Byron Hua Kwan

Eradication of large established tumors in mice by combination immunotherapy that engages innate and adaptive immune responses

Checkpoint blockade with antibodies specific for cytotoxic T lymphocyte–associated protein (CTLA)-4 or programmed cell death 1 (PDCD1; also known as PD-1) elicits durable tumor regression in metastatic cancer, but these dramatic responses are confined to a minority of patients. This suboptimal outcome is probably due in part to the complex network of immunosuppressive pathways present in advanced tumors, which are unlikely to be overcome by intervention at a single signaling checkpoint. Here we describe a combination immunotherapy that recruits a variety of innate and adaptive immune cells to eliminate large tumor burdens in syngeneic tumor models and a genetically engineered mouse model of melanoma; to our knowledge tumors of this size have not previously been curable by treatments relying on endogenous immunity. Maximal antitumor efficacy required four components: a tumor-antigen-targeting antibody, a recombinant interleukin-2 with an extended half-life, anti-PD-1 and a powerful T cell vaccine. Depletion experiments revealed that CD8+ T cells, cross-presenting dendritic cells and several other innate immune cell subsets were required for tumor regression. Effective treatment induced infiltration of immune cells and production of inflammatory cytokines in the tumor, enhanced antibody-mediated tumor antigen uptake and promoted antigen spreading. These results demonstrate the capacity of an elicited endogenous immune response to destroy large, established tumors and elucidate essential characteristics of combination immunotherapies that are capable of curing a majority of tumors in experimental settings typically viewed as intractable.

Antigen specificity can be irrelevant to immunocytokine efficacy and biodistribution

Significance Cytokines (potent immunostimulatory proteins) exert powerful antitumor effects but often cause severe whole-body inflammation when used as cancer therapies. Contrary to the current paradigm that fusion to antitumor antibodies can constrain cytokine activity to tumors, we have found that, for some immunocytokines incorporating the cytokine IL-2, the cytokine moiety overrides antibody-mediated targeting, localizing the fusion protein to IL-2 receptor-expressing cells rather than tumor cells. Although the IL-2 immunocytokines did not selectively home to tumors, they persisted longer in circulation than free IL-2, such that a nontoxic immunocytokine dose could synergize with tumor-specific antibody to cure mice with aggressive solid tumors. Cytokine therapy can activate potent, sustained antitumor responses, but collateral toxicity often limits dosages. Although antibody–cytokine fusions (immunocytokines) have been designed with the intent to localize cytokine activity, systemic dose-limiting side effects are not fully ameliorated by attempted tumor targeting. Using the s.c. B16F10 melanoma model, we found that a nontoxic dose of IL-2 immunocytokine synergized with tumor-specific antibody to significantly enhance therapeutic outcomes compared with immunocytokine monotherapy, concomitant with increased tumor saturation and intratumoral cytokine responses. Examination of cell subset biodistribution showed that the immunocytokine associated mainly with IL-2R–expressing innate immune cells, with more bound immunocytokine present in systemic organs than the tumor microenvironment. More surprisingly, immunocytokine antigen specificity and Fcγ receptor interactions did not seem necessary for therapeutic efficacy or biodistribution patterns because immunocytokines with irrelevant specificity and/or inactive mutant Fc domains behaved similarly to tumor-specific immunocytokine. IL-2–IL-2R interactions, rather than antibody–antigen targeting, dictated immunocytokine localization; however, the lack of tumor targeting did not preclude successful antibody combination therapy. Mathematical modeling revealed immunocytokine size as another driver of antigen targeting efficiency. This work presents a safe, straightforward strategy for augmenting immunocytokine efficacy by supplementary antibody dosing and explores underappreciated factors that can subvert efforts to purposefully alter cytokine biodistribution.

