CD40 Agonist Research Articles

Abstract 4026: The combination of DLL4/VEGF-a blockade and immunomodulation can eliminate MHC class I negative tumors in mice

Abstract CTX-009 is a bispecific antibody targeting VEGF-A and DLL4 which is undergoing clinical investigation in patients with multiple tumor types, including biliary tract and colon cancers. In biliary tract cancers, checkpoint inhibitor therapy has been shown to improve outcomes when combined with chemotherapy. In an effort to increase the breadth and duration of response of CTX-009, we set out to investigate the combined anti-tumor efficacy of this bispecific antibody and immunotherapy in preclinical models. Here we report the evaluation of the combination of the mouse surrogates of CTX-009 and CTX-471, an agonistic anti-mouse CD137 antibody, or monoclonal antibodies that disrupt the PD-1/PD-L1 signaling axis. Coupling VEGF-A/DLL4 targeting with either CD137 agonism or PD-1/PD-L1 blockade markedly increased the anti-tumor efficacy of the individual treatments alone in multiple syngeneic mouse tumor models. Tumor growth inhibition was accompanied by pharmacodynamic evidence consistent with enhancement of anti-tumor immunity. Notably, the combination of CTX-009 and CTX-471 retained potent anti-tumor activity even in a tumor line (MC38) which was made MHC I deficient through the deletion of the beta 2 microglobulin gene. This system may model the clinically relevant unmet need characterized by tumor escape through the loss of antigen presentation machinery such as HLA LOH. Interestingly, the model cell lines CT26 and MC38 failed to thrive in the B2M-deficient setting and depletion of NK cells markedly increased their ability to grow. This implies that NK cells might be involved in controlling tumor growth in this setting, and that the combined action of CTX-471 and CTX-009 in the tumor stroma enhances NK cell activity. The findings presented here suggest that CTX-009 may provide enhanced clinical benefit when combined with immunomodulatory agents, such as agonistic anti-CD137 antibodies or PD-1/PD-L1 blockers in tumors where immune checkpoint inhibitors have failed. Citation Format: Diana I. Albu, Rachael Duffy, Amy Daniel Ulumben, Jason Kong, Jessica Zolotarevsky, Melissa Brundin, Mei Su, Ruturaj Jadhav, Kris F. Sachsenmeier, Bing Gong, Thomas J. Schuetz, Alberto Visintin. The combination of DLL4/VEGF-a blockade and immunomodulation can eliminate MHC class I negative tumors in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4026.

Abstract 5305: M9657, a tumor-targeted anti-CD137 agonist, induced significant antitumor immunity in combination with anti-PD-1 antibody

Abstract Background: M9657, a first-in-class tumor-targeted conditional immune agonist, was developed to boost antitumor immune responses in the tumor microenvironment (TME) by targeting CD137. The immune agonism potency of M9657 was validated through in vitro assays and in vivo efficacy studies in syngeneic tumor models using the surrogate antibody FS122m. Combining CD137 agonists and antiPD(L)-1 antibodies presents a novel therapeutic strategy to overcome immune checkpoint inhibitor (ICI) resistance and enhance antitumor activity via complementary mechanisms. Anti-PD-(L)-1 antibodies can reverse the suppressive immune signals, and CD137 agonists can boost T cell activation, improving antigen presentation and cytokine release. Methods: The antitumor immunity of M9657 + pembrolizumab (anti-PD-1 antibody) combination therapy was assessed using in-vitro luciferase and ex-vivo functional assays. In vivo antitumor efficacy was investigated with the M9657 surrogate (FS122m) and anti-mPD-1 in syngeneic tumor models expressing Mesothelin (MSLN). Results: The combination of M9657 and pembrolizumab displayed dose-dependent immune agonism potency and significantly enhanced tumor cell cytotoxicity and CD8+ T cell cytokine release relative to single agent treatments. In various syngeneic tumor models, the combination of FS122m with antimPD1 significantly increased antitumor efficacy relative to monotherapies, with more mice achieving complete tumor regression and prolonged median survival. Tumor-cured mice after combination treatment exhibited resistance to the primary tumor rechallenge, indicating the generation of tumor antigen-specific antitumor immunity and long-term immune protectivity. Conclusion: The combination of M9657 with anti-PD-(L)1 could overcome primary ICI therapy resistance and improve the antitumor efficacy of current anti-PD-(L)1 therapy in the clinic. Our findings support a combination strategy to combine M9657 with anti-PD-(L)1 in a clinical setting, potentially leading to enhanced antitumor immunity and improved patient outcomes. Future studies should investigate the safety and efficacy of this combination therapy in human patients. Sponsored by the healthcare business of Merck KGaA, Darmstadt, Germany (CrossRef Funder ID: 10.13039/100009945) Citation Format: Chunxiao Xu, Sireesha Yalavarthi, Hong Zhang, Uma Mahesh Gundra, Clotilde Bourin, Laura Helming. M9657, a tumor-targeted anti-CD137 agonist, induced significant antitumor immunity in combination with anti-PD-1 antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5305.

Abstract 5302: CB699: A novel mesothelin-binding Humabody® CD40 and CD137 dual-agonist for enhancing immune cell responses against MSLN+ tumors

