Monique J. Kauke

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.

Durable antitumor responses to CD47 blockade require adaptive immune stimulation

Significance Therapeutic antitumor antibodies are widely used clinically. CD47 is an antiphagocytic ligand expressed by tumors that binds the inhibitory receptor signal regulatory protein alpha (SIRPα) on phagocytic cells. Interruption of CD47–SIRPα interactions in immunodeficient mice bearing human tumors enhances therapeutic antitumor antibody responses by promoting phagocytosis of antibody-bound tumor cells. Here, we use a novel anti-CD47 single domain antibody, derived from an alpaca, in an immunocompetent mouse model of melanoma and find that, in contrast to immunodeficient models, CD47 blockade alone is insufficient to enhance the effects of antimelanoma antibodies. However, when combined with blockade of programmed death-ligand 1 (PD-L1), an immune receptor that inhibits antitumor T cell responses, we find synergistic activity, suggesting a role for both innate and adaptive inhibitory pathways in the response to therapeutic antibodies. Therapeutic antitumor antibodies treat cancer by mobilizing both innate and adaptive immunity. CD47 is an antiphagocytic ligand exploited by tumor cells to blunt antibody effector functions by transmitting an inhibitory signal through its receptor signal regulatory protein alpha (SIRPα). Interference with the CD47–SIRPα interaction synergizes with tumor-specific monoclonal antibodies to eliminate human tumor xenografts by enhancing macrophage-mediated antibody-dependent cellular phagocytosis (ADCP), but synergy between CD47 blockade and ADCP has yet to be demonstrated in immunocompetent hosts. Here, we show that CD47 blockade alone or in combination with a tumor-specific antibody fails to generate antitumor immunity against syngeneic B16F10 tumors in mice. Durable tumor immunity required programmed death-ligand 1 (PD-L1) blockade in combination with an antitumor antibody, with incorporation of CD47 antagonism substantially improving response rates. Our results highlight an underappreciated contribution of the adaptive immune system to anti-CD47 adjuvant therapy and suggest that targeting both innate and adaptive immune checkpoints can potentiate the vaccinal effect of antitumor antibody therapy.

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.

Chaperone proteins as single component reagents to assess antibody nonspecificity

ABSTRACT Early stage assays that evaluate monoclonal antibody drug-like properties serve as valuable tools for selection of lead candidates. One liability for clinical development, off-target reactivity, is often assessed by binding to a mixture or panel of noncognate proteins. While robust, these mixes are often ill-defined, and can suffer from issues such as lot-to-lot variability. In this study, we discovered in immunoprecipitation experiments that certain chaperones are present in one of these mixtures;we then explored the use of recombinant chaperone proteins as well-characterized agents to predict antibody nonspecificity. Antibody binding to the heat shock proteins HSP70, HSP90, or trigger factor all served as predictors of cross-interaction propensity, with HSP90 providing the greatest ability to predict antibody clearance rates in mouse. Individual chaperone binding correlates surprisingly closely with binding to complex cell extracts, with the exception of a few “false negatives” (assuming a complex cell extract as the “true” value). As defined reagents, these chaperone reagents present advantages for high throughput assays of nonspecificity.

Cytosolic delivery of siRNA by ultra-high affinity dsRNA binding proteins

Abstract Protein-based methods of siRNA delivery are capable of uniquely specific targeting, but are limited by technical challenges such as low potency or poor biophysical properties. Here, we engineered a series of ultra-high affinity siRNA binders based on the viral protein p19 and developed them into siRNA carriers targeted to the epidermal growth factor receptor (EGFR). Combined in trans with a previously described endosome-disrupting agent composed of the pore-forming protein Perfringolysin O (PFO), potent silencing was achieved in vitro with no detectable cytotoxicity. Despite concerns that excessively strong siRNA binding could prevent the discharge of siRNA from its carrier, higher affinity continually led to stronger silencing. We found that this improvement was due to both increased uptake of siRNA into the cell and improved pharmacodynamics inside the cell. Mathematical modeling predicted the existence of an affinity optimum that maximizes silencing, after which siRNA sequestration decreases potency. Our study characterizing the affinity dependence of silencing suggests that siRNA-carrier affinity can significantly affect the intracellular fate of siRNA and may serve as a handle for improving the efficiency of delivery. The two-agent delivery system presented here possesses notable biophysical properties and potency, and provide a platform for the cytosolic delivery of nucleic acids.

A Raf-Competitive K-Ras Binder Can Fail to Functionally Antagonize Signaling

Mutated in approximately 30% of human cancers, Ras GTPases are the most common drivers of oncogenesis and render tumors unresponsive to many standard therapies. Despite decades of research, no drugs directly targeting Ras are currently available. We have previously characterized a small protein antagonist of K-Ras, R11.1.6, and demonstrated its direct competition with Raf for Ras binding. Here we evaluate the effects of R11.1.6 on Ras signaling and cellular proliferation in a panel of human cancer cell lines. Through lentiviral transduction, we generated cell lines that constitutively or through induction with doxycycline express R11.1.6 or a control protein YW1 and show specific binding by R11.1.6 to endogenous Ras through microscopy and co-immunoprecipitation experiments. Genetically encoded intracellular expression of this high-affinity Ras antagonist, however, fails to measurably disrupt signaling through either the MAPK or PI3K pathway. Consistently, cellular proliferation was unaffected as well. To understand this lack of signaling inhibition, we quantified the number of molecules of R11.1.6 expressed by the inducible cell lines and developed a simple mathematical model describing the competitive binding of Ras by R11.1.6 and Raf. This model supports a potential mechanism for the lack of biological effects that we observed, suggesting stoichiometric and thermodynamic barriers that should be overcome in pharmacologic efforts to directly compete with downstream effector proteins localized to membranes at very high effective concentrations. Mol Cancer Ther; 17(8); 1773–80. ©2018 AACR.

Artificial Anti-Tumor Opsonizing Proteins with Fibronectin Scaffolds Engineered for Specificity to Each of the Murine FcγR Types

We have engineered a panel of novel Fn3 scaffold-based proteins that bind with high specificity and affinity to each of the individual mouse Fcγ receptors (mFcγR). These binders were expressed as fusions to anti-tumor antigen single-chain antibodies and mouse serum albumin, creating opsonizing agents that invoke only a single mFcγR response rather than the broader activity of natural Fc isotypes, as well as all previously reported Fc mutants. This panel isolated the capability of each of the four mFcγRs to contribute to macrophage phagocytosis of opsonized tumor cells and in vivo tumor growth control with these monospecific opsonizing fusion proteins. All activating receptors (mFcγRI, mFcγRIII, and mFcγRIV) were capable of driving specific tumor cell phagocytosis to an equivalent extent, while mFcγRII, the inhibitory receptor, did not drive phagocytosis. Monospecific opsonizing fusion proteins that bound mFcγRI alone controlled tumor growth to an extent similar to the most active IgG2a murine isotype. As expected, binding to the inhibitory mFcγRII did not delay tumor growth, but unexpectedly, mFcγRIII also failed to control tumor growth. mFcγRIV exhibited detectable but lesser tumor-growth control leading to less overall survival compared to mFcγRI. Interestingly, in vivo macrophage depletion demonstrates their importance in tumor control with mFcγRIV engagement, but not with mFcγRI. This panel of monospecific mFcγR-binding proteins provides a toolkit for isolating the functional effects of each mFcγR in the context of an intact immune system.

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.