Naveen K. Mehta

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.

Strong Enrichment of Aromatic Residues in Binding Sites from a Charge-neutralized Hyperthermostable Sso7d Scaffold Library

The Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus is an attractive binding scaffold because of its small size (7 kDa), high thermal stability (Tm of 98 °C), and absence of cysteines and glycosylation sites. However, as a DNA-binding protein, Sso7d is highly positively charged, introducing a strong specificity constraint for binding epitopes and leading to nonspecific interaction with mammalian cell membranes. In the present study, we report charge-neutralized variants of Sso7d that maintain high thermal stability. Yeast-displayed libraries that were based on this reduced charge Sso7d (rcSso7d) scaffold yielded binders with low nanomolar affinities against mouse serum albumin and several epitopes on human epidermal growth factor receptor. Importantly, starting from a charge-neutralized scaffold facilitated evolutionary adaptation of binders to differentially charged epitopes on mouse serum albumin and human epidermal growth factor receptor, respectively. Interestingly, the distribution of amino acids in the small and rigid binding surface of enriched rcSso7d-based binders is very different from that generally found in more flexible antibody complementarity-determining region loops but resembles the composition of antibody-binding energetic hot spots. Particularly striking was a strong enrichment of the aromatic residues Trp, Tyr, and Phe in rcSso7d-based binders. This suggests that the rigidity and small size of this scaffold determines the unusual amino acid composition of its binding sites, mimicking the energetic core of antibody paratopes. Despite the high frequency of aromatic residues, these rcSso7d-based binders are highly expressed, thermostable, and monomeric, suggesting that the hyperstability of the starting scaffold and the rigidness of the binding surface confer a high tolerance to mutation.

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.

Enhancement of Peptide Vaccine Immunogenicity by Increasing Lymphatic Drainage and Boosting Serum Stability

Augmented antitumor vaccines were synthesized by conjugating albumin-binding moieties to peptide antigens. This platform improved vaccine stability and lymphatic distribution, leading to augmented and extended antigen presentation in lymph nodes and enhanced CD8+ T-cell priming. Antitumor T-cell responses have the potential to be curative in cancer patients, but the induction of potent T-cell immunity through vaccination remains a largely unmet goal of immunotherapy. We previously reported that the immunogenicity of peptide vaccines could be increased by maximizing delivery to lymph nodes (LNs), where T-cell responses are generated. This was achieved by conjugating the peptide to 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-PEG (DSPE-PEG) to promote albumin binding, which resulted in enhanced lymphatic drainage and improved T-cell responses. Here, we expanded upon these findings and mechanistically dissected the properties that contribute to the potency of this amphiphile-vaccine (amph-vaccine). We found that multiple linkage chemistries could be used to link peptides with DSPE-PEG, and further, that multiple albumin-binding moieties conjugated to peptide antigens enhanced LN accumulation and subsequent T-cell priming. In addition to enhancing lymphatic trafficking, DSPE-PEG conjugation increased the stability of peptides in serum. DSPE-PEG peptides trafficked beyond immediate draining LNs to reach distal nodes, with antigen presented for at least a week in vivo, whereas soluble peptide presentation quickly decayed. Responses to amph-vaccines were not altered in mice deficient in the albumin-binding neonatal Fc receptor (FcRn), but required Batf3-dependent dendritic cells (DCs). Amph-peptides were processed by human DCs equivalently to unmodified peptides. These data define design criteria for enhancing the immunogenicity of molecular vaccines to guide the design of next-generation peptide vaccines. Cancer Immunol Res; 6(9); 1025–38. ©2018 AACR.

Abstract A42: Combination immunotherapy of an autochthonous murine lung cancer model expressing human CEA as a tumor-associated self-antigen

