Richard D. Huhn

CD40 Agonists Alter Tumor Stroma and Show Efficacy Against Pancreatic Carcinoma in Mice and Humans

CD40 immunotherapy shows efficacy in treating pancreatic cancer in mice and humans by eliciting antitumor immunity. Immunosuppressive tumor microenvironments can restrain antitumor immunity, particularly in pancreatic ductal adenocarcinoma (PDA). Because CD40 activation can reverse immune suppression and drive antitumor T cell responses, we tested the combination of an agonist CD40 antibody with gemcitabine chemotherapy in a small cohort of patients with surgically incurable PDA and observed tumor regressions in some patients. We reproduced this treatment effect in a genetically engineered mouse model of PDA and found unexpectedly that tumor regression required macrophages but not T cells or gemcitabine. CD40-activated macrophages rapidly infiltrated tumors, became tumoricidal, and facilitated the depletion of tumor stroma. Thus, cancer immune surveillance does not necessarily depend on therapy-induced T cells; rather, our findings demonstrate a CD40-dependent mechanism for targeting tumor stroma in the treatment of cancer.

Clinical Activity and Immune Modulation in Cancer Patients Treated With CP-870,893, a Novel CD40 Agonist Monoclonal Antibody

PURPOSE The cell-surface molecule CD40 activates antigen-presenting cells and enhances immune responses. CD40 is also expressed by solid tumors, but its engagement results in apoptosis. CP-870,893, a fully human and selective CD40 agonist monoclonal antibody (mAb), was tested for safety in a phase I dose-escalation study. PATIENTS AND METHODS Patients with advanced solid tumors received single doses of CP-870,893 intravenously. The primary objective was to determine safety and the maximum-tolerated dose (MTD). Secondary objectives included assessment of immune modulation and tumor response. RESULTS Twenty-nine patients received CP-870,893 in doses from 0.01 to 0.3 mg/kg. Dose-limiting toxicity was observed in two of seven patients at the 0.3 mg/kg dose level (venous thromboembolism and grade 3 headache). MTD was estimated as 0.2 mg/kg. The most common adverse event was cytokine release syndrome (grade 1 to 2) which included chills, rigors, and fever. Transient laboratory abnormalities affecting lymphocytes, monocytes, platelets, D-dimer and liver function tests were observed 24 to 48 hours after infusion. Four patients with melanoma (14% of all patients and 27% of melanoma patients) had objective partial responses at restaging (day 43). CP-870,893 infusion resulted in transient depletion of CD19+ B cells in blood (93% depletion at the MTD for < 1 week). Among B cells remaining in blood, we found a dose-related upregulation of costimulatory molecules after treatment. CONCLUSION The CD40 agonist mAb CP-870,893 was well tolerated and biologically active, and was associated with antitumor activity. Further studies of repeated doses of CP-870,893 alone and in combination with other antineoplastic agents are warranted.

Immune modulation with weekly dosing of an agonist CD40 antibody in a phase I study of patients with advanced solid tumors

Background: Single-dose infusion of the agonistic anti-CD40 monoclonal antibody (mAb) CP-870,893 accomplishes immune activation and clinical responses in patients with advanced cancers, but repeat dosing of this agent has not been reported. Patients and methods: Patients with advanced solid tumor malignancies received weekly intravenous infusions of CP-870,893 in four dose level cohorts. Safety and immune pharmacodynamics were assessed. Results: Twenty-seven patients were enrolled. The most common adverse event was transient, infusion-related cytokine release syndrome (CRS). Dose-limiting toxicities included grade 3 CRS and grade 3 urticaria; the maximum tolerated dose (MTD) was estimated to be 0.2 mg/kg. Seven patients (26%) had stable disease as the best clinical response; no partial or complete responses were observed. At the MTD, patient B lymphocytes exhibited persistently increased expression of costimulatory and adhesion molecules without resetting to baseline between doses. In 4 of 8 patients (50%) evaluated at the MTD, there were marked declines in total CD3+ T lymphocytes, as well as CD4+ and CD8+ subsets. Conclusions: Weekly infusions of the agonist CD40 antibody CP-870,893 were well-tolerated, but there was little clinical activity in advanced cancer patients. Correlative studies demonstrate chronic B cell activation and in some patients, T cell depletion. Longer dosing intervals may be desirable for optimal immune pharmacodynamics.

