Agonistic CD40 Antibody Research Articles

A bispecific CD40 agonistic antibody allowing for antibody-peptide conjugate formation to enable cancer-specific peptide delivery, resulting in improved T proliferation and anti-tumor immunity in mice

Current antibody-based immunotherapy depends on tumor antigen shedding for proper T cell priming. Here we select a novel human CD40 agonistic drug candidate and generate a bispecific antibody, herein named BiA9*2_HF, that allows for rapid antibody-peptide conjugate formation. The format is designed to facilitate peptide antigen delivery to CD40 expressing cells combined with simultaneous CD40 agonistic activity. In vivo, the selected bispecific antibody BiA9*2_HF loaded with peptide cargos induces improved antigen-specific proliferation of CD8+ (10-15 fold) and CD4+ T cells (2-7 fold) over control in draining lymph nodes. In both virus-induced and neoantigen-based mouse tumor models, BiA9*2_HF demonstrates therapeutic efficacy and elevated safety profile, with complete tumor clearance, as well as measured abscopal impact on tumor growth. The BiA9*2_HF drug candidate can thus be utilized to tailor immunotherapeutics for cancer patients.

Abstract PR-11: Fc-optimized agonistic CD40 antibody induces tumor rejection and systemic antitumor immunity - From the bench to the bedside and back

Abstract PR-11: Fc-optimized agonistic CD40 antibody induces tumor rejection and systemic antitumor immunity - From the bench to the bedside and back

The novel and potent CD40 agonist KHK2840 augments the antitumor efficacy of anti-PD-1 antibody and paclitaxel.

Lack of tumor-reactive cytotoxic T lymphocytes (CTLs)limits the antitumor efficacy of immune checkpoint inhibitors (ICIs). CD40 agonists have been expected to overcome this limitation by generating tumor-reactive CTLs. However, the clinical efficacy of CD40 agonistic antibodies is not as good as in non-clinical studies. The novel human CD40 (hCD40) agonist KHK2840 is a fully human anti-CD40 IgG2 agonistic antibody that is Fc-engineered to minimize complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity. Compared to other hCD40 agonists, KHK2840 exhibited the most potent hCD40 agonistic signal in tumor-bearing hCD40 transgenic mice and human peripheral blood B cells. Moreover, KHK2840 enhanced the antitumor efficacy of the antiprogrammed cell death 1 antibody and paclitaxel. Comprehensive immune profiling revealed that the antitumor immune response of the triple combination involved tumor-draining lymph nodes in addition to tumor microenvironments. This suggests that a coordinated antitumor immune response between tumors and lymph nodes may underlie the synergistic antitumor efficacy of the triple combination therapy. Finally, a toxicology study in cynomolgus monkeys demonstrated that KHK2840 activated the CD40 signal with tolerable toxicological properties. These results indicate that KHK2840 is a novel and potent hCD40 agonistic antibody for cancer immunotherapy, which is expected to augment the antitumor efficacy of ICIs and chemotherapy.

Abstract PR-10: Phase 1b study of maintenance soluble beta-glucan (Odetiglucan) in combination with a CD40 agonistic monoclonal antibody (CDX-1140) in patients with metastatic pancreatic adenocarcinoma that had not progressed on first-line chemotherapy

Abstract Background: Metastatic pancreatic ductal adenocarcinoma (mPDAC) has a dismal 5-year overall survival (OS) rate of 3%. Current standard of care provides only modest anti-cancer impact. Preclinical models show that combinations of myeloid agonists can induce strong anti-tumor activity in PDAC tumors resistant to immunotherapy, including immune checkpoint blockade (anti-CTLA4, anti-PD1). This study combines odetiglucan, a beta glucan pathogen-associated molecular pattern (PAMP), with CDX-1140, a fully human Ig2K agonistic monoclonal CD40 antibody, in the maintenance setting after cytotoxic chemotherapy induction. Preliminary findings are presented as the study was closed early for financial reasons. Methods: Patients with mPDAC who had not progressed after 4-8 months of 1L chemotherapy were enrolled and received weekly odetiglucan (4 mg/kg) plus every 3-week dosing of CDX-1140 (1.5 mg/kg). The primary endpoints were safety, MTD and RP2D, with secondary endpoints including DOR, PFS, ORR, and OS. Tissue and blood samples were collected at baseline and during treatment to evaluate immune pharmacodynamic responses. Results: A total of 5 patients received treatment on study including 4 patients who were efficacy evaluable and 1 patient who withdrew due to adverse event (arthralgia) after beginning treatment. Treatment-related emergent adverse events were seen in all patients with most common AEs including grade 1-2 arthralgia, chills, and fatigue. Grade 3 AEs included arthralgia (n=1), fatigue (n=1), and leukopenia (n=2). The ORR included 1 PR, 1 SD, and 2 PD. Two patients remained on treatment for >100 days. The pharmacodynamic profile was consistent with the mode of action of odetiglucan and CDX-1140, including a reduction in peripheral Dectin-1 expressing monocytes and transient decreases in peripheral B cells. Patients with disease control (PR or SD, n=2) compared to patients with progression (PD, n=2) showed expansion of peripheral Ki67+ CD8+ T cells following treatment. Analysis of a paired tumor biopsy from 1 patient with PD confirmed penetration of odetiglucan into a liver metastasis with expansion of CD11b+ macrophages and a reduction of cytokeratin+ tumor content, but without significant changes in T cell infiltration. Conclusions: The combination of odetiglucan and CDX-1140 was feasible and safe when administered as maintenance therapy in patients with mPDAC whose disease had not progressed on 1L chemotherapy. Treatment produced encouraging activity with changes in immunological correlates associated with disease control. These early results support further investigation combining odetiglucan with a CD40 agonist in mPDAC. Citation Format: Mark H O'Hara, Max Wattenberg, Ignacio Garrido-Laguna, Eileen M O'Reilly, Michael Yellin, Tibor Keler, Michele Gargano, Nick Niles, Jeremy Drees, Nandita Bose, Gregory L Beatty. Phase 1b study of maintenance soluble beta-glucan (Odetiglucan) in combination with a CD40 agonistic monoclonal antibody (CDX-1140) in patients with metastatic pancreatic adenocarcinoma that had not progressed on first-line chemotherapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr PR-10.

