as well as untreated, distant tumors. Accordingly, tumor antigen-specific T-cell responses were measured in both treated and untreated tumors, validating the development of systemic anti-tumor immunity. Cured mice resisted tumor challenge, indicating the presence of systemic immune memory. Cumulatively these results demonstrate reversal of adaptive immune resistance following induction of innate and adaptive immunity by NIR-PIT, resulting in high rates of tumor rejection and/or significant tumor growth control in highly antigenic syngeneic models of cancer. Citation Format: Tadanobu Nagaya, Jay Friedman, Yasuhiro Maruoka, Fusa Ogata, Shuhei Okuyama, Paul E. Clavijo, Peter L. Choyke, Clint Allen, Hisataka Kobayashi. Host immunity following near infrared photoimmunotherapy is enhanced with PD-1 checkpoint blockade to eradicate established highly antigenic tumors [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 3206.

Host Immunity Following Near-Infrared Photoimmunotherapy Is Enhanced with PD-1 Checkpoint Blockade to Eradicate Established Antigenic Tumors.

Near-infrared photoimmunotherapy (NIR-PIT) induces immunogenic cell death but has mostly failed to induce durable antitumor responses in syngenic tumor mouse models. We hypothesized that adaptive immune resistance could be limiting durable responses after treatmemt with NIR-PIT. We investigated the effects of combining NIR-PIT targeting cell-surface CD44 and PD-1 blockade in multiple syngeneic tumor models. In two of three models, NIR-PIT monotherapy halted tumor growth, enhanced dendritic cell tumor infiltration, and induced de novo tumor antigen-specific T-cell responses absent at baseline. The addition of PD-1 blockade reversed adaptive immune resistance, resulting in both enhanced preexisting tumor antigen-specific T-cell responses and enhanced de novo T-cell responses induced by NIR-PIT. Enhanced immune responses correlated with shared tumor antigen expression, suggesting that antigenicity is a major determinant of response to combination NIR-PIT and PD-1 blockade. Combination treatment induced complete rejection of MC38 tumors treated with NIR-PIT, as well as untreated, distant tumors. Accordingly, tumor antigen-specific T-cell responses were measured in both treated and untreated tumors, validating the development of systemic antitumor immunity. Mice that cleared tumors resisted subsequent tumor challenge, indicating the presence of systemic immune memory. Cumulatively, these results demonstrate reversal of adaptive immune resistance following induction of innate and adaptive immunity by NIR-PIT, resulting in high rates of tumor rejection and/or significant tumor growth control in antigenic syngeneic models of cancer.

Abstract B133: Implantable synthetic immune hydrogel for spatiotemporal modulation of tumor-derived immunosuppression and systemic antitumor immunity

Abstract The development of biomaterial-based immune niches that can shape tumor-induced immunosuppressive factors will play an important role to improve current cancer immunotherapy. Herein, we developed an engineered 3-dimensional porous scaffold for creating synergistic action between myeloid-derived suppressor cell (MDSC)-depleting drugs, which can revert an immunosuppressive tumor microenvironment to one supporting antitumor immunity, and cancer vaccines (whole tumor lysate-based antigens and nano-adjuvants carrying Toll-like receptor 3 agonists), which can induce tumor antigen-specific T-cell responses. The local implantation of the scaffold (called immuneCare-DISC, iCD) as a post-surgical treatment in large established 4T1 breast tumors induced systemic antitumor immunity and prevented relapse and metastasis, resulting in survival of 100%. These therapeutic results were obtained from the depletion of MDSCs by control releasing of gemcitabine and the recruitment/activation of dendritic cells, increased infiltrating of T-cells and enhanced level of IFN-γ production by sustained releasing of cancer vaccine from the iCD. This synthetic immune niche iCD as a spatiotemporal modulation of tumor-induced immunosuppression and cancer vaccine-induced immune stimulation is proposed to generate favorable environment to prevent recurrence and metastasis following the surgical resection. Citation Format: Chanyoung Song, Il Woo Shin, Long Ren, Yong Taik Lim. Implantable synthetic immune hydrogel for spatiotemporal modulation of tumor-derived immunosuppression and systemic antitumor immunity [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 B133.

