Cytotoxic T-lymphocyte associated antigen 4 (CTLA-4) is an inhibitory checkpoint receptor that is expressed by activated conventional T cells and regulatory T cells and negatively regulates T cell activation. CTLA-4 plays a key role in restraining the adaptive immune response of T cells against a variety of antigens, including self-antigens. However, in the tumor microenvironment, the co-inhibitory CTLA-4 receptor contributes to tumor immune escape. CTLA-4 shares its two ligands, CD80 and CD86, which are largely expressed on antigen-presenting cells, with CD28, a co-activator of T cells; however, CTLA-4 binds CD80 and CD86 with higher affinity than CD28. Blockade of CTLA-4 elicits an effective antitumor immunity in preclinical animal models and has demonstrated favorable clinical results in several solid malignancies. REGN4659 is a human IgG1 antibody that binds to the extracellular domain of human and monkey CTLA-4 with high affinity and specificity. REGN4659 blocks CTLA-4 interaction with CD80 and CD86 ligands and enhances the cytokine release of activated primary human T cells in vitro. The effects of treatment with REGN4659 on the subcutaneous growth of a mouse adenocarcinoma tumors expressing chicken ovalbumin (MC38.Ova) were evaluated in immunocompetent mice genetically engineered to express a human CTLA-4 chimeric protein from the endogenous mouse locus (CTLA-4 hum/hum knock-in). Prophylactic treatment with REGN4659 significantly suppressed MC38.Ova tumor growth in CTLA-4 knock-in mice in a dose dependent manner, and improved mouse survival. Because patients treated systemically with CTLA-4 blockade have suffered from immune-related adverse events, we investigated the efficacy of a peritumoral low-dose administration of a mouse-reactive surrogate anti-CTLA-4 antibody in an attempt to minimize systemic exposure. Peritumoral injection of anti-CTLA-4 antibody was as efficacious as intraperitoneal delivery at inducing anti-tumor immunity. Peritumoral anti-CTLA-4 antibody delivery was associated with lower circulating antibody levels but nonetheless caused systemic antitumor immunity as revealed by growth retardation of concomitantly implanted distant tumors. The mechanism of action and robust anti-tumor efficacy of REGN4659 supports its clinical development for the treatment of human cancers. Citation Format: Elena Burova, Omaira Allbritton, Susannah Brydges, William Poueymirou, Robert Durso, Douglas MacDonald, Ella Ioffe, Markus Mohr, William Olson, Gavin Thurston. Antitumor activity of REGN4659, a fully human anti-CTLA-4 monoclonal antibody, against MC38.Ova tumors grown in immunocompetent human CTLA-4 knock-in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3824.