While cancer immunotherapies like checkpoint inhibitors have resulted in unprecedented clinical success, they only benefit a subset of patients. To improve therapeutic outcomes for greater numbers of patients, one strategy is to rationally combine different immunotherapy modalities. We recently demonstrated that attaching albumin-binding lipophilic tails to peptide antigens or molecular adjuvants (creating amphiphile vaccines) results in enhanced T-cell responses. Additionally, we observed significant tumor regression upon combining tumor-antigen targeting antibodies with extended half-life IL-2 (exPK-IL-2) in mouse models of melanoma and prostate cancer. In the present work, we combined both approaches to treat a spontaneous model of lung adenocarcinoma expressing carcinoembryonic antigen (CEA), an oncofetal protein expressed in some human lung cancers. KrasLSL-G12D/+;;p53fl/fl mice were crossed with transgenic mice expressing human-CEA to generate a CEA-tolerant background. Lung tumors were induced by infection with a lentivirus expressing Cre recombinase and human CEA. Due to the extended latency of tumor initiation in this model, we also generated a CEA-expressing cell line from these mice to test the efficacy of different combination immunotherapy regimens. We discovered that weekly treatments combining a CEA-targeting amphiphile-vaccine, exPK-IL-2, and an anti-CEA antibody with checkpoint inhibitors (anti-PD-1 and -CTLA4) resulted in sustained tumor regression in 50% of mice bearing established tumors. We are currently testing this combination immunotherapy on autochthonous lung tumors. Our results suggest that breaking tolerance to a tumor-associated self-antigen (CEA) and combining immunotherapies to recruit both innate and adaptive immune effectors can have a potent therapeutic effect in intractable tumors like lung cancer. Citation Format: Kavya Rakhra, Eric F. Zhu, Wuhbet Abraham, Kelly D. Moynihan, Naveen Mehta, Karl D. Wittrup, Darrell J. Irvine. Combination immunotherapy of an autochthonous murine lung cancer model expressing human CEA as a tumor-associated self-antigen. [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 A42.

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.

Abstract B120: Characterization and combination immunotherapy treatment of an inducible autochthonous murine lung cancer model expressing human carcinoembryonic antigen (CEA) as a tumor-associated self-antigen

Previous work from our lab has demonstrated that a combination of anti-tumor antibody and an IL-2 fusion protein that exhibits extended serum half-life elicits an immune response that can effectively control a wide variety of tumor models. We have since then combined this therapeutic regimen with a vaccine exhibiting efficient lymph node trafficking that can generate an impressive population of tumor-antigen specific CD8+ T-cells but by itself does not provide good anti-tumor efficacy. The efficacy of this combination immunotherapy is further boosted by immune checkpoint blockade, leading to a robust four-component therapy: 1) anti-tumor antigen antibody; 2) IL-2 fusion protein; 3) anti-tumor antigen vaccine; 4) anti-PD-1 antibody. Although this four-pronged approach is demonstrably effective in the syngeneic subcutaneous melanoma model B16F10, we wish to test its efficacy in a more physiological model. To this end, we have turned to a model developed by the Jacks Lab, known as the KP model: an inducible lung tumor model where lentivirus-driven integration and expression of Cre is able to activate oncogenic Kras and completely remove p53 function. Because our therapeutic regimen requires a targetable tumor-associated antigen with respect to both the antibody and vaccine, we chose to induce expression of human carcinoembryonic antigen (CEA) in these tumors, as CEA has a well-studied structure and biology, and frequently expresses aberrantly in many forms of human adenocarcinomas. Additionally, our lab has previously engineered an antibody targeting CEA possessing picomolar affinity. Finally, to remove any endogenous immunological response against human CEA as a foreign antigen in our mouse system, we have crossed the KP model with a mouse model transgenic for human CEA, which in the literature has been described to have identical spatiotemporal expression of CEA as found in humans and should allow for central tolerance of this antigen. In the course of this work, we have successfully introduced human CEA into our lentivirus constructs and shown tumorigenesis by these constructs in the KP model coincides with expression of tumor-associated CEA, as detected by qPCR. On the therapeutic side, we have tailored the vaccine to successfully drive an anti-CEA CD8+ T-cell response. Performing preliminary therapeutic experiments in a transplant model of the KP tumor with our four-component therapy, we saw tumor control compared to untreated tumors. Upon interrogating the CD8+ T-cell response against CEA, we found 1-15% of CD8+ T-cells in the blood respond to CEA stimulation by intracellular cytokine staining. With regards to the lung tumor model, in the course of establishing the system we have also observed that the growth kinetics of tumors expressing CEA lags behind those tumors without CEA, even in the transgenic background. Preliminary immunophenotyping work by flow cytometry suggests that tumors with CEA seem to have a reduced myeloid-derived suppressor cell (MDSC) population and a higher CD8a+ dendritic cell (DC) population compared to tumors without CEA, suggesting that the former may have a less immunosuppressive tumor microenvironment that is better able to prime an anti-tumor CD8+ T-cell response. We will be planning to conduct therapeutic trials in the more physiological lung tumor KP model in the near future, as well as investigate the differences in the immune response with tumors expressing or lacking CEA. Citation Format: Eric F. Zhu, Kavya Rakhra, Naveen Mehta, Kelly D. Moynihan, Cary F. Opel, Darrell J. Irvine, K. Dane Wittrup. Characterization and combination immunotherapy treatment of an inducible autochthonous murine lung cancer model expressing human carcinoembryonic antigen (CEA) as a tumor-associated self-antigen. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B120.