Abstract B061: Phase 1b study of maintenance soluble beta-glucan (Odetiglucan) in combination with a CD40 agonistic monoclonal antibody (CDX-1140) in patients with metastatic pancreatic adenocarcinoma that had not progressed on first-line chemotherapy

Abstract Background: Metastatic pancreatic ductal adenocarcinoma (mPDAC) has a dismal 5-year overall survival (OS) rate of 3%. Current standard of care provides only modest anti-cancer impact. Preclinical models show that combinations of myeloid agonists can induce strong anti- tumor activity in PDAC tumors resistant to immunotherapy, including immune checkpoint blockade (anti-CTLA4, anti-PD1). This study combines odetiglucan, a beta glucan pathogen- associated molecular pattern (PAMP), with CDX-1140, a fully human Ig2K agonistic monoclonal CD40 antibody, in the maintenance setting after cytotoxic chemotherapy induction. Preliminary findings are presented as the study was closed early for financial reasons. Methods: Patients with mPDAC who had not progressed after 4-8 months of 1L chemotherapy were enrolled and received weekly odetiglucan (4 mg/kg) plus every 3-week dosing of CDX-1140 (1.5 mg/kg). The primary endpoints were safety, MTD and RP2D, with secondary endpoints including DOR, PFS, ORR, and OS. Tissue and blood samples were collected at baseline and during treatment to evaluate immune pharmacodynamic responses. Results: A total of 5 patients received treatment on study including 4 patients who were efficacy evaluable and 1 patient who withdrew due to adverse event (arthralgia) after beginning treatment. Treatment-related emergent adverse events were seen in all patients with most common AEs including grade 1-2 arthralgia, chills, and fatigue. Grade 3 AEs included arthralgia (n=1), fatigue (n=1), and leukopenia (n=2). The ORR included 1 PR, 1 SD, and 2 PD. Two patients remained on treatment for >100 days. The pharmacodynamic profile was consistent with the mode of action of odetiglucan and CDX-1140, including a reduction in peripheral Dectin-1 expressing monocytes and transient decreases in peripheral B cells. Patients with disease control (PR or SD, n=2) compared to patients with progression (PD, n=2) showed expansion of peripheral Ki67+ CD8+ T cells following treatment. Analysis of a paired tumor biopsy from 1 patient with PD confirmed penetration of odetiglucan into a liver metastasis with expansion of CD11b+ macrophages and a reduction of cytokeratin+ tumor content, but without significant changes in T cell infiltration. Conclusions: The combination of odetiglucan and CDX-1140 was feasible and safe when administered as maintenance therapy in patients with mPDAC whose disease had not progressed on 1L chemotherapy. Treatment produced encouraging activity with changes in immunological correlates associated with disease control. These early results support further investigation combining odetiglucan with a CD40 agonist in mPDAC. Citation Format: Mark H O'Hara, Max Wattenberg, Ignacio Garrido-Laguna, Eileen M O'Reilly, Michael Yellin, Tibor Keler, Michele Gargano, Nick Niles, Jeremy Drees, Nandita Bose, Gregory L Beatty. Phase 1b study of maintenance soluble beta-glucan (Odetiglucan) in combination with a CD40 agonistic monoclonal antibody (CDX-1140) in patients with metastatic pancreatic adenocarcinoma that had not progressed on first-line chemotherapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B061.