Abstract INTRODUCTION: CD137 is a TNF receptor superfamily (TNFRSF9) costimulatory receptor expressed by T cells and NK cells. Clustering-mediated CD137 signaling enhances immune cell survival, proliferation, cytokine production and memory formation. CD40 (previously TNFRSF5) is a related superfamily member expressed primarily on B cells and antigen-presenting cells (APCs) which upon clustering is also a key modulator of T cell responses. Mesothelin (MSLN) is highly upregulated on tumor cells in ovarian and pancreatic cancer and select other solid tumour histologies. To avoid systemic toxicity, CB699 is designed to be immunologically active in the presence of any two of its three binding targets, thus enhancing both immune-immune and tumour-immune crosstalk. We present here the preclinical pharmacology of CB699. EXPERIMENTAL PROCEDURES: Binding of CB699 to CD40, CD137 or MSLN was measured by surface plasmon resonance and by flow cytometry. CD137 and/or CD40 agonism, immune cell activation and cytokine secretion were evaluated in vitro using reporter gene assays and primary cell coculture assays in the presence/absence of MSLN-expressing tumor cells. The ability of CB699 to enhance tumor cell killing was characterized in 3D spheroid cocultures. CB699 toxicology and toxicokinetics was assessed in a dose range-finding study in cynomolgus macaques. CB699 was also assessed in vivo with a human CD34+ cell engrafted mouse model. RESULTS: CB699 binds to primary and immortalized cells that express CD40, CD137 or MSLN, with low nanomolar affinity. CB699 also binds to cells from primary disaggregated human solid tumors. CB699 induces CD137 reporter cell activity in cocultures containing cells expressing either CD40 or MSLN. Additionally, CB699 induces CD40 reporter cell activity in cocultures containing cells expressing either CD137 or MSLN. CB699 induces IL-2 secretion and upregulation of CD86 on B cells in primary cocultures containing healthy human PBMCs and MSLN expressing cancer cells. Enhanced tumor cell killing was observed in spheroid cultures containing CB699 and was coupled with immune cell expansion. CB699 showed significant pharmacology in vivo with immune cell engrafted mice. Finally, CB699 was well tolerated in a dose range finding non-human primate toxicology study. CONCLUSIONS: CB699 is a novel MSLN-binding CD137 and CD40 dual agonist that selectively enhances immune cell activity by enhancing both T cell and APC signaling axes. CB699 is positioned to enter clinical development. Citation Format: Andrew J. Pierce, Phillip M. Brailey, Sophie Archer, Holly Spencer, Minjung Song, Timothy Wong, Phillip D. Bartlett, Kieran Williams, Lukas Jasaitis, Philip Bland-Ward. CB699: A novel mesothelin-binding Humabody® CD40 and CD137 dual-agonist for enhancing immune cell responses against MSLN+ tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5302.

Abstract 117: Improved PBMC humanized mouse models for immuno-oncology drugs evaluation

Abstract It has been demonstrated that anti-PD(L)1 therapy could significantly improve survival outcomes for patients with metastatic solid cancers, such as melanoma, non-small cell lung cancer and MSI-H colorectal cancer. However, more than half of these patients (NCT02563002, NCT02142738, NCT02563002) do not respond to immune checkpoint blockade (ICB) therapy, and 90% of colorectal cancer patients have MSS/pMMR characteristics and almost no response at all, which drives scientists to design novel drug candidates urgently and integrate tumor and tumor microenvironment (TME) signals systemically to rejuvenate exhausted immune system and overcome ICB resistance. LIDE has developed both traditional PBMC humanized mice model, and human immune cells and cancer co-inoculated model for IO drugs functional evaluation in the last 5-years by using unique PBMC donor selection and standard cancer biobank (PDX patient-derived xenografts, CDX cell derived xenograft) establishment. Cancer biopsies with MDM2 amplification or DNMT3A alternation have been shown resistant to PD1-Ab therapy but sensitive to chemical combo-therapy, and pMMR cancer PDX models seem responsible well to EGFR targeted, CD39-CD73-Adenosine targeted therapy. Human IL15 transgenic mice are used as one useful tool to investigate in vivo function of NK cells, NK engagers/modulators as well as drugs with ADCC (Antibody dependent cell-mediated cytotoxicity) effect. The activated immune cells and cancer co-transfer model, can significantly delay the graft-versus-host-reaction for another month compared with traditional PBMC humanized model, and archives maximal therapeutic effect of the testing drugs by pre-activating T cells with specific tumor antigens. Dendritic cells or macrophages could be successfully introduced into this co-inoculated system to work together with T cells, mimic human tumor tissue complexity, to help determine the overall effect induced by integrated signals. It seems CD40 agonist or CD47 antagonist could well synergies with anti-PD(L)1 to control melanoma and triple negative breast cancer development, respectively. This optimized PBMC humanized cancer-bearing mice models have been tested for various cancer targets, e.g. PD(L)1, Tim3, Lag3, Tigit, PVRIG, TGFβ1, CD73/CD39, CD47/Sirpa, DLL3/CD3, Claudin18.2/4-1BB, B7-H3, GITR, to better understand the interaction between cancer cells and immune cells. Citation Format: Bin Xie, Xin Hou, Pengfei Yang, Yanbing Zhou, Danyi Wen. Improved PBMC humanized mouse models for immuno-oncology drugs evaluation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 117.

Abstract 5310: KK2269, an epithelial cell adhesion molecule-targeted CD40 agonist, demonstrates antitumor effects in combination with standard therapies for NSCLC

Abstract CD40 is a key molecule expressed on antigen presenting cells (APCs), whose signaling activates APCs and generates antitumor immune responses. Although CD40 agonists have been investigated in clinical trials, systemic toxicities have limited their full potential in providing adequate antitumor effects. To overcome this limitation, we developed KK2269, a bispecific antibody, using REGULGENT™ technology. KK2269 binds to CD40 and tumor-expressed epithelial cell adhesion molecule (EpCAM) to allow activation of CD40+ APCs only at EpCAM+ tumor sites, thus avoiding systemic toxicity. EpCAM is overexpressed in many types of epithelial cancers, including non-small cell lung cancer (NSCLC). KK2269 exerts synergistic antitumor activity and tumor-specific immune responses in combination with docetaxel, a common chemotherapy for NSCLC, in an EpCAM-expressing tumor mouse model. Hence, we aimed to evaluate KK2269 for the treatment of NSCLC. Docetaxel is used as a combination therapy with ramucirumab, an anti-VEGF receptor 2 (VEGFR2) antibody, for previously treated advanced NSCLC. In addition, many clinical studies are evaluating the add-on effects of PD-1/PD-L1 blockade combined with standard chemotherapies (including docetaxel) in NSCLC, which may become the standard of care for advanced NSCLC in the future. Therefore, we investigated the antitumor effect of KK2269 in combination with docetaxel + anti-mouse VEGFR2 (study 1) and with docetaxel + anti-mouse PD-1 (study 2) using a mouse EpCAM-expressing B16F10 tumor-bearing human CD40 transgenic mouse model. The prolongation of TTP5X (the day on which the tumor volume is observed to be ≥5-fold of that observed on day 0) was evaluated. In study 1, the median TTP5X (95% confidence interval [CI]) with the control, docetaxel + anti-VEGFR2, and triple combination (KK2269, docetaxel, anti-VEGFR2) was 10.0 days (7.0, 10.0), 26.0 days (17.0, 28.0), and 52.0 days (42.0, not calculable), respectively. The TTP5X was significantly prolonged with the triple combination compared with the control (p=0.000007, log-rank test) and docetaxel + anti-VEGFR2 (p=0.000004, log-rank test). In study 2, the median TTP5X (95% CI) was 7.0 days (not calculable), 9.0 days (6.0, 11.0), 16.0 days (7.0, 18.0), and 26.5 days (7.0, 32.0) with the control, KK2269, docetaxel + anti-PD-1, and triple combination (KK2269, docetaxel, anti-PD-1), respectively. TTP5X was significantly prolonged with the triple combination compared with the control, KK2269, and docetaxel + anti-PD-1 (p<0.0001, p=0.0003, and p=0.0023, respectively, log-rank test). Thus, a significant antitumor effect was demonstrated when KK2269 was added to the standard treatment of NSCLC (docetaxel + anti-VEGFR2 antibody) and to docetaxel + anti-PD-1 antibody. Our findings strongly suggest that KK2269 provides improved treatment options for patients with NSCLC. Citation Format: Yuta Tezuka, Kyoko Kawasaki, Yoshiki Sumitomo, Masato Saito, Akari Yao, Munetoshi Ando. KK2269, an epithelial cell adhesion molecule-targeted CD40 agonist, demonstrates antitumor effects in combination with standard therapies for NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5310.