Abstract B057: Enhancing immunotherapy for triple-negative and HER2+ breast cancer using EpCAM aptamer-siRNA mediated gene knockdown

Abstract Triple-negative (TNBC) and HER2+ breast cancers (BCs) are especially aggressive tumors, which are prone to relapse and metastasize post chemo- or targeted therapy. Immunotherapy provides a promising, but unproven, alternative approach for treating poor-prognosis BCs. BCs have relatively low nonsynonymous mutation rates, suggesting that many BCs will respond poorly to immunotherapy. Novel strategies to increase BC cell immunogenicity and improve tumor-antigen specific T-cell responses are needed to enhance the efficacy of BC immune therapy. We aim to enhance the immunogenicity of breast tumor by taking advantage of the unique strength of EpCAM-aptamer conjugated small interfering RNAs (AsiCs), which can knock down gene expression selectively in EpCAM+ BC cells, to make aggressive BCs visible to T-cells and improve T-cell tumor recruitment and function. EpCAM is a tumor-specific antigen expressed at several logs higher levels on all epithelial cancers and cancer stem cells relative to normal epithelia. The EpCAM aptamer our lab developed binds with high affinity to both mouse and human EpCAM. Using this platform, we investigated the use of EpCAM-AsiCs for BC immune modulation by targeting genes controlling different functional processes: 1) knocking down the nonsense-mediated mRNA decay (NMD) gene UPF2, to elicit tumor neoantigen expression to improve BC tumor recognition by the immune system; 2) knocking down CD47 to promote phagocytosis of cancer cells and enhance cross-presentation of tumor antigens; 3) knocking down DNA repair enzyme PARP1 to produce more DNA damage and consequent genetic mutations, thereby introducing tumor-specific neoantigens to the immune system; 4) knocking down PLK1 which is essential for cell mitosis, and MCL1, which is a critical survival factor in TNBC, to directly kill tumor cells. The increased tumor cell death induced by PLK1 and MCL1 knockdown could potentially promote tumor antigen cross-presentation to CD8+ T-cells. Our data indicate that each of these AsiCs potently silences gene expression within mouse or human BC cell lines in vitro, and efficiently reduce target gene expression in EpCAM+ tumor cells in vivo. When treating mice bearing 4T1E (4T1 cell line with high EpCAM expression) tumors, EpCAM-AsiCs targeting UPF2, CD47, PARP1, PLK1 and MCL1 each significantly suppressed 4T1E tumor growth, increased CD8+ T-cell tumor infiltration, CD8+ T-cell/regulatory T-cells (Treg) ratio, the effector functions and degranulation capacity of CD8+ TILs compared to those of the control group. The capacity of these CD8+ TILs to directly kill tumor cells was also improved. Tumor-associated macrophages (TAMs) from CD47-AsiC treated tumors showed improved phagocytic capacity as determined by in vitro and in vivo phagocytosis assays. CD47-AsiC also increased the ratio of M1 (tumor suppressive) to M2 (tumor promoting) TAMs. Dendritic cells (DCs) from CD47-AsiC treated tumors show enhanced CD40 and CD86 expression, suggesting an improved immunostimulatory capacity. Using antibody depletion assay, we further show that CD4+ T-cells, CD8+ T-cells and macrophages all contribute to the tumor inhibition capacity of CD47-AsiC; and TAMs/DC play an important role in stimulating the functions of CD8+ TILs. In addition, we show that CD47-AsiC outperforms the anti-CD47 antibody in suppressing 4T1E tumor progression. Finally, we show that UPF2, CD47, PARP1, PLK1 and MCL1 AsiCs work in synergy to further delay tumor growth, and even lead to tumor regression, compared to single AsiC treatment. The combination therapy significantly increased the amount of CD8+ TILs, reduced the Tregs and myeloid-derived suppressor cells (MDSCs) in the tumor, and improved the functions of both CD4+ and CD8+ TILs. Our data suggest that these novel EpCAM-AsiCs, with their small size and high selectivity, have great potential to improve therapeutic efficacy and reduce toxicity for BC patients compared to current checkpoint inhibitors. Citation Format: Ying Zhang, Judy Lieberman. Enhancing immunotherapy for triple-negative and HER2+ breast cancer using EpCAM aptamer-siRNA mediated gene knockdown [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 B057.

Mucosal HPV E6/E7 Peptide Vaccination in Combination with Immune Checkpoint Modulation Induces Regression of HPV+ Oral Cancers.