CD40 agonist engineered immunosomes modulated tumor microenvironment and showed pro-immunogenic response, reduced toxicity, and tumor free survival in mice bearing glioblastoma

CD40 agonist antibodies (αCD40) have shown promising anti-tumor response in both preclinical and early clinical studies. However, its systemic administration is associated with immune- and hepato-toxicities which hampers its clinical usage. 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 a multifunctional Immunosome where αCD40 is conjugated on the surface and RRX-001, a small molecule immunomodulator was encapsulated inside it. Immunosomes showed higher tumor accumulation till 96 h of administration and displayed sustained release of αCD40 in vivo. Immunosomes significantly delayed tumor growth and showed tumor free survival in mice bearing GL-261 glioblastoma by increasing the population of CD45+CD8+ T cells, CD45+CD20+ B cells, CD45+CD11c+ DCs and F4/80+CD86+ cells in TME. Immunosome significantly reduced the population of T-regulatory cells, M2 macrophage, and MDSCs and lowered the PD-L1 expression. Moreover, Immunosomes significantly enhanced the levels of Th1 cytokines (IFN-γ, IL-6, IL-2) over Th2 cytokines (IL-4 and IL-10) 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. In addition, Immunosomes treated long-term surviving mice showed tumor specific immune memory response which prevented tumor growth upon rechallenge. Our results suggested that this novel formulation can be further explored in clinics to improve in vivo anti-tumor efficacy of αCD40 with long-lasting tumor specific immunity while reducing the associated toxicities.

TRLS-06. PHASE 1 STUDY OF THE CD40 AGONISTIC MONOCLONAL ANTIBODY APX005M (SOTIGALIMAB) IN PEDIATRIC, ADOLESCENT, AND YOUNG ADULT PATIENTS WITH RECURRENT OR PROGRESSIVE CNS TUMORS AND NEWLY DIAGNOSED BRAIN STEM GLIOMA: A PEDIATRIC BRAIN TUMOR CONSORTIUM STUDY (PBTC-051; NCT03389802)

Abstract BACKGROUND CD40 is a co-stimulatory receptor present on antigen-presenting cells. CD40 agonists are hypothesized to reverse cancer-induced immune suppression and induce tumor cell killing. The PBTC completed a first-in-pediatrics phase 1 study (PBTC-051) of the CD40 agonistic monoclonal antibody APX005M (sotigalimab). METHODS Patients 1-21.99 years old with recurrent/progressive/refractory malignant non-brainstem central nervous system (CNS) tumors were eligible for stratum 1, and those with post-radiation pre-progression diffuse intrinsic pontine glioma (DIPG) for stratum 2. Patients received APX005M (sotigalimab) via IV on day 1 of each 21 day cycle. A 3 + 3 statistical design was used for dose escalation through 3 planned dose levels (DL). Patients could receive APX005M (sotigalimab) for 36 cycles or until disease progression, unacceptable toxicity, or death. RESULTS 31 eligible patients enrolled (20 on stratum 1, 11 on stratum 2) between 2018-2023; 29 were evaluable for dose finding. The most common diagnosis in stratum 1 was ependymoma (35%). DL3 (0.6 mg/kg) was the recommended phase 2 dose (RP2D) for stratum 1 and DL2 (0.3 mg/kg) for stratum 2. Five patients had dose limiting toxicities—two stratum 1 patients at DL3 and three stratum 2 patients at DL3. The most common grade 3+ adverse events at least possibly attributable to APX005M (sotigalimab) were lymphopenia, neutropenia, leukopenia, and increased ALT. Median number of cycles was 2 (1-36) for stratum 1 and 3 (1-19) for stratum 2. No patients had an objective response to APX005M. 6 month progression-free survival (PFS) for stratum 2 patients (calculated from start of treatment) was 30.7±12.8%; median PFS was 3.7 months. CONCLUSIONS APX005M was well-tolerated in pediatric patients with both recurrent/refractory/progressive CNS tumors and post-radiation pre-progression DIPG. A RP2D was established for both populations. Future studies of this agent in pediatric neuro-oncology patients should prioritize combination therapy.

Intratumoral Fc-optimized agonistic CD40 antibody induces tumor rejection and systemic antitumor immunity in patients with metastatic cancer.

While CD40 agonism is an attractive approach for activating antigen-presenting cells and initiating antitumor responses, previous attempts have encountered limited clinical efficacy coupled with toxicity. We previously demonstrated that interactions between the antibody Fc domain and the inhibitory receptor FcγRIIB are critical for enhanced antitumor activity. Here, we present the results of a phase 1 study on intratumoral administration of an anti-CD40 agonistic antibody (2141-V11) Fc-engineered to enhance FcγRIIB binding. Primary endpoints included safety, maximum tolerated dose (MTD), and recommended phase 2 dose. Secondary objectives included preliminary clinical activity and correlative studies from biospecimens. 2141-V11 was well-tolerated without dose-limiting toxicities and MTD was not reached. In ten evaluable patients with metastatic cancer, the overall response rate was 20%, with complete responses in two patients (melanoma and breast carcinoma) and stable disease in six patients. 2141-V11 induced tumor regression in injected and non-injected lesions, with increased leukocyte infiltration and tertiary lymphoid structures (TLS) formation in post-treatment biopsies. In a humanized mouse model for CD40 and FcγRs, 2141-V11 induced TLS formation in mice bearing orthotopic breast carcinoma, correlating with local and abscopal antitumor effects, systemic immune activation, and immune memory. These findings support the safety and efficacy of 2141-V11, warranting phase 2 studies and suggesting a unique mechanism of action for this Fc-enhanced immunotherapy (NCT04059588).