Integrin-targeted cancer immunotherapy elicits protective adaptive immune responses

Certain RGD-binding integrins are required for cell adhesion, migration, and proliferation and are overexpressed in most tumors, making them attractive therapeutic targets. However, multiple integrin antagonist drug candidates have failed to show efficacy in cancer clinical trials. In this work, we instead exploit these integrins as a target for antibody Fc effector functions in the context of cancer immunotherapy. By combining administration of an engineered mouse serum albumin/IL-2 fusion with an Fc fusion to an integrin-binding peptide (2.5F-Fc), significant survival improvements are achieved in three syngeneic mouse tumor models, including complete responses with protective immunity. Functional integrin antagonism does not contribute significantly to efficacy; rather, this therapy recruits both an innate and adaptive immune response, as deficiencies in either arm result in reduced tumor control. Administration of this integrin-targeted immunotherapy together with an anti–PD-1 antibody further improves responses and predominantly results in cures. Overall, this well-tolerated therapy achieves tumor specificity by redirecting inflammation to a functional target fundamental to tumorigenic processes but expressed at significantly lower levels in healthy tissues, and it shows promise for translation.

Directed evolution of broadly crossreactive chemokine-blocking antibodies efficacious in arthritis

Chemokine receptors typically have multiple ligands. Consequently, treatment with a blocking antibody against a single chemokine is expected to be insufficient for efficacy. Here we show single-chain antibodies can be engineered for broad crossreactivity toward multiple human and mouse proinflammatory ELR+ CXC chemokines. The engineered molecules recognize functional epitopes of ELR+ CXC chemokines and inhibit neutrophil activation ex vivo. Furthermore, an albumin fusion of the most crossreactive single-chain antibody prevents and reverses inflammation in the K/BxN mouse model of arthritis. Thus, we report an approach for the molecular evolution and selection of broadly crossreactive antibodies towards a family of structurally related, yet sequence-diverse protein targets, with general implications for the development of novel therapeutics.CXCR2 antagonism has been shown to be anti-arthritic, but anti-chemokine therapies usually fail in the clinic owing to redundancy in chemokine-receptor interactions. Here the authors develop single-chain antibodies with multiple chemokine specificities to achieve high affinity and broad specificity to mouse and human CXC chemokines with efficacy in a K/BxN serum transfer model of arthritis.

Abstract A52: Eradication of large established tumors with combination immunotherapy engaging innate and adaptive immunity

Checkpoint blockade against CTLA-4 or PD-1 has demonstrated that an endogenous immune response can be stimulated to elicit durable regressions in advanced cancer, but these dramatic responses are currently confined to a minority of patients. This outcome is probably due in part to the complex network of immunosuppressive pathways present in advanced tumors, which are unlikely to be overcome by intervention at a single signaling checkpoint, requiring a counter-directed network of pro-immunity signals. Here we demonstrate a combination immunotherapy that recruits a diverse set of innate and adaptive immune effectors, enabling robust elimination of tumor burdens that to our knowledge have not previously been curable by treatments relying on endogenous immunity. Maximal anti-tumor efficacy required four components: a tumor antigen targeting antibody, an extended half-life IL-2, anti-PD-1, and a powerful T-cell vaccine. This combination elicited durable cures in a majority of animals, formed immunological memory in multiple transplanted tumor models, and induced sustained tumor regression in an autochthonous BRrafV600E/Pten-/- melanoma model. Multiple innate immune cell subsets, CD8+ T-cells, and cross-presenting dendritic cells were critical to successful therapy. Treatment induced high levels of intratumoral inflammatory cytokines and immune cell infiltration, enhanced antibody-mediated tumor antigen uptake, and promoted antigen spreading. These results demonstrate the capacity of an elicited endogenous immune response to destroy large, established tumors and elucidate essential characteristics of combination immunotherapies capable of curing a majority of tumors in experimental settings typically viewed as intractable. Citation Format: Kelly Dare Moynihan, Cary Opel, Gregory Szeto, Alice Tzeng, Zhu Eric, Jesse Engreitz, Williams Robert, Kavya Rakhra, Michael Zhang, Adrienne Rothschilds, Sudha Kumari, Ryan L. Kelly, Byron Kwan, Wuhbet Abraham, Kevin Hu, Naveen Mehta, Monique Kauke, Heikyung Suh, Douglas A. Lauffenburger, K. Dane Wittrup, Darrell J. Irvine. Eradication of large established tumors with combination immunotherapy engaging innate and adaptive immunity. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A52.