Combination anti-CD137 and anti-CD40 antibody therapy in murine myc-driven hematological cancers.

In order to stimulate antigen presentation and T cell activity against cancer, we treated three different tumor models in mice with the monoclonal antibodies anti-CD40 plus anti-CD137 (BiMab). In a subcutaneous transplantable MC38 colon cancer model, there was significant enhancement in the survival of mice following BiMab treatment. Anti-CD40 has shown considerable success against lymphoma in previous studies by other investigators, and we also showed in this study that, in a model of Eμ-Myc lymphoma, there was a statistically significant enhancement of survival of mice following BiMab treatment. Following the success of the BiMab treatment in the previous two models, we wished to determine if it would be successful in a mouse model of multiple myeloma. Firstly, we tested a transplantable model of disease in which multiple myeloma cells derived from Vk*MYC mice were injected intravenously. A minor proportion of anti-CD137 and BiMab treated mice experienced prolongation of life beyond 250 days. Then we tested the therapy in a spontaneously occurring multiple myeloma model, in Vk*MYC transgenic mice. The majority of mice treated survived longer than control mice, although statistical significance was not demonstrated.

Abstract 3688: Imprime PGG, a novel innate immune therapeutic in phase 2 clinical development, induces mobilization of monocytes and focalized recruitment of innate immune cells to tumor sites

Immune checkpoint inhibitors (CPI) have shown compelling clinical efficacy in multiple tumor types, though only in a minority of treated patients. Significant research and clinical development are focused on expanding CPI efficacy. Imprime PGG is a novel, IV administered 1,3/1,6 β-glucan PAMP (pathogen-associated molecular pattern) that activates innate immune effector cells to enhance tumor killing, to repolarize the suppressive myeloid cells of the tumor microenvironment and to activate the antigen presentation capability of dendritic cells, macrophages and monocytes. In multiple preclinical models, Imprime enhances the anti-tumor efficacy of CPIs. Imprime is now in multiple phase 2 clinical studies in combination with the CPI, pembrolizumab. We sought to understand more precisely how Imprime activates the innate immune system to enable a concerted innate and adaptive anti-cancer immune response. Using multispectral fluorescence IHC we now show that Imprime induces focalized recruitment of innate immune cells to tumor bearing tissue. In the B16F10 experimental metastasis model, Imprime dosed in combination with the tumor-targeting antibody TA-99 can nearly completely repress the outgrowth of pulmonary metastases across a 19 day time course. At 24h post-Imprime treatment, the presence of Ly6G+ neutrophils was evident throughout the lung tissue. At later time points (72h and beyond) the formation of immune cell clusters was readily evident in lungs from Imprime treated mice and rarer in control mice or mice treated only with TA-99. These immune cell clusters were predominately localized to arterioles near B16 tumor sites and comprised of multiple immune cell subtypes including macs, B cells, T cells as well as a monocyte population that are CD11b+, Ly6G- and F4/80- and strongly positive for MHCII. Consistent with these preclinical findings, IV administration of Imprime to healthy human volunteers increased neutrophil and monocyte mobilization into peripheral blood 2-3 fold 4h post infusion. Imprime treatment also resulted in a significantly increased subset of CD16+ monocytes that are known to have higher antigen presentation capability and express higher levels of the activation markers CD86, PD-L1, and HLA-DR (MHCII). Furthermore, RNA expression profiling of whole blood from Imprime-treated volunteers shows increased expression of the CCL3, CCL4, IL-1β and TNF-α, functional mediators produced by these monocyte populations. Together, these data show that Imprime drives the concerted activation of multiple innate immune subtypes and promotes the appearance of unique monocyte populations that may be critical for an Imprime-induced anti-cancer immune response. Citation Format: Steven Leonardo, Nadine Ottosson, Keith Gorden, Takashi Kangas, Xiaohong Qiu, Ross Fulton, Benjamin Harrison, Adria Jonas, Richard Walsh, Katie Ertelt, Jamie Lowe, Richard Huhn, Jeremy Graff, Nandita Bose, Mark T. Uhlik. Imprime PGG, a novel innate immune therapeutic in phase 2 clinical development, induces mobilization of monocytes and focalized recruitment of innate immune cells to tumor sites [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3688. doi:10.1158/1538-7445.AM2017-3688

Abstract B008: Imprime PGG, an intravenously administered beta glucan PAMP activates the innate immune system: A phase I clinical study to evaluate immunopharmacodynamic responses

Imprime PGG (Imprime), in combination with both tumor-targeting and anti-angiogenic antibodies, has shown promising efficacy in multiple phase 2 clinical trials. In numerous pre-clinical in vivo tumor models, Imprime also enhances the efficacy of immune checkpoint inhibitor antibodies in addition to tumor-targeting and anti-angiogenic antibodies. Imprime is a yeast-derived, soluble β-1,3/1,6 glucan that acts as a Pathogen Associated Molecular Pattern (PAMP) to trigger activation of innate immune effector cells (macrophages, monocytes, neutrophils, dendritic cells (DC)), which orchestrate a coordinated anti-cancer immune response with cells of the adaptive immune system. Ex vivo studies with human whole blood have shown that Imprime forms an immune complex with endogenous anti-beta glucan antibodies (ABA) to trigger a constellation of innate immune functions. These include complement activation via the classical complement pathway, select chemokine production, phenotypic activation and enhanced tumor cell killing by neutrophils and macrophages. Imprime also activates antigen-presenting cells (e.g. macrophages, DC), enabling T cell expansion and activation. In vivo, intravenous (IV) injection of Imprime in C57BL/6 mice increases select chemokine expression, triggers neutrophil and monocyte mobilization into circulation and secondary lymphoid organs, and also enhances DC maturation and antigen-specific T-cell priming. In this study, we show that the immunopharmacodynamic (IPD) responses elicited by IV administration of Imprime in healthy human subjects are consistent with the innate immune responses observed in ex vivo human and in vivo mouse studies. Healthy human volunteers (18-65 yr) were administered single (Cohort 1) or multiple (once weekly for 3 wks-Cohort 2) doses of Imprime PGG (4 mg/kg) by IV infusion over 2-3 hrs. Physical examination with vital signs, adverse event solicitation and timed blood sampling for IPD changes were performed. IPD endpoints included complement protein levels, circulating blood cell lineage counts, ABA concentrations, circulating immune complex (CIC) levels, cytokine and chemokine concentrations, as well as binding and activation of blood leukocytes. Cohort 1 and 2 results show that the complement activation proteins C5a and SC5b-9 were significantly increased in the plasma at the end of infusion (EOI) of Imprime. The formation of Imprime:ABA complexes was evident in a substantial drop of free ABA and a concomitant increase in CIC in the serum also at the EOI. IL-8 and MCP-1 were consistently detected between EOI and 1 hr post infusion. Additional chemokines, including MIP-1α, MIP-1β, and IP-10 were also detected in some of the subjects. A significant increase in the neutrophil and monocyte counts was seen in the blood after infusion. Cellular analyses showed Imprime binding to neutrophils, monocytes, and subsets of DC (classical and inflammatory) 15-30 mins after the start of infusion. Additionally, 24 hrs after completion of Imprime administration, a population of non-classical monocytes (CD14/CD16 positive), which are known to have higher antigen presentation capability and thus express higher levels of the activation markers CD86, PD-L1, and HLA-DR, was observed. Importantly, these IPD responses were evident only in subjects with higher ABA levels. Collectively, these data provide the first evidence that, when dosed IV in healthy human subjects, Imprime elicits a constellation of innate immune activating events that are consistent with efficacy in preclinical tumor models. Importantly, these human data also provide the first evidence linking pre-treatment ABA levels and Imprime induced IPD changes, suggesting the plausibility of using pre-treatment ABA levels in the selection of patients most likely to benefit from Imprime-based therapy. Citation Format: Nadine C. Ottoson, Richard D. Huhn, Jamie Lowe, Ben Harrison, Jose Iglesias, Blaine Rathmann, Takashi Kangas, Lindsay R. Wurst, Xiaohong Qiu, Anissa Chan, Adria Bykowski Jonas, Kathryn Fraser, Richard M. Walsh, Katie Ertelt, Steven M. Leonardo, Ross Fulton, Keith Gorden, Mark A. Matson, Mark Uhlik, Jeremy Graff, Nandita Bose. Imprime PGG, an intravenously administered beta glucan PAMP activates the innate immune system: A phase I clinical study to evaluate immunopharmacodynamic responses [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B008.