High-risk human papillomavirus (HPV)-associated squamous cell carcinomas of the oropharynx (SCCOP) are among the fastest growing cancers. After standard-of-care treatment, however, patients with HPV+ SCCOP have better overall and disease-specific survival than patients with HPV- SCCOP, suggesting the importance of HPV-specific immunity. We reasoned that therapeutic vaccination targeting the HPV-16 E6 and E7 oncogenes could elicit high-affinity, high-frequency tumor antigen-specific T-cell responses, which could then be augmented and shielded from suppression in the tumor microenvironment by immune checkpoint modulation. In this study, we used a preclinical syngeneic mouse model of oral cancer comprised of mouse tonsil-derived epithelial cells stably expressing HPV-16 E6 and E7 genes along with H-ras oncogene (mEER) to identify combinations of vaccination and checkpoint antibodies capable of promoting tumor regression. Intranasal HPV E6/E7 peptide vaccination and single checkpoint antibodies failed to elicit responses in more than half of animals; however, 4-1BB agonist antibody along with either CD40 agonist antibody or CTLA-4 blockade eliminated the majority of established mEER tumors. The combination of intranasal HPV peptide vaccine and α4-1BB and αCTLA-4 antibodies produced curative efficacy and a better safety profile against orally implanted mEER tumors. Correlates of protective immunity included enhanced intratumoral levels of CD8 T cells relative to immunosuppressive regulatory T cells and myeloid-derived suppressor cells. Overall, our results demonstrate combination vaccine-immunotherapy modalities as novel treatment options for HPV+ SCCOP.Significance: Combinations of vaccine and checkpoint modulation are effective and safe treatment options for HPV+ oral cancers. Cancer Res; 78(18); 5327-39. ©2018 AACR.

Using Antigen-Specific B Cells to Combine Antibody and T Cell–Based Cancer Immunotherapy

Cancer immunotherapy by therapeutic activation of T cells has demonstrated clinical potential. Approaches include checkpoint inhibitors and chimeric antigen receptor T cells. Here, we report the development of an alternative strategy for cellular immunotherapy that combines induction of a tumor-directed T-cell response and antibody secretion without the need for genetic engineering. CD40 ligand stimulation of murine tumor antigen-specific B cells, isolated by antigen-biotin tetramers, resulted in the development of an antigen-presenting phenotype and the induction of a tumor antigen-specific T-cell response. Differentiation of antigen-specific B cells into antibody-secreting plasma cells was achieved by stimulation with IL21, IL4, anti-CD40, and the specific antigen. Combined treatment of tumor-bearing mice with antigen-specific CD40-activated B cells and antigen-specific plasma cells induced a therapeutic antitumor immune response resulting in remission of established tumors. Human CEA or NY-ESO-1-specific B cells were detected in tumor-draining lymph nodes and were able to induce antigen-specific T-cell responses in vitro, indicating that this approach could be translated into clinical applications. Our results describe a technique for the exploitation of B-cell effector functions and provide the rationale for their use in combinatorial cancer immunotherapy. Cancer Immunol Res; 5(9); 730-43. ©2017 AACR.

Highlights of This Issue

Legat and colleagues report a clinical trial in 16 advanced-stage melanoma patients vaccinated with five tumor peptides, Montanide and IMP321/LAG-3Ig, a non-TLR agonist with interesting adjuvant properties including dendritic cell activation. The treatment was well tolerated. Combinatorial immune monitoring revealed multiple tumor antigen-specific CD8 and CD4 T-cell responses in most patients, supporting the further use of these vaccine components for the development of combination immunotherapies.Zabor and colleagues analyzed individual patient tumor data from a phase III clinical trial of vemurafenib versus dacarbazine in melanoma patients to identify criteria for tumor measures that correlated with OS. Correlation between OS and early response was weak no matter how the response was defined; however, a strong correlation was seen between OS and progression when defined as ≥50% increase in sum of tumor diameters or appearance of new tumors. These data suggest that tumor progression is a better surrogate endpoint for OS than tumor response, such as RECIST.Zhuang and colleagues studied whether targeting IL-1a inhibits NF-κB activity and suppresses PDAC cell growth. Previously, studies show that KRASG12V induces expression of IL-1a in genetically engineered mouse models that express mutant KRAS and p53 and in cell lines expressing KRASG12V with p16 knockdown. The authors also observed a correlation between mutant KRAS and IL-1a expression in PDAC patient samples. The experimental evidence suggests a combination of gemcitabine and IL-1 receptor antagonist can reduce PDAC cell growth by inhibiting IL-1a-induced NF-κB activity, thus suggesting a new therapeutic approach for PDAC.Poulain and colleagues analyzed the genomic landscape of CXCR4 somatic mutation in Waldenström macroglobulinemia (WM) combining deep sequencing and SNP array. The study showed existence of intraclonal (variation in coexpression of MYD88 and CXCR4 mutations) and interclonal (mutually exclusive BCR and CXCR4 mutations in MYD88L265P WM) heterogeneity of the molecular spectrum of CXCR4 mutation. Furthermore, CXCR4 mutated WM displayed a specific clinicobiologic and genomic and transcriptomic signature. This dataset further suggests the genomic heterogeneity of WM beyond the current indolent versus symptomatic classification.