Abstract 2465: A novel agonist of CD137 as a single agent shows cancer immunoprevention efficacy against various tumor types that is not dependent on the trained immunity

Abstract Introduction: We recently demonstrated that pretreatment of mice with SA-4-1BBL, a novel oligomeric form of CD137 agonist, as a single agent prevents the development of various tumor types in mice. The tumor immunoprevention efficacy is long-lasting (>14 weeks) and contingent upon CD4+ immune cells, NK cells, and IFN-γ, but not CD8+ T and B cells. Trained immunity mediated by previously activated myeloid cells has been shown to have efficacy against cancer. The objective of this study was twofold: i) assess the role of trained immunity and ii) elucidate the mechanistic underlying of cancer immunoprevention. Study Design: Wild-type C57BL/6 mice and various transgenic (CD4+ OT-II.Rag+/+, CD4+ OT-II.Rag−/−, CD8+ OT-I/Rag2−/- KO, and Rag1−/−) mice on C57BL/6 background were pretreated subcutaneously with SA-4-1BBL or an agonistic Ab that does not have immunoprevention efficacy (3H3) twice two weeks apart. Mice were challenged two weeks later with a cervical cancer cell line (TC-1) and monitored for tumor development. A group of mice was also treated with monophosphoryl lipid A (MPLA), an agonist of TLR4 involved in trained immunity, using the above scheme. Mice treated with saline served as the control throughout all studies. Deep immunophenotyping was conducted to assess immune correlates of cancer immunoprevention. Results: Pretreatment with SA-4-1BBL prevented tumor growth in wild-type mice (> 60%), whereas all MPLA treated mice developed tumor. SA-4-1BBL did not show tumor immunoprevention efficacy in mice lacking adaptive immune cells (Rag1−/- KO), OT-II cells on Rag−/- or Rag+/+ background, and OT-I on Rag2−/− background. Deep immunophenotyping revealed a significant increase in the absolute number of CD4+ T central memory and CD4+ T stem cell-like memory cells, NK cells, NKT cells, and various myeloid cells in mice treated with SA-4-1BBL as compared with those treated with the 3H3 Ab or saline treated controls. Conclusions: These results demonstrate that cancer immunoprevention efficacy of SA-4-1BBL requires a diverse T cell receptor repertoire as OT-II mice on Rag+/+ background, restricted to a TCRβ chain, but have all other myeloid and NK cells, do not show efficacy. Furthermore, treatment with MPLA involved in trained immunity has no efficacy. Altogether, these observations do not implicate trained immunity as a mechanism of cancer immunoprevention. Elucidation of the mechanistic basis of SA-4-1BBL immunoprevention may have significant clinical implications. Citation Format: Feyza Nur Arguc, Mohammad Tarique, Vahap Ulker, Esma S. Yolcu, Haval Shirwan. A novel agonist of CD137 as a single agent shows cancer immunoprevention efficacy against various tumor types that is not dependent on the trained immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2465.

Abstract 6597: CD40 agonism induces CD4 T cell mediated rejection of major histocompatibility complex class I deficient pancreatic tumors

Abstract Cytotoxic CD8 T cells are the canonical mediators of tumor clearance by the adaptive immune system. As such, current immunotherapies, including immune checkpoint blockade (ICB), target exhausted CD8 T cells with the hope of reinvigorating the CD8 T cells to drive tumor clearance. However, many tumors, including pancreatic ductal adenocarcinoma (PDAC), can evade CD8 T cell detection via downregulation of the antigen presentation molecule, major histocompatibility complex I (MHC I) as either primary or acquired resistance to immunotherapy. We have previously reported that agonistic CD40 antibody (αCD40), which promotes the maturation and licensing of dendritic cells to drive T cell priming, synergizes with ICB to induce tumor rejection dependent on both CD4 and CD8 T cells in mouse models of PDAC. Given the potential mechanisms of resistance to CD8 T cell immunity, we hypothesized that interrogation of the mechanisms underlying αCD40-induced CD4 T cell rejection of PDAC will reveal targets for improving CD4 T cell-directed immunotherapies. Here, we show that implantation of an MHC I deficient PDAC cell line derived from the genetically engineered KrasG12DTrp53R172HPdx1-Cre (KPC) mouse model results in normal tumor establishment and progression. Upon treatment with combined αCD40 and ICB, tumor growth was significantly decreased in a CD4, but not CD8, T cell-dependent manner. Using genetic knockout mice, we determined that tumor regression was perforin-independent and identified host expression of IFNγ as requisite for therapeutic efficacy. To identify the cellular subset mediating CD4 T cell-rejection of the tumor, we assessed the major histocompatibility complex II (MHC II) positive cells in the tumor microenvironment (TME), which possess the ability to directly synapse with CD4 T cells to drive anti-tumorigenic T cell programs. We determined that macrophage and myeloid cells were dispensable for treatment efficacy, however non-immune cells in the TME can also interact with CD4 T cells through expression of MHC II. Interrogation of stromal cells reveals an increase in the proportion of MHC II+ subsets after combined αCD40/ICB treatment, with a concomitant decrease in the proportion of MHC II+ tumor cells. These data support the notion that CD4 T cell control of tumors may be mediated by both direct and indirect mechanisms of tumor killing. Findings from this work will elucidate non-canonical mechanisms of CD4 T cell-mediated rejection of tumors and may be leveraged to discover novel translational targets for immunotherapy. Citation Format: Margaret E. Haerr, Samuel I. Kim, Charu Arora, Kyle P. Gribbin, Robert H. Vonderheide, Katelyn T. Byrne. CD40 agonism induces CD4 T cell mediated rejection of major histocompatibility complex class I deficient pancreatic tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6597.