Abstract Immune checkpoint blockade has revolutionized cancer immunotherapy, and combination treatment with antibodies against multiple inhibitory receptors promises to improve efficacy substantially. However, the spatiotemporal and combinatorial expression profile of various checkpoint receptors on effector T cells and the effect of combined blockade on immune function remain poorly defined. In this study we set out to determine the co-expression kinetics and functional impact of multiple checkpoint receptors (PD-1, LAG-3, TIM-3, and BTLA) on tumor antigen-specific effector T cells in controlled in vitro culture systems and in the tumor environment. To elicit tumor antigen-specific responses we immunized mice with irradiated MC38 tumor cells engineered to express chicken ovalbumin (OVA; MC38.OVA) and used pentamer reagents to identify OVA257-264-specific CD8 T cells. Repeated stimulation with irradiated MC38.OVA cells or irradiated OVA-pulsed splenocytes ex vivo resulted in robust proliferation and activation of antigen-specific T cells as determined by BrdU incorporation and production of IFN-γ. Most OVA-specific cells expressed PD-1 ex vivo and maintained PD-1 expression during repeated cycles of in vitro stimulation. In contrast, antigen-specific T cells did not express TIM-3 or LAG-3 ex vivo, yet both receptors were induced on the majority of PD1 positive, but not PD-1 negative, cells upon repeated stimulation ex vivo, resulting in double and triple positive cells. The frequency of BTLA expressing cells remained low under all conditions. The addition of PD-1 and TIM-3 blocking antibodies during ex vivo restimulation enhanced T cell activation, demonstrating that these receptors mediate inhibitory function. Consistent with our in vitro data demonstrating that repeated antigen stimulation promotes the coordinated expression of checkpoint receptors, we found that the vast majority of MC38.OVA or Colon26 tumor infiltrating T cells (TILs) express PD-1, and most PD-1 positive cells also co-express TIM-3 and LAG-3, whereas these receptors are largely absent from T cells in secondary lymphoid organs. Importantly, combination treatment of tumor-bearing mice with anti-PD-1 and anti-TIM-3, or anti-PD-1 and anti-LAG-3 antibodies enhanced the immune control of tumor growth in both models as compared to the respective monotherapies. Finally, in vitro activation of human PBMC-derived T cells resulted in a similar sequential upregulation of checkpoint inhibitors ultimately resulting in their co-expression. Here, we have established a culture system to delineate the hierarchical expression of inhibitor checkpoint receptors on tumor antigen-specific murine T cells during repeated antigen stimulation. Defining the inhibitory checkpoint receptor landscape will guide the process of identifying the most promising combinations of checkpoint blockers in cancer immunotherapy. Citation Format: Robert J. Durso, Jerry Pei, Michelle Russell, Pratha Budhani, Elena Burova, Chandrika Taduriyasas, Ella Ioffe, Markus Mohrs, Gavin Thurston, Jie Dai. Multiple immune checkpoint receptors are co-expressed on tumor antigen-specific T cells and contribute to tumor immune evasion [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 B129.

PSMA-VRP is a propagation defective, viral replicon vector system encoding PSMA under phase I evaluation for patients with castration resistant metastatic prostate cancer (CRPC). The product is derived from an attenuated strain of the alphavirus, Venezuelan Equine Encephalitis (VEE) virus, and incorporates multiple redundant safety features. In this first in human trial, two cohorts of 3 patients with CRPC metastatic to bone were treated with up to five doses of either 0.9 × 107 IU or 0.36 × 108 IU of PSMA-VRP at weeks 1, 4, 7, 10 and 18, followed by an expansion cohort of 6 patients treated with 0.36 × 108 IU of PSMA-VRP at weeks 1, 4, 7, 10 and 18. No toxicities were observed. In the first dose cohort, no PSMA specific cellular immune responses were seen but weak PSMA-specific signals were observed by ELISA. The remaining 9 patients, which included the higher cohort and the extension cohort, had no PSMA specific cellular responses. PSMA-VRP was well-tolerated at both doses. While there did not appear to be clinical benefit nor robust immune signals at the two doses studied, neutralizing antibodies were produced by both cohorts suggesting that dosing was suboptimal.

SOSIP gp140 trimers represent a soluble, stabilized, proteolytically cleaved form of the HIV-1 envelope (Env) glycoproteins. SOSIP gp140 derived from a subtype A HIV-1 isolate, KNH1144, forms exceptionally stable trimers that resemble virion-associated Env in antigenicity and topology. Here, we used electron microscopy to demonstrate that KNH1144 SOSIP gp140 trimers bound three soluble CD4 molecules in a symmetrical orientation similar to that seen for native Env. We compared the immunogenicities of KNH1144 SOSIP gp140 trimers and gp120 monomers in rabbits and found that the trimers were superior at eliciting neutralizing antibodies (NAbs) to homologous virus as well as neutralization-sensitive subtype B and C viruses. The NAb specificities for SOSIP antisera mapped in part to the CD4 binding site on gp120. We also observed adjuvant-dependent induction of antibodies to the residual levels of host cell proteins (HCPs) contained in the purified Env preparations. When present, HCP antibodies enhanced pseudovirus infection. Our findings are relevant for the further development of Env-based vaccines for HIV-1.