Combining CD40 agonist mitazalimab with mFOLFIRINOX in previously untreated metastatic pancreatic ductal adenocarcinoma (OPTIMIZE-1): a single-arm, multicentre phase 1b/2 study

Current systemic therapies for metastatic pancreatic ductal adenocarcinoma are associated with poor outcomes with a 5-year overall survival rate under 5%. We aimed to assess the safety and antitumour activity of mitazalimab, a human CD40 agonistic IgG1 antibody, with modified FOLFIRINOX (mFOLFIRINOX; fluorouracil, leucovorin, oxaliplatin, and irinotecan), in chemotherapy-naive patients with metastatic pancreatic ductal adenocarcinoma. OPTIMIZE-1 was a single-arm, multicentre, phase 1b/2 study which enrolled adults with histologically-confirmed metastatic pancreatic ductal adenocarcinoma and European Cooperative Oncology Group performance status 0 or 1 in 14 university hospitals in Belgium, France, and Spain. The primary endpoint of phase 1b was to determine the recommended phase 2 dose of intravenous mitazalimab (450 μg/kg or 900 μg/kg) when combined with intravenous mFOLFIRINOX (oxaliplatin 85 mg/m2, leucovorin 400 mg/m2, irinotecan 150 mg/m2, fluorouracil 2400 mg/m2). In the first 21-day treatment cycle, mitazalimab was administered on days 1 and 10, and mFOLFIRINOX on day 8. In subsequent 14-day cycles mitazalimab was administered 2 days after mFOLFIRINOX. The phase 2 primary endpoint was objective response rate. Activity and safety analyses were conducted on the full analysis set (all patients who received the combination of mitazalimab at the recommended phase 2 dose and mFOLFIRINOX for at least two treatment cycles) and safety set (all patients who received any study treatment), respectively. Enrolment is complete, and data represents a primary analysis of the ongoing trial. The trial is registered at Clinicaltrials.gov (NCT04888312). Between Sept 29, 2021, and March 28, 2023, 88 patients were screened and 70 patients were enrolled (40 [57%] were female and 30 [43%] were male). In phase 1b, 900 μg/kg mitazalimab was determined as the recommended phase 2 dose. Overall, five patients received 450 μg/kg mitazalimab; 65 received 900 μg/kg mitazalimab. No dose-limiting toxicities were observed at 450 μg/kg, and one dose-limiting toxicity was observed at 900 μg/kg. 57 patients were evaluated for activity, and all 70 patients were included in the safety set. At data cutoff on Nov 14, 2023, median follow-up was 12·7 months (95% CI 11·1-15·7). Of the 57 patients, 29 (51%) remained on study and 18 (32%) remained on treatment. The primary endpoint (objective response rate >30%) was met (objective response rates in 23 [40%]; one-sided 90% CI ≥32 of 57 patients). The most common grade 3 or worse adverse events were neutropenia (18 [26%] of 70 patients), hypokalaemia (11 patients [16%]), and anaemia and thrombocytopenia (eight patients [11%]). Serious adverse events were reported in 29 (41%) of 70 patients, the most common being vomiting (five [7%] of 70 patients), decreased appetite (four [6%]), and diarrhoea and cholangitis (three [4%] of 70 patients for each), none considered related to mitazalimab. No treatment-related deaths were reported. Mitazalimab with mFOLFIRINOX demonstrated manageable safety and encouraging activity, warranting continued development in a phase 3, randomised, controlled trial. The results from OPTIMIZE-1 pave the way for further exploration and confirmation of a novel immunotherapy treatment regimen for metastatic pancreatic ductal adenocarcinoma, which is a complex and aggressive cancer with very low survival rates and restricted treatment options. Alligator Bioscience.

Reversible Chemical Modification of Antibody Effector Function Mitigates Unwanted Systemic Immune Activation.

Antibody effector functions including antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP) are mediated through the interaction of the antibody Fc region with Fcγ receptors present on immune cells. Several approaches have been used to modulate antibody Fc-Fcγ interactions with the goal of driving an effective antitumor immune response, including Fc point mutations and glycan modifications. However, robust antibody-Fcγ engagement and immune cell binding of Fc-enhanced antibodies in the periphery can lead to the unwanted induction of systemic cytokine release and other dose-limiting infusion-related reactions. Creating a balance between effective engagement of Fcγ receptors that can induce antitumor activity without incurring systemic immune activation is an ongoing challenge in the field of antibody and immuno-oncology therapeutics. Herein, we describe a method for the reversible chemical modulation of antibody-Fcγ interactions using simple poly(ethylene glycol) (PEG) linkers conjugated to antibody interchain disulfides with maleimide attachments. This method enables dosing of a therapeutic with muted Fcγ engagement that is restored in vivo in a time-dependent manner. The technology was applied to an effector function enhanced agonist CD40 antibody, SEA-CD40, and experiments demonstrate significant reductions in Fc-induced immune activation in vitro and in mice and nonhuman primates despite showing retained efficacy and improved pharmacokinetics compared to the parent antibody. We foresee that this simple, modular system can be rapidly applied to antibodies that suffer from systemic immune activation due to peripheral FcγR binding immediately upon infusion.

Abstract LT08: Extracellular Vesicle Surface Display Enhances the Therapeutic Efficacy and Safety Profile of Cancer Immunotherapy

Abstract As our understanding of the complex interactions between the immune system and cancer has progressed, so has the identification of novel targets and ligands that can modulate effective anti-cancer immune responses. While immunotherapy has emerged as a mainstay in cancer therapy, its efficacy has been limited by the potential for off-target toxicity against normal body cells. In this study, we present a nanoparticle-based delivery system that enhances the signaling efficacy of immunomodulatory ligands while concurrently limiting systemic toxicity. We demonstrate that extracellular vesicles (EVs), lipid bilayer enclosed nanoparticles released by cells, can be efficiently engineered to display multiple immunomodulatory ligands on their surface. Display of immunomodulatory ligands such as agonistic CD137 and CD40 antibodies on the EV surface significantly enhanced their therapeutic efficacy over equivalent doses of free ligands by inducing ligand multimerization, efficient receptor crosslinking and the formation of immune synapses. EVs conjugated with a complementary combination of immune stimulatory ligands and immune checkpoint inhibitors were able to shift the tumor immune milieu towards an anti-tumorigenic phenotype in a lung metastatic melanoma allograft and suppress tumor progression to a greater extent than an equivalent dose of free ligands. We also demonstrate the ability of EVs to co-deliver intraluminally loaded TLR agonists which synergize with surface displayed agonistic CD40 antibodies to induce effective anti-tumor responses and tumor regression in a metastatic autochthonous cell-derived model of PDAC. Remarkably, we also observed that EV-associated immunotherapeutics were limited in their biodistribution to the tumor microenvironment, resulting in significantly lower off-target toxicity than treatment with free ligands. In summary, we present an EV-based delivery approach that allows the use of potent cancer immunotherapy combinations to achieve superior anti-tumor responses at significantly lower doses with less side-effects than is possible with conventional administration methods for cancer immunotherapy. Citation Format: Migara Kavishka Jayasinghe, Dawn Liu Xiao Tian, Yock Sin Lay, Chang Yu Lee, Brendon Zhi Jie Yeo, Dong Van Hoang, Rebecca Carissa Prajogo, Boya Peng, Thach Tuan Pham, Minh Thi Nguyet Le. Extracellular Vesicle Surface Display Enhances the Therapeutic Efficacy and Safety Profile of Cancer Immunotherapy [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr LT08.

Abstract CT192: A first-in-human phase 1 study of TQB2916, a novel CD40 agonist antibody for advanced malignancies

Abstract Background: CD40 receptor is a member of the tumor necrosis factor receptor family that plays a prominent role in immune responses. TQB2916, a humanized IgG2 CD40 monoclonal antibody, has demonstrated CD40 agonistic activity and promising anti-tumor activity in preclinical studies. A first-in-human study of TQB2916 single agent in advanced solid tumors and lymphomas is ongoing (NCT05213767). Methods: Patients with an ECOG performance status of 0 or 1, adequate hematologic and organ function and refractory to standard therapies met inclusion criteria. TQB2916 was given intravenously every 3 weeks until progression or unacceptable toxicity. Bayesian optimal interval (BOIN) design was used to determine the maximum tolerated dose (MTD) and recommended Phase 2 dose (RP2D). The objective is to assess the safety, tolerability, pharmacokinetics, and preliminary anti-tumor activity of TQB2916. Results: From April 2022 to November 2023, 18 patients with solid tumors and 2 with lymphoma have been treated with TQB2916 monotherapy, and were assigned to 7 cohorts (0.5 mg n=1, 2.5 mg n=1, 12.5 mg n=3, 60 mg n=3, 200 mg n=7, 300 mg n=2, and 400 mg n=3). Three dose limiting toxicities (DLTs) were observed: one with grade 3 pneumonia and one with grade 4 lipase/amylase increase in the 400 mg cohort; one with grade 3 pancreatitis in the 300 mg cohort. All patients experienced at least one treatment-related adverse event (TRAE). The most common TRAEs were lipase increase (n=15, 75%), amylase increase (n=14, 70%), lymphocyte count decrease (n=13, 65%), alanine aminotransferase (ALT) increase (n=12, 60%), alkaline phosphatase (ALP) increase (n=12, 60%), aspartate aminotransferase (AST) increase (n=11, 55%), hypoalbuminemia (n=11, 55%), and anorexia (n=11, 55%). Serious TRAEs occurred in 5 (25%) patients. Most of TRAEs were of grade 1 or grade 2 and manageable. Sixteen patients were evaluated for efficacy, and 3 of them achieved stable disease as per immune response evaluation criteria in solid tumors (iRECIST) and lymphoma response to immunomodulatory therapy criteria (LYRIC). The pharmacokinetics were well-behaved with increasing exposure dose-proportionally and no accumulation occurred after repeated dosing. The Cmax, AUC(0-t) and T1/2 of 400 mg single dosing were 109±21.8 μg/ml, 611±228 d*µg/mL and 5.54±3.37 d. Dose-dependent occupancy of CD40 was detected of 0.5 mg and above doses. Reduction in peripheral B cells and increasing in cytokines secretion were also observed. Conclusions: Pharmacodynamics analysis suggested that TQB2916 treatment achieved CD40 occupancy and immune activation with the changes in cytokines. Considering the safety profile and efficacy, 200 mg was determined as the preliminary expansion dose as it was well tolerated. Studies of TQB2916 in combination with immune checkpoint inhibitors and/or other anti-cancer therapies are ongoing. Citation Format: Yi Ba, Huilai Zhang, Ting Deng, Tao Ning, Qian Fan. A first-in-human phase 1 study of TQB2916, a novel CD40 agonist antibody for advanced malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT192.

Abstract 2366: Balancing efficacy and toxicity of a fibroblast activation protein dependent tetravalent CD40 agonist antibody for cancer therapy

Abstract Background: CD40 agonist antibodies have been found to augment tumor antigen presentation and have shown potential as anti-tumor treatments in human clinical trials. The clinical success of CD40 agonist antibodies has been restricted due to their limited efficacy and on-target, off-tumor toxicity. We hypothesized that toxicity associated with CD40 agonist antibodies could be overcome by activating CD40 specifically through tumor-specific antigens. Additionally, limited efficacy can be improved by rational design of CD40 bispecific antibodies (BsAbs). Herein, we present FAPxCD40, a trivalent CD40 agonist BsAb targeting fibroblast activation protein (FAP) possessing an optimized therapeutic window. Method: We generated a series of FAPxCD40 bispecific antibodies with 2, 4, and 6 CD40 valences by using a high-affinity anti-FAP antibody and a crosslink-dependent CD40 agonist VHH antibody. These BsAbs were evaluated using multiple immune cell functional assays, including assays for dendritic cell (DC) maturation, B cell maturation, and CMV-reactive T cell recall. The in vivo therapeutic window of the FAPxCD40 bispecifics was compared in CD40 humanized mice with subcutaneously transplanted FAP-positive MC38 tumor xenografts. Results: We developed FAPxCD40 BsAbs with varying valences, including bivalent FAPxCD40-2, tetravalent FAPxCD40-4, and hexavalent FAPxCD40-6. FAPxCD40-2 showed strict FAP-dependent CD40 agonist activity in both DC and B cell maturation assays, while its CD40 agonist activity upon FAP-crosslinking was modest. FAPxCD40-4 exhibited efficient CD40 agonist activity upon FAP-crosslinking, but however, it also exhibited some undesired FAP-independent CD40 agonist activity. FAPxCD40-6 efficiently clustered CD40 but marginally benefited from FAP binding, making it unsuitable as a choice. In the humanized CD40 mice model, FAPxCD40-2 effectively eliminated systemic toxicity at all tested doses. Its antitumor activity, however, is modest with TGI (tumor growth inhibition) rate of 32.8 % at a dose of 6.96 mpk. Further escalation of the FAPxCD40-2 dosage to 13.92 mpk did not result in enhanced antitumor activity with TGI rate of 46.9 %, indicating that its CD40 agonist activity may have reached a plateau. In contrast, FAPxCD40-4 broke this plateau and induce a promising antitumor response at a dose of 1.3 mpk, resulting in a 92% TGI rate. Furthermore, no liver enzyme upregulation or platelet count decrease was observed under these conditions, indicating an ideal therapeutic window. In addition, during nonclinical toxicology studies conducted on cynomolgus monkeys, it was found that doses of FAPxCD40-4 up to 99 mg/kg administered weekly for a total of four doses were well-tolerated. Conclusion: Our study demonstrates that FAPxCD40-4, a tetravalent FAPxCD40 BsAb, exhibits efficient CD40 agonist activity with an optimized therapeutic window. Citation Format: Simeng Chen, Yuan Lin, Xiaoru Zhou, Xing Sun, Changyong Yang, Cheng Liao. Balancing efficacy and toxicity of a fibroblast activation protein dependent tetravalent CD40 agonist antibody for cancer therapy [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 2366.

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.

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.

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.

Vaccination against neoantigens induced in cross-priming cDC1 in vivo

The conventional type 1 dendritic cells (cDC1) play a pivotal role in protective immunity against pathogens and cancer. However, their low frequency in the blood and tissues limits their use in immune therapy. We have recently described a method to vaccinate against neoantigens that are induced in tumor cells by targeted delivery of a TAP siRNA to dendritic cells using a TLR9 binding CpG oligonucleotide. Since TLR9 is also expressed in immune suppressive myeloid populations TLR9 targeting could reduce the effectiveness of this approach. Here, we describe a modular multivalent antibody platform to target the TAP siRNA to resident Clec9a expressing cDC1 and show that it leads to selective and sustained TAP downregulation in cDC1 and inhibits tumor growth in mice more effectively than CpG targeted siRNA. To induce DC maturation an agonistic CD40 antibody was administered to the siRNA treated mice. To obviate the need for a second drug formulation and reduce the risk of toxicity, we exploited the multivalent nature of this targeting platform to co-deliver the TAP siRNA and a DC maturation agent, a CpG containing oligonucleotide, to cDC1 in vivo and show that it was more effective than Clec9a targeting of TAP siRNA in combination with CD40 antibody. This study describes a way to manipulate the function of cDC1 cells in vivo using a broadly applicable antibody-based targeting platform to deliver multiple biological agents to specific cells in vivo to potentiate (immune) therapy and to probe the biology of specific cell types in their natural settings.

Potentiating Local and Abscopal Antitumor Efficacy through Radiation with FAP-CD40 DARPin and Anti-PD1 Therapy

Although the advent of Immune Checkpoint Inhibitors (ICI) has changed the facet of oncology, about 70% of patients develop resistance. CD40 is a potent costimulatory molecule that drives dendritic cells (DCs) and tumor-associated macrophages (TAMs) to prime T-cells. Clinically, agonistic CD40 antibodies have demonstrated antitumor activity, but dose-limiting toxicities have impaired efficacy. Radiation (RT), especially at higher doses, activates DCs and TAMs, but also upregulates Fibroblast Activation Protein (FAP) in the tumor microenvironment (TME). The upregulated FAP expression can be harnessed for the design of targeted drug delivery to the TME. The FAP-CD40 DARPin is a molecule that utilizes FAP and CD40 binding domains to selectively cross-link and activate CD40 in the tumor, thus avoiding systemic CD40 activation. This study aimed to evaluate if RT combined with a murine FAP-CD40 (mFAP-CD40) and ICI could improve survival and local and abscopal responses in a pre-clinical lung adenocarcinoma model. Human FAPxCD40 (MP0317) is being evaluated in Phase I trials. The 344SQ-P tumor cells were bilaterally injected into the right and left hind legs of 129Sv/Ev mice to establish primary and secondary tumors respectively. When primary tumors reached ∼7mm in diameter, they were irradiated with 12 Gy x 3 fractions, while secondary tumors were monitored. mFAP-CD40 (5mg/kg) was injected intraperitoneally at 2, 7, and 11 days after the last fraction of RT. A backbone of α-PD1 ICI was given twice per week starting with RT for 5 shots total. Mice were euthanized when tumors reached 14mm in diameter. Lungs were collected at experimental endpoints, fixed, and enumerated for metastases. For FAP detection in the TME, we first conducted an IHC analysis on 344SQ-P tumors harvested 11 days post RT. Although 5 Gy x 3 was superior to 12 Gy x 3 in upregulating FAP expression compared to unirradiated controls (P = 0.0088), we chose the 12 Gy x 3 dose for subsequent experiments due to its superiority in releasing tumor antigens, priming T-cells, and promoting abscopal responses. In our bilaterally established murine groups, we have recorded the following median survival days: Control = 23; RT = 26; RT+α-PD1 = 33; mFAP-CD40+α-PD1 = 23; and RT+mFAP-CD40+α-PD1 (Triple therapy) = 44 days. These results were further buttressed by the difference in average tumor growth observed between groups; whereby there was a significant delay in tumor growth of both primary (P<0.0001) and secondary tumors (P<0.0001) of the Triple therapy group compared to either RT+α-PD1 or mFAP-CD40+α-PD1 dual therapies. In addition, the Triple therapy cohort had a significantly lower count of lung metastases vs. control (P = 0.0007) and vs. mFAP-CD40+α-PD1 (P = 0.0246) cohorts. mFAP-CD40 DARPin with RT and α-PD1 proved efficacious to control primary and secondary tumors in a murine lung carcinoma model with no detected toxicities.

Abstract PO-078: Tissue-resident memory CD8+ T cells correlate with anti-PD-1 response in head and neck cancer and expand upon cDC1 activation in tumor-draining lymph nodes to overcome PD-1 resistance

Abstract Introduction: Tumor infiltrating tissue-resident memory CD8+ T (TRM) cells correlate with immunotherapy response against several solid tumor types. Recent data from neoadjuvant immunotherapy studies highlight CD8+ TRM cells as early responders in head and neck squamous carcinoma (HNSCC). Here, we defined anti-tumor CD8+ TRM cell representation in neoadjuvant anti-PD1 clinical trial HNSCC tumors and examined their development in anti-PD1 responsive and resistant HNSCC mouse models. Methods: CD8+ CD103+ TRM cells were quantified using CODEX (CO-detection by inDEXing) in tumor tissue microarray cores from 28 HNSCC patients treated with neoadjuvant anti-PD1 on a previously reported clinical trial (NCT02296684). Anti-PD1 sensitive MOC1 and isogenic anti-PD1 resistant MOC1-esc1 cells were engineered to express ovalbumin derived SIINFEKL peptide (OVA). OVA-specific CD8+ TRM induction and activation from endogenous T cells and following OT-1 adoptive transfer was evaluated in orthotopic (buccal) MOC1/MOC1esc1-OVA tumor bearing mice by ELISA and flow cytometry employing SIINFEKL-H2Kbdextramers. In vitro OVA-directed T cell activity was evaluated using co-culture of OT-1 T cells and MOC1/MOC1OVA/MOC1esc1OVA cells, OVA peptide, or IL-2. OT-1 CD8+ T cells were transferred into MOC1OVA or MOC1-esc1OVA-bearing mice and the dynamics of TRM development were evaluated using flow cytometry. Conventional Dendritic cells 1 (cDC1s) from tumor-draining lymph nodes (TDLNs) were cocultured with OT-1 cells and evaluated for antigen-specific TRM development. Results: CD8+ CD103+ TRM cells were more frequent in baseline tumors that developed pathological tumor response following neoadjuvant pembrolizumab compared to baseline nonresponding tumors (p=0.011). In mouse orthotopic models, MOC1-esc1OVA cells maintained in vivo PD-1 resistance despite eliciting SIINFEKL dextramer+ antigen-specific T cells in the tumors that expanded in response to PD-1 blockade. Further characterization of these tumor antigen specific T cells showed that compared to MOC1OVA, MOC1-esc1OVA had reduced CD69 and CD103 expression (MOC1OVA, Average 17.55% (14.1-19.4%); MOC1-esc1OVA, Average 11.84% (9.42-15.8%); p=0.025) and reduced response to SIINFEKL restimulation (MOC1OVA, Average 1093.3 pg/mL (860.6-1240.9 pg/mL); MOC1-esc1OVA, Average 533.3 pg/mL (254.5-749.0 pg/mL); p=0.006). A time course analysis in vivo showed that OVA-specific TRM cells were generated in TDLN and migrated into tumor. Compared to MOC1-OVA, MOC1-esc1OVA TDLN cDC1s had reduced CD40 expression and fewer TRM cells. Anti-CD40 blocking monoclonal antibodies reduced OVA-specific TRM cells in MOC1OVA while CD40 agonist antibodies increased antitumor TRMs and reduced PD1-resistant tumor growth compared to the control IgG groups. Conclusion: These findings highlight TRM CD8_+ T cells as a correlate of PD-1 blockade response in HNSCC patient tissues and mouse models and delineate cDC1 CD40 function in the TDLN as critical for effective antitumor TRM development. Citation Format: Michihisa Kono, Shin Saito, Geoff Ivison, Ayano Kondo, Aaron Mayer, Jonathan D. Schoenfeld, Ann Marie Egloff, Ravindra Uppaluri. Tissue-resident memory CD8+ T cells correlate with anti-PD-1 response in head and neck cancer and expand upon cDC1 activation in tumor-draining lymph nodes to overcome PD-1 resistance [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-078.

IL-15 synergizes with CD40 agonist antibodies to induce durable immunity against bladder cancer

CD40 is a central costimulatory receptor implicated in productive antitumor immune responses across multiple cancers, including bladder cancer. Despite strong preclinical rationale, systemic administration of therapeutic agonistic antibodies targeting the CD40 pathway has demonstrated dose-limiting toxicities with minimal clinical activity, emphasizing an important need for optimized CD40-targeted approaches, including rational combination therapy strategies. Here, we describe a role for the endogenous IL-15 pathway in contributing to the therapeutic activity of CD40 agonism in orthotopic bladder tumors, with upregulation of transpresented IL-15/IL-15Rα surface complexes, particularly by cross-presenting conventional type 1 DCs (Dendritic Cells), and associated enrichment of activated CD8 T cells. In bladder cancer patient samples, we identify DCs as the primary source of IL-15, although they lack high levels of IL-15Rα at baseline. Using humanized immunocompetent orthotopic bladder tumor models, we demonstrate the ability to therapeutically augment this interaction through combined treatment with anti-CD40 agonist antibodies and exogenous IL-15, including the fully-human Fc-optimized antibody 2141-V11 currently in clinical development for the treatment of bladder cancer. Collectively, these data reveal an important role for IL-15 in mediating antitumor CD40 agonist responses in bladder cancer and provide key proof-of-concept for combined use of Fc-optimized anti-CD40 agonist antibodies and agents targeting the IL-15 pathway. These data support expansion of ongoing clinical studies evaluating anti-CD40 agonist antibodies and IL-15-based approaches to develop combinations of these promising therapeutics for the treatment of patients with bladder cancer.