Abstract 2376: Mitazalimab, a potent CD40 agonist in combination with FOLFIRINOX demonstrates changes consistent with increased immune activation in TME and peripheral blood in a preclinical pancreatic cancer tumor model

Abstract Mitazalimab is a human CD40 FcγR-conditional agonistic IgG1 antibody developed for cancer immunotherapy. Immune-excluded tumors, such as pancreatic ductal adenocarcinoma (PDAC), are defined by low infiltration of immune cells. In PDAC, effector CD8+ T cells are excluded from the tumor by its desmoplastic stroma which surrounds cancer cell islets and hosts immunosuppressive macrophages. CD40 is expressed on key myeloid cell populations and targeting this receptor has a two-fold mode of action. First, mitazalimab activates macrophages, skewing them towards a tumoricidal phenotype that can potentially reverse the suppressive tumor microenvironment found in PDAC and sensitizes the tumor for chemotherapy by stromal degradation. Second, mitazalimab activates antigen-presenting cells, resulting in priming and expansion of tumor-specific T cells with the potential to generate an effective immune response that could provide immunological memory resulting in long term benefits to patients. Mitazalimab is currently being investigated in a phase 2 study in patients with metastatic PDAC (OPTIMIZE-1) in combination with modified FOLFIRINOX (NCT04888312). The treatment starts with a dose of mitazalimab administered one week prior to chemotherapy in the first cycle to leverage the mode of action and increase the efficacy of the combination therapy. Interim results of the study demonstrated encouraging anti-tumor activity with good durability of responses. To study the changes induced by mitazalimab and FOLFIRINOX in the tumor microenvironment, we evaluated the combination in human CD40 transgenic mice using the KPCY pancreatic tumor model. Tumor, tumor draining lymph nodes, blood and spleen were analyzed for changes in the immune compartment using multiplex assays at different time points. The results showed distinct immunophenotypical changes induced by mitazalimab, FOLFIRINOX and the combination. The combination of mitazalimab and FOLFIRINOX resulted in an increased tumoricidal immune profile, including a marked increase in effector CD8+ T cells compared to the individual therapies. Overall, these preclinical data support the chosen dosing regimen of mitazalimab and modified FOLFIRINOX in the OPTIMIZE-1study and underline the synergistic potential of this combination for an improved antitumor response. Citation Format: Karin Enell Smith, David Gomez Jimenez, Mia Thagesson, Anneli Nilsson, Yago Pico de Coaña, Sumeet Ambarkhane, Peter Ellmark. Mitazalimab, a potent CD40 agonist in combination with FOLFIRINOX demonstrates changes consistent with increased immune activation in TME and peripheral blood in a preclinical pancreatic cancer tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2376.

Abstract 1543: 40-color spectral flow cytometry for comprehensive immunophenotyping of murine cancers

Abstract Introduction: High-dimensional immunoprofiling is essential for studying host response to cancer immunotherapy. Synergistic agents often induce systemic changes in leukocyte abundance/function due to targeting, dosage, or delivery, necessitating multi-tissue analyses to decipher immune response mechanisms. We set out to dissect leukocyte composition in lymphoid tissues and tumors by using spectral cytometry, a multiplexed, high-throughput technology. Methods: We engineered a 40-color spectral flow cytometry panel to immunophenotype all major leukocytes and their functional subsets. Naïve and tumor-burdened C57BL/6 mice treated with combinations of agonist CD40 (aCD40) and checkpoint inhibitors were sacrificed, and tissues were processed. Stained cells were acquired on a 5-laser CyTEK Aurora. Unmixing accuracy and resolution was validated via 780 NxN plots, and high-dimensional analysis was performed. Results: Spectral cytometry affords resolution of 43+ parameters compared to 26 or less in conventional systems. We first designed a 25-marker backbone panel to identify all immune lineages. Additional channels were assigned to functional markers, allowing for profiling of migration, maturation, and activation in tumor-infiltrating leukocytes (TILs). In naïve secondary lymphoid tissues (SLTs), 35 distinct CD45+ populations were resolved. Germinal center B cells (17% vs. ~0.3% of CD45+ in other SLTs) and T follicular cells (31% vs. ~1-5% of CD4+ T cells in other SLTs) were mostly found in the Peyer’s patches, representing a hypothesized model tissue for studying tertiary immune structures in tumors. Peripheral blood contained the highest frequency of NK cells (4%), monocytes (6%), basophils (0.3%), eosinophils (4%), and neutrophils (7%) as a percentage of CD45+ cells. The spleen held the largest reservoir of CD4/8 central/effector memory cells (5% of CD45+) while ~80% of all T cells in the lymph nodes were naïve. In primary lymphoid tissues (PLTs), 12 distinct thymocyte and 7 B cell populations were resolved. Wanderlust trajectory analysis revealed CD24 and CD127 as key developmental regulators in lymphocyte differentiation. Autofluorescence extraction was essential for myeloid phenotyping. With this approach, we found monocytes comprised ~60% of TILs following aCD40 treatment (~8% in non-treatment and checkpoint only controls) and had altered macrophage differentiation fates. aCD40-treated tumors also contained higher frequencies of activated CD40/80/86+ DCs (~80/86/60% vs. ~60/51/50% of DCs in NTCs). Conclusion: This work describes a novel tool that efficiently resolves leukocytes on the axes of time, tissue, and treatment. Applications range from serial monitoring of circulating blood, endpoint analysis, validation of multiplexed-IHC, and cell sorting. PLT and SLT data has been made publicly available to serve as a training tool and reference proteomic atlas in future work. Citation Format: Aris John Kare, Lisa Nichols, Ricardo Zermeno, Marina N. Raie, Spencer K. Tumbale, Katherine W. Ferrara. 40-color spectral flow cytometry for comprehensive immunophenotyping of murine cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1543.