The Env glycoproteins gp120 and gp41 are used in humoral immunity-based vaccines against human immunodeficiency virus (HIV-1) infection. One among many obstacles to such a vaccine is the structural defenses of Env glycoproteins that limit their immunogenicity. For example, gp120 mannose residues can induce immunosuppressive responses in vitro, including IL-10 expression, via mannose C-type lectin receptors on antigen-presenting cells. Here, we have investigated whether mannose removal alters gp120 immunogenicity in mice. Administering demannosylated gp120 (D-gp120) in the T(H)2-skewing adjuvant Alum induced approximately 50-fold higher titers of anti-gp120 IgG, compared to unmodified gp120. While the IgG subclass profile was predominantly T(H)2-associated IgG1, Abs of the T(H)1-associated IgG2a and IgG3 subclasses were also detectable in D-gp120 recipients. Immunizing with D-gp120 also improved T-cell responses. Giving an IL-10 receptor blocking MAb together with unmodified gp120 in Alum increased the anti-gp120 IgG titer, implicating IL-10 as a possible mediator of auto-suppressive responses to gp120.

L-SIGN is a C-type lectin that is expressed on liver sinusoidal endothelial cells. Capture of Hepatitis C virus (HCV) by this receptor results in trans -infection of hepatoma cells. L-SIGN alleles have been identified that encode between three and nine tandem repeats of a 23 residue stretch in the juxtamembrane oligomerization domain. Here, it was shown that these repeat-region isoforms are expressed at the surface of mammalian cells and variably bind HCV envelope glycoprotein E2 and HCV pseudoparticles. Differences in binding were reflected in trans -infection efficiency, which was highest for isoform 7 and lowest for isoform 3. These findings provide a molecular mechanism whereby L-SIGN polymorphism could influence the establishment and progression of HCV infection.

2572 Background and Methods The development of vaccination strategies targeted to tumor antigens holds great promise for cancer therapy. Prostate-specific membrane antigen (PSMA) is an attractive candidate self-antigen for vaccination based on its expression signature. PSMA is abundantly expressed on the surface of prostate cancer cells and on the neovasculature of a wide array of other tumors, but PSMA has limited expression in normal extraprostatic tissues. In prostate cancer, PSMA expression increases with disease progression, becoming highest in high-grade, metastatic, hormone-refractory disease. Our main goals are to overcome tolerance to PSMA by active immunization and to induce recognition and rejection of tumor cells expressing PSMA. We are investigating non-replicating vaccine replicon particles (VRP) based on the alphavirus, Venezuelan equine encephalitis virus. VRP have demonstrated potency in animal models, and infect cells in the lymph nodes where the replicon drives production of large quantities of the heterologous antigen. Robust immune responses can be further amplified by VRP-induced cell apoptosis leading to cross-priming. We are also evaluating a complementary purified protein vaccine based on a novel recombinant soluble PSMA (rsPMSA) immunogen both alone and with VRP in prime-boost regimens. Results When used as single immunogens, VRP encoding full-length human PSMA elicited vigorous, durable Th1-biased cellular and humoral responses in normal BALB/c mice, whereas rsPSMA vaccines induced high levels of Th2-biased antibodies. We have now extended this analysis to human HLA-A2 transgenic mice, a more pertinent model for quantification of human Class I-restricted cellular responses, and to prime-boost regimens. Mice were immunized with VRP alone or in combination with rsPSMA boosts. PSMA-specific immune responses were measured with a panel of cellular and humoral immunoassays, including ELISPOT and intracellular cytokine staining for IFN-γ and IL-4 in T cell subsets. Conclusions Based on these translational findings, the rsPSMA protein vaccine has been advanced into a dose-escalating Phase I clinical trial in advanced prostate cancer, while the VRP-PSMA vaccine is in late-stage preclinical testing. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration AlphaVax, PSMA Development Company, LLC

Target cell tropism of enveloped viruses is regulated by interactions between viral and cellular factors during transmission, dissemination, and replication within the host. Binding of viral envelope glycoproteins to specific cell-surface receptors determines susceptibility to viral entry. However, a number of cell-surface molecules bind viral envelope glycoproteins without mediating entry. Instead, they serve as capture receptors that disseminate viral particles to target organs or susceptible cells. We and others recently demonstrated that the C type lectins L-SIGN and DC-SIGN capture hepatitis C virus (HCV) by specific binding to envelope glycoprotein E2. In this study, we use an entry assay to demonstrate that HCV pseudoviruses captured by L-SIGN+ or DC-SIGN+ cells efficiