Leslie L. Sharp

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.

Radiotherapy Increases the Permissiveness of Established Mammary Tumors to Rejection by Immunomodulatory Antibodies

It is becoming increasingly evident that radiotherapy may benefit from coincident or subsequent immunotherapy. In this study, we examined whether the antitumor effects of radiotherapy, in established triple-negative breast tumors could be enhanced with combinations of clinically relevant monoclonal antibodies (mAb), designed to stimulate immunity [anti-(α)-CD137, α-CD40] or relieve immunosuppression [α-programmed death (PD)-1]. While the concomitant targeting of the costimulatory molecules CD137 and CD40 enhanced the antitumor effects of radiotherapy and promoted the rejection of subcutaneous BALB/c-derived 4T1.2 tumors, this novel combination was noncurative in mice bearing established C57BL/6-derived AT-3 tumors. We identified PD-1 signaling within the AT-3 tumors as a critical limiting factor to the therapeutic efficacy of α-CD137 therapy, alone and in combination with radiotherapy. Strikingly, all mice bearing established orthotopic AT-3 mammary tumors were cured when α-CD137 and α-PD-1 mAbs were combined with single- or low-dose fractionated radiotherapy. CD8+ T cells were essential for curative responses to this combinatorial regime. Interestingly, CD137 expression on tumor-associated CD8+ T cells was largely restricted to a subset that highly expressed PD-1. These CD137+PD-1High CD8+ T cells, persisted in irradiated AT-3 tumors, expressed Tim-3, granzyme B and Ki67 and produced IFN-γ ex vivo in response to phorbol 12-myristate 13-acetate (PMA) and ionomycin stimulation. Notably, radiotherapy did not deplete, but enriched tumors of functionally active, tumor-specific effector cells. Collectively, these data show that concomitant targeting of immunostimulatory and inhibitory checkpoints with immunomodulatory mAbs can enhance the curative capacity of radiotherapy in established breast malignancy.

Targeting of 4-1BB by monoclonal antibody PF-05082566 enhances T-cell function and promotes anti-tumor activity

Abstract4-1BB (CD137, TNFRSF9) is a costimulatory receptor expressed on several subsets of activated immune cells. Numerous studies of mouse and human T cells indicate that 4-1BB promotes cellular proliferation, survival, and cytokine production. 4-1BB agonist mAbs have demonstrated efficacy in prophylactic and therapeutic settings in both monotherapy and combination therapy tumor models and have established durable anti-tumor protective T-cell memory responses. PF-05082566 is a fully human IgG2 that binds to the extracellular domain of human 4-1BB with high affinity and specificity. In preclinical studies, this agonist antibody demonstrated its ability to activate NF-κB and induce downstream cytokine production, promote leukocyte proliferation, and inhibit tumor growth in a human PBMC xenograft tumor model. The mechanism of action and robust anti-tumor efficacy of PF-05082566 support its clinical development for the treatment of a broad spectrum of human malignancies.

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 3836: Novel conditionally active biologic anti-Axl antibody-drug conjugate demonstrates anti-tumor efficacy and improved safety profile

Axl is a TAM family receptor tyrosine kinase that has been implicated in the pathogenesis of many cancer types. The high level of expression on the cancer cell surface has made it an attractive target for antibody therapeutics. However, Axl is expressed on many normal tissues and has been implicated in wide ranging requisite biological processes including response of endothelial cells to vascular injury, hematopoiesis, and regulation of immune responses. This normal tissue expression may limit Axl as a target for antibody-drug-conjugates (ADC). Conditionally Active Biologics (CAB) technology is a proprietary platform that selects antibodies that bind to target antigen in the context of diseased tissues, but not normal tissues, by taking advantage of the unique cancer microenvironment that is produced largely as a result of the Warburg effect. Using our CAB technology, we have identified anti-Axl Abs that reversibly bind to recombinant Axl and Axl expressing cells under conditions that are present in the tumor microenvironment but not in normal tissues. CAB-Axl antibodies were then conjugated to a model toxin payload to generate CAB-Axl-ADCs. The CAB-Axl-ADCs were active against Axl positive human tumor xenografts with tumor stasis observed at 1mg/kg weekly and tumor regressions observed at 1 mg/kg twice a week dose levels. A non-specific IgG-ADC showed minimal efficacy at the same dose levels. Single dose studies in cynomolgus macaques have demonstrated that CAB-Axl-ADC has reduced liver toxicity and immune system effects compared to Axl-ADCs that bind to Axl under normal conditions. In conclusion, our data is consistent with our work on CAB-EGFR antibodies, and suggests that ADCs generated using the CAB technology provides biologics with increased therapeutic index. Specifically, the CAB-Axl-ADC is an excellent candidate for evaluation as a treatment for human cancers that are Axl positive. Citation Format: Cathy Chang, Gerhard Frey, William J. Boyle, Leslie L. Sharp, Jay M. Short. Novel conditionally active biologic anti-Axl antibody-drug conjugate demonstrates anti-tumor efficacy and improved safety profile. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3836.

Abstract 361: PET imaging of antitumor responses with anti-4-1BB (anti-CD137)

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Agonistic antibodies to 4-1BB (CD137), a member of the tumor necrosis factor family expressed on activated T cells, are being tested for immunotherapy in patients with advanced cancers. Being able to study the mechanism of 4-1BB action in vivo using non-invasive approaches would help guiding its clinical development. We used positron emitting tomography (PET) with [18F]FDG to study how 4-1BB mAb modulates the immune system in a mouse model for cancer treatment. The administration of 1mg/kg of the 4-1BB mAb to Balb/c mice induced reproducible CT26 tumor regressions and improved survival (n=8 mice per group, p = 0.006 for survival by log rank test). The mean tumor size (mm) was also strikingly different (5.8 ± 0.20 4-1BB mAb group vs 8.3 ± 0.19 control, on day 7 after 4-1BB mAb; p < 0.0009). All mice responded to the treatment and complete tumor shrinkage was achieved in 75% of cases, 14 days after 4-1BB mAb administration. Using microPET imaging there was markedly increased mean signal (% ID/g) of [18F]FDG at the tumor site and draining lymph nodes (18.4 ± 1.6 4-1BB mAb group vs. 10.3 ± 0.418 control group). Due to the shrinkage of the tumor in the treated group, we hypothesized this higher PET signal is the result of higher activity of immune cells in the site. In a metabolic probe in vitro uptake assay, there was a 7-fold increase in uptake of [3H]DDG in lymphocytes extracted from tumor-draining lymph nodes and a 2-fold increase in tumor infiltrating lymphocytes (TILS) of mice treated with 4-1BB mAb compared to untreated mice, supporting the in vivo PET data. In conclusion, increased uptake of [18F]FDG by PET scans visualizes 4-1BB mAB-induced anti-tumor immune responses and can be used as a pharmacodynamic readout to guide the development of this class of antibodies in the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 361. doi:1538-7445.AM2012-361

Abstract 4384: Targeting of 4-1BB by monoclonal antibody, PF-05082566, enhances T cell function and promotes antitumor activity

4-1BB (CD137, TNFRSF9) is a costimulatory receptor expressed in an activation induced manner on several subsets of immune cells. Numerous studies of mouse and human T cells indicate that 4-1BB promotes enhanced cellular proliferation, survival, and cytokine production. 4-1BB agonist mAbs have demonstrated efficacy in prophylactic and therapeutic settings in both monotherapy and combination therapy tumor models and have established durable anti-tumor protective T cell memory responses. PF-05082566 is a fully human IgG2 which binds to the extracellular domain of human 4-1BB with high affinity and specificity. In preclinical studies this agonist antibody demonstrated its ability to activate NF-κB and induce downstream cytokine production, promote leukocyte proliferation, and inhibit tumor growth in a human PBMC xenograft tumor model. The mechanism of action and robust anti-tumor efficacy of PF-05082566 support its clinical development for the treatment of a broad spectrum of human malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4384. doi:1538-7445.AM2012-4384

Abstract 3811: Robust anti-tumor activity through targeting integrin α5β1 and eliciting ADCC with a dual functional monoclonal antibody PF-04605412

Integrin α5β1 and its ligand fibronectin are significantly and coordinately over-expressed in tumor associated blood vessels and cancer cells. These molecules have been reported to mediate angiogenesis, tumor survival and metastasis. α5β1 has also been implicated as a poor prognostic marker in a number of human tumor types including NSCLC and ovarian carcinoma. Thus, α5β1 is a potential target for cancer therapy. Accumulating evidence suggests an important role of innate immune effector cells in tumor killing via antibody dependent cellular cytotoxicity (ADCC). Taking advantage of both of these mechanisms, we generated an α5 integrin-specific neutralizing monoclonal antibody (mAb, PF-04605412) with high affinity for Fcγ receptors. In vitro, PF-04605412 potently inhibited α5β1-mediated intracellular signaling, cell adhesion, migration, endothelial cell tubulogenesis, and induced apoptosis of endothelial and tumor cells. Compared to a wild type IgG1 of identical antigen specificity (wt IgG1), PF-04605412 induced a significantly greater tumor and endothelial cell cytotoxicity. ADCC activity correlated with the abundance of NK cells within PBMCs of human donors, but was independent of donor FcγRIIIa polymorphism. In in vivo studies using human xenograft, immuno-xenograft and syngeneic tumor models, PF-04605412 (and an anti-murine α5 surrogate mAb) displayed robust and dose-dependent anti-angiogenesis and anti-tumor efficacy including tumor growth delay, regression and metastasis inhibition, all of which were superior to effects observed with an IgG2 or wt IgG1 of identical antigen specificity. Western blotting and immunohistochemistry analysis of tumor samples showed that anti-tumor efficacy was associated with reduction of total and phosphorylated focal adhesion kinase and positively correlated with α5 expression, infiltrating macrophages, markers of effector cell activity, and apoptosis. Furthermore, depletion of host macrophages abrogated anti-tumor efficacy, indicating a critical contribution of ADCC/phagocytosis to the anti-tumor activity of PF-04605412. The PK-PD relationship, efficacy of PF-04605412 in combination with anti-angiogenic and tumor targeting agents will also be reported. The distinct dual mechanism of action and robust anti-tumor efficacy of PF-04605412 supports the clinical development of this agent for the treatment of human cancers. Short Title: Dual functional mAb PF-04605412 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3811.