Abstract 5312: Agonist CD40 engineered smart immunosomes induced anti-tumor immunity, reduced associated toxicities and showed tumor free survival in murine glioblastoma model

Abstract CD40 agonist antibodies (αCD40) have proven great potential as an anti-cancer immunotherapeutic by showing promising anti-tumor response in both preclinical and early clinical studies. However, it has been observed that the systemic administration of αCD40, even at low dose, is associated with serious immune- and hepato- toxicities. In addition, αCD40 showed low tumor retention and induced PD-L1 expression which makes tumor microenvironment (TME) immunosuppressive. To overcome these issues, in this study, we have developed an immunosome where αCD40 is conjugated on the surface and VJ23, a small molecule immune-modulator was encapsulated inside it . Immunosome showed higher tumor accumulation till 96 hr of administration and displayed sustained release of αCD40 in vivo. Immunosome significantly delayed tumor growth and showed tumor free survival in mice bearing GL-261 glioblastoma tumor by increasing the population of CD45+CD8+ T cells, CD45+CD20+ B cells, CD45+CD11c+ DCs and F4/80+CD86+M1 macrophage in TME. In addition, Immunosome significantly reduced the population of T-regulatory cells, M2 macrophage, and MDSCs and lowered the expression of PD-L1 to modulate immunosuppressive TME. Moreover, Immunosomes significantly enhanced the level of pro-inflammatory cytokines (IFN-γ, IL-6, IL-2) and lowered anti-inflammatory cytokines (IL-4 and IL-10) level which supported anti-tumor response. Most interestingly, Immunosomes averted the in vivo toxicities associated with free αCD40 by lowering the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), IL-6, IL-1α and reduced the degree of liver damage. Our results suggested that this novel formulation can be further explored in clinics to improve the in vivo anti-tumor efficacy of αCD40 while reducing the associated toxicities. Citation Format: Vidit Gaur, Witty Tyagi, Sanjeev Das, Surajit Ganguly, Jayanta Bhattacharyya. Agonist CD40 engineered smart immunosomes induced anti-tumor immunity, reduced associated toxicities and showed tumor free survival in murine glioblastoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5312.

Machine Learning Links T-cell Function and Spatial Localization to Neoadjuvant Immunotherapy and Clinical Outcome in Pancreatic Cancer.

Tumor molecular data sets are becoming increasingly complex, making it nearly impossible for humans alone to effectively analyze them. Here, we demonstrate the power of using machine learning (ML) to analyze a single-cell, spatial, and highly multiplexed proteomic data set from human pancreatic cancer and reveal underlying biological mechanisms that may contribute to clinical outcomes. We designed a multiplex immunohistochemistry antibody panel to compare T-cell functionality and spatial localization in resected tumors from treatment-naïve patients with localized pancreatic ductal adenocarcinoma (PDAC) with resected tumors from a second cohort of patients treated with neoadjuvant agonistic CD40 (anti-CD40) monoclonal antibody therapy. In total, nearly 2.5 million cells from 306 tissue regions collected from 29 patients across both cohorts were assayed, and over 1,000 tumor microenvironment (TME) features were quantified. We then trained ML models to accurately predict anti-CD40 treatment status and disease-free survival (DFS) following anti-CD40 therapy based on TME features. Through downstream interpretation of the ML models' predictions, we found anti-CD40 therapy reduced canonical aspects of T-cell exhaustion within the TME, as compared with treatment-naïve TMEs. Using automated clustering approaches, we found improved DFS following anti-CD40 therapy correlated with an increased presence of CD44+CD4+ Th1 cells located specifically within cellular neighborhoods characterized by increased T-cell proliferation, antigen experience, and cytotoxicity in immune aggregates. Overall, our results demonstrate the utility of ML in molecular cancer immunology applications, highlight the impact of anti-CD40 therapy on T cells within the TME, and identify potential candidate biomarkers of DFS for anti-CD40-treated patients with PDAC.

Systemic immune response to a CD40 agonist antibody in nonhuman primates.

The cell surface molecule CD40 is a member of the tumor necrosis factor receptor superfamily and is broadly expressed by immune cells including B cells, dendritic cells, and monocytes, as well as other normal cells and some malignant cells. CD40 is constitutively expressed on antigen-presenting cells, and ligation promotes functional maturation, leading to an increase in antigen presentation and cytokine production, and a subsequent increase in the activation of antigen-specific T cells. It is postulated that CD40 agonists can mediate both T cell-dependent and T cell-independent immune mechanisms of tumor regression in mice and patients. In addition, it is believed that CD40 activation also promotes apoptotic death of tumor cells and that the presence of the molecule on the surface of cancer cells is an important factor in the generation of tumor-specific T cell responses that contribute to tumor cell elimination. Notably, CD40 agonistic therapies were evaluated in patients with solid tumors and hematologic malignancies with reported success as a single agent. Preclinical studies have shown that subcutaneous administration of CD40 agonistic antibodies reduces systemic toxicity and elicits a stronger and localized pharmacodynamic response. Two independent studies in cynomolgus macaque (Macaca fascicularis) were performed to further evaluate potentially immunotoxicological effects associated with drug-induced adverse events seen in human subjects. Studies conducted in monkeys showed that when selicrelumab is administered at doses currently used in clinical trial patients, via subcutaneous injection, it is safe and effective at stimulating a systemic immune response.

Neoadjuvant CD40 Agonism Remodels the Tumor Immune Microenvironment in Locally Advanced Esophageal/Gastroesophageal Junction Cancer.

Sotigalimab is an agonistic anti-CD40 mAb that can modulate antitumor immune responses. In a phase II clinical trial of sotigalimab combined with neoadjuvant chemoradiation (CRT) in locally advanced esophageal/gastroesophageal junction (E/GEJ) cancer with the primary outcome of efficacy as measured by pathologic complete response (pCR) rate, the combination induced pCR in 38% of treated patients. We investigated the mechanism of action of sotigalimab in samples obtained from this clinical trial. Tumor biopsies and peripheral blood samples were collected at baseline, following an initial dose of sotigalimab, and at the time of surgery after CRT completion from six patients. High dimensional single-cell techniques were used, including combined single-cell RNA-sequencing and proteomics (CITEseq) and multiplexed ion beam imaging, to analyze immune responses. Sotigalimab dramatically remodeled the immune compartment in the periphery and within the tumor microenvironment (TME), increasing expression of molecules related to antigen processing and presentation and altering metabolic pathways in myeloid cells. Concomitant with these changes in myeloid cells, sotigalimab treatment primed new T cell clonotypes and increased the density and activation of T cells with enhanced cytotoxic function. Sotigalimab treatment also induced a decrease in the frequency of Tregs in the TME. These findings indicate that a single dose of sotigalimab leads to enhanced antigen presentation that can activate T cells and induce new T cell clones. This restructuring of the TME provides elements which are critical to the development of effective antitumor immune responses and improved clinical outcomes.

Expanding immunotherapy options in cholangiocarcinoma: Role of CD27 agonist in combination with PD-L1 and MEK inhibition on antitumor effect and CD8+ T cells.

518 Background: Despite addition of PD-L1 therapy to unresectable cholangiocarcinoma (CCA) standard of care, there remains an unmet need for novel therapies. Our recent phase II trial of dual PD-L1 inhibition (PD-L1i) and MEK inhibition (MEKi) significantly improved progression-free survival (PFS). While MEKi enhances tumor antigen presentation and T cell tumor infiltration, it also impairs T cell priming and activation with notable CD27 upregulation. Our work reveals that CD27 agonists (CD27Ag) foster T cell stem-like and resident memory phenotypes, negating inhibitory effect of MEKi on CD8+ T cells. We hypothesized that addition of a CD27Ag to PD-L1i/MEKi combination therapy would rescue T cell activation and improve antitumor efficacy. Methods: Immune cells from pmel-1 mice were isolated, activated with gp100, and cultured in vitro with 100 IU/mL IL-2 and single, dual, or triple therapy with CD27Ag, cobimetinib (MEKi) and/or αPD-L1. Day 3 flow cytometry characterized CD8+ T cell phenotype and cytokine expression. Additionally, syngeneic murine CCA cell line URCCA4.3 (KrasG21D, tp53-/-) was injected into C57BL/6 mice, subcutaneously or intrahepatic, followed by ~3 weeks of treatment with mono, dual, or triple therapy. For CD8 depletion studies, CD8-depleting antibody was given on the day prior to and following surgery and every 3-4 days afterwards. Tumor and tumor-draining lymph nodes were harvested at study endpoint. Lymphocytes were characterized by flow cytometry. Results: MEKi, alone or with αPD-L1, reduced frequency of activated T cells in vitro (dose-dependent effect on CD69+). CD27Ag combined with MEKi maintained prominent T cell activation. Triple therapy PD-L1i/MEKi/CD27Ag significantly reduced tumor growth rate in mice versus mono or dual therapies (p<0.05). Tumor-infiltrating lymphocytes comprised a stem-like (Tcf-1+), effector memory (CD44+CD62L-), and tissue resident memory (CD103+CD69+) phenotype in triple therapy versus single or dual therapy (p<0.01, p<0.0001, p<0.0001 respectively). All groups had similar numbers of CD8+ stem-like cells (Tcf-1+Tim-3-) in draining lymph nodes, but these cells were only present in tumor from the triple therapy arm (p<0.05). Cell depletion studies showed that CD8+ T cells were required for efficacy of triple therapy in vivo. Finally, an aggressive orthotopic liver tumor model showed improved median survival in triple therapy mice versus control (37.5 days versus 29.5 days, p<0.01). Conclusions: CD27 agonism prevents the impairment of T cell activation that was mediated by MEKi. Moreover, this CD27Ag/MEKi/PDL1i triple therapy bolstered trafficking of T cells with stem-like, effector, or resident memory properties, in turn enhancing the antitumor response. This therapy is being evaluated in metastatic CCA patients in an ongoing Phase II clinical trial.

Abstract C038: Interim pharmacodynamic analyses of mitazalimab in combination with FOLFIRINOX in first-line metastatic pancreatic ductal adenocarcinoma (mPDAC) identify CD4 effector T cells as a correlate of treatment outcomes

Abstract CD40 agonists are being investigated in combination with standard of care chemotherapy as an approach to trigger anti-tumor immunity and improve outcomes for patients with cancer. Preclinical models suggest the importance of appropriate sequencing of chemotherapy and a CD40 agonist. Herein, we report interim pharmacodynamic analyses and their association with outcomes from a Phase 2 study evaluating mitazalimab, a CD40 agonist, in combination with modified (m) FOLFIRINOX chemotherapy as first line therapy in patients with metastatic pancreatic ductal adenocarcinoma (mPDAC). Patients with newly diagnosed mPDAC were treated with mitazalimab on day 1 prior to beginning an every 2-week regimen on day 8 of mFOLFIRINOX followed by mitazalimab 48 hours later. Tumor response was determined by RECIST v1.1 imaging. Peripheral blood was analyzed for selected cytokines and chemokines and by flow cytometry to assess changes in leukocyte subsets including B cells, monocytes, T cells, and NK cells. The neutrophil-to-lymphocyte ratio was determined from complete blood counts. The analysis included 23 patients with associated response data and indicated a mitazalimab-specific contribution to tumor response. Mitazalimab triggered an expected immune response characterized by transient cytokine (IL-8, IP-10, MCP-1, and MIP1b) release and B cell depletion. Chemotherapy provoked a reduction in classical monocytes and proliferating CD4+ T cells. Both mitazalimab and chemotherapy caused a transient reduction in circulating dendritic cells and NK cells. Tumor response was associated with an expansion in the frequency of effector CD4 T cells (p < 0.0001) at day 8 after receiving mitazalimab but did not correlate with neutrophil-to-lymphocyte ratio (p = 0.851). In conclusion, mitazalimab and FOLFIRINOX modulate immune responses in patients with mPDAC in different ways. Interim results of pharmacological analyses identify CD4 effector T cells as a correlate of treatment outcomes suggesting the contribution of mitazalimab to tumor responses. Citation Format: Max M. Wattenberg, Karin Enell Smith, Yago Pico de Coana, David Gomez Jimenez, Malin Carlsson, Sumeet Vijay Ambarkhane, Peter Ellmark, Gregory L. Beatty. Interim pharmacodynamic analyses of mitazalimab in combination with FOLFIRINOX in first-line metastatic pancreatic ductal adenocarcinoma (mPDAC) identify CD4 effector T cells as a correlate of treatment outcomes [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C038.

Abstract PR06: CD40 agonist mitazalimab in combination with mFOLFIRINOX in patients with metastatic pancreatic ductal adenocarcinoma (mPDAC): Interim efficacy results of the OPTIMIZE-1 phase 1b/2 study

Abstract Mitazalimab is a human CD40 agonistic IgG1antibody. Targeting CD40 kickstarts the cancer immunity cycle by licensing dendritic cells leading to tumor specific T cell priming and activation. Furthermore, in PDAC CD40 agonists on myeloid cells promote degradation of the desmoplastic tumor stroma, improving influx of T cells and sensitizing the tumor to chemotherapeutic agents. OPTIMIZE-1 (NCT04888312) is a Phase 1b/2, open label, multicenter study designed to evaluate safety, tolerability, and efficacy of mitazalimab in combination with mFOLFIRINOX (Oxaliplatin 85 mg/m2, leucovorin 400 mg/m2, irinotecan 150 mg/m2, 5-FU 2.4 g/m2) in patients with previously untreated mPDAC. In the first part of the study (phase 1b), 900 µg/kg mitazalimab was selected as the recommended phase 2 dose (RP2D). In the first 21-day treatment cycle, mitazalimab is administered IV at RP2D on day 1 and 10 and mFOLFIRINOX infusion started on day 8. In subsequent cycles, treatment follows a 14-day cycle where mitazalimab is administered 2 days after mFOLFIRINOX. The primary endpoint of this ongoing study is overall response rate (ORR) per RECIST v1.1. Secondary and exploratory endpoints include duration of response, progression free and overall survival, safety, PK and PD biomarker assessments. As of May 24, 2023, study enrolment was complete, 65 patients were treated with mFOLFIRINOX and mitazalimab 900 µg/kg (RP2D). Interim efficacy analysis comprised of 57 evaluable patients. Median follow up was 6.5 months and median exposure to treatment was 5 months. 28 patients (49%) remain on treatment, 18 (32%) continued treatment beyond 6 months, the longest being 17 months (ongoing). ORR was 43.9% (25 patients with partial response), an additional 19 patients achieved stable disease, resulting in a 77.2% disease control rate. Median time to response was 2.2 months and median duration of response was 8.7 months. The most common grade (Gr) ≥3 TEAEs were neutropenia (21.5%), anemia (12.3%), thrombocytopenia (12.3%) and hypokalemia (12.3%). Mitazalimab related Gr >3 AEs occurring in more than one patient were fatigue in 3 (4.6%) patients, increased ALT in 3 (4.6%) patients, diarrhea in 2 (3.1%) patients, asthenia in 2 (3.1%) patients, increased AST in 2 (3.1%) patients, and hypokalemia in 2 (3.1%) patients. 25 patients (38.5%) experienced infusion reactions (all Gr 1-2, managed with symptomatic treatment, none amounting to cytokine release syndrome) and 7 (10.8%) patients discontinued treatment due to TEAEs (pneumonia, gastric obstruction, neuropathy, bacteremia, ileal obstruction, stroke, skin reaction). In this ongoing phase 1b/2 trial, 900 µg/kg mitazalimab has shown no evidence of additive toxicity when administered in combination with mFOLFIRINOX, allowing extended treatment until progression. Interim efficacy results demonstrate encouraging antitumor activity in mPDAC with good durability of responses, meriting continued development. Citation Format: Jean-Luc Van Laethem, Karen P. Geboes, Philippe A. Cassier, Ivan Borbath, Aurélien Lambert, Emmanuel Mitry, Hans Prenen, Jean-Frédéric Blanc, Lorenzo Pilla, Mercedes Rodriguez Garrote, Roberto A. Pazo Cid, Inmaculada Gallego, Jaime Feliú, Karin Nordbladh, Peter Ellmark, Malin Carlsson, Yago Pico de Coaña, Sumeet V. Ambarkhane, Teresa Macarulla. CD40 agonist mitazalimab in combination with mFOLFIRINOX in patients with metastatic pancreatic ductal adenocarcinoma (mPDAC): Interim efficacy results of the OPTIMIZE-1 phase 1b/2 study [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr PR06.

Abstract B033: Tumor cell-intrinsic mPGES-1-PGE2-EP4 signaling is a major immunosuppressive pathway in pancreatic cancer

Abstract Pancreatic adenocarcinoma (PDA) is the 4th leading cause of cancer-related deaths in the US partly because of its resistance to conventional chemotherapies. Immunotherapies that improve survival in many cancers, almost universally fail in PDA. The resistance to immune checkpoint blockade is at least partially explained by the lack of T cells in the tumor microenvironment (TME) in PDA. As demonstrated previously, tumor cell-derived prostaglandin E2 (PGE2) is one of the tumor intrinsic factors that creates/supports immunosuppressive TME in PDA. The current study investigates how depleting tumor cell-derived PGE2 or disrupting its autocrine signaling through the cognate EP4 receptors on tumor cells reverses the T cell low, myeloid cell rich PDA TME and suppresses the tumor growth. Pharmacological blockade of EP4 receptors combined with anti-PD-1 and agonistic CD40 antibodies induced complete tumor regressions with a lasting vaccine effect. Knockout (KO) of Ptges, the gene encoding PGE2 synthesis enzyme mPGES-1, or EP4 receptor gene, Ptger4, in KrasG12D/P53R172H/Yfp/PdxCre (KPCY) pancreatic tumor cells was sufficient to abolish the growth of implanted tumors (p<0.0001 both). The rejection of the primary KO and rechallenge control tumors was T cell-dependent as it did not happen in hosts depleted of CD4+ and CD8+ T cells. Both KO tumor cells were more susceptible to TNF-a killing while their TMEs were enriched with central memory T cells and phagocytic macrophages and depleted of granulocytic myeloid-derived suppressor cells (gMDSC). Further analysis of Ptger4 tumors revealed changes in CCL2, CXCL9, and GM-CSF production probably causing the changes in TME. In addition, in both Ptges and Ptger4 KO tumors, the adenosine synthesis pathway was significantly downregulated probably due to decreased TGF-b signaling in tumor cells. When cultured ex vivo in hypoxic conditions, Ptger4 KO tumors made significantly less immunosuppressive adenosine (p<0.001). Consequently, 57% of Ptger4 KO tumors in adenosine deaminase inhibitor treated hosts accumulated enough adenosine to suppress the anti-tumor immune response and grow out. The study demonstrates a primary role for the tumor cell-intrinsic mPGES1-PGE2-EP4 signaling in creating immune-suppressive TME in PDA, suggesting EP4 blockade as a novel approach in the treatment of pancreatic cancer. Citation Format: Nune Markosyan, Il-Kyu Kim, Charu Arora, Liz Quinones, Noah Cheng, Ben Z. Stanger, Robert H. Vonderheide. Tumor cell-intrinsic mPGES-1-PGE2-EP4 signaling is a major immunosuppressive pathway in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B033.

Genetically engineered IgG1 and nanobody oligomers acquire strong intrinsic CD40 agonism

ABSTRACT Most anti-CD40 antibodies show robust agonism only upon binding to FcγR+ cells, such as B cells, macrophages, or DCs, but a few anti-CD40 antibodies display also strong intrinsic agonism dependent on the recognized epitope and/or isotype. It is worth mentioning, however, that also the anti-CD40 antibodies with intrinsic agonism can show a further increase in agonistic activity when bound by FcγR-expressing cells. Thus, conventional antibodies appear not to be sufficient to trigger the maximum possible CD40 activation independent from FcγR-binding. We proved here the hypothesis that oligomeric and oligovalent anti-CD40 antibody variants generated by genetic engineering display high intrinsic, thus FcγR-independent, agonistic activity. We generated tetra-, hexa- and dodecavalent variants of six anti-CD40 antibodies and a CD40-specific nanobody. All these oligovalent variants, even when derived of bivalent antagonistic anti-CD40 antibodies, showed strongly enhanced CD40 agonism compared to their conventional counterparts. In most cases, the CD40 agonism reached the maximum response induced by FcγR-bound anti-CD40 antibodies or membrane CD40L, the natural engager of CD40. In sum, our data show that increasing the valency of anti-CD40 antibody constructs by genetic engineering regularly results in molecules with high intrinsic agonism and level out the specific limitations of the parental antibodies.

Antigen-presenting B cells promote TCF-1+ PD1- stem-like CD8+ T-cell proliferation in glioblastoma.

Understanding the spatial relationship and functional interaction of immune cells in glioblastoma (GBM) is critical for developing new therapeutics that overcome the highly immunosuppressive tumor microenvironment. Our study showed that B and T cells form clusters within the GBM microenvironment within a 15-μm radius, suggesting that B and T cells could form immune synapses within the GBM. However, GBM-infiltrating B cells suppress the activation of CD8+ T cells. To overcome this immunosuppression, we leveraged B-cell functions by activating them with CD40 agonism, IFNγ, and BAFF to generate a potent antigen-presenting B cells named BVax. BVax had improved antigen cross-presentation potential compared to naïve B cells and were primed to use the IL15-IL15Ra mechanism to enhance T cell activation. Compared to naïve B cells, BVax could improve CD8 T cell activation and proliferation. Compared to dendritic cells (DCs), which are the current gold standard professional antigen-presenting cell, BVax promoted highly proliferative T cells in-vitro that had a stem-like memory T cell phenotype characterized by CD62L+CD44- expression, high TCF-1 expression, and low PD-1 and granzyme B expression. Adoptive transfer of BVax-activated CD8+ T cells into tumor-bearing brains led to T cell reactivation with higher TCF-1 expression and elevated granzyme B production compared to DC-activated CD8+ T cells. Adoptive transfer of BVax into an irradiated immunocompetent tumor-bearing host promoted more CD8+ T cell proliferation than adoptive transfer of DCs. Moreover, highly proliferative CD8+ T cells in the BVax group had less PD-1 expression than those highly proliferative CD8+ T cells in the DC group. The findings of this study suggest that BVax and DC could generate distinctive CD8+ T cells, which potentially serve multiple purposes in cellular vaccine development.

Development of a c-MET x CD137 bispecific antibody for targeted immune agonism in cancer immunotherapy

BackgroundTargeting the costimulatory receptor CD137 has shown promise as a therapeutic approach for cancer immunotherapy, resulting in anti-tumor efficacy demonstrated in clinical trials. However, the initial CD137 agonistic antibodies, urelumab and utomilumab, faced challenges in clinical trials due to the liver toxicity or lack of efficacy, respectively. Concurrently, c-MET has been identified as a highly expressed tumor-associated antigen (TAA) in various solid and soft tumors. MethodsIn this study, we aimed to develop a bispecific antibody (BsAb) that targets both c-MET and CD137, optimizing the BsAb format and CD137 binder for efficient delivery of the CD137 agonist to the tumor microenvironment (TME). We employed a monovalent c-MET motif and a trimeric CD137 Variable Heavy domain of Heavy chain (VHH) for the BsAb design. ResultsOur results demonstrate that the c-MET x CD137 BsAb provides co-stimulation to T cells through cross-linking by c-MET-expressing tumor cells. Functional immune assays confirmed the enhanced efficacy and potency of the c-MET x CD137 BsAb, as indicated by activation of CD137 signaling, target cell killing, and cytokine release in various tumor cell lines. Furthermore, the combination of c-MET x CD137 BsAb with Pembrolizumab showed a dose-dependent enhancement of target-induced T cell cytokine release. ConclusionOverall, the c-MET x CD137 BsAb exhibits a promising developability profile as a tumor-targeted immune agonist by minimizing off-target effects while effectively delivering immune agonism. It has the potential to overcome resistance to anti-PD-(L)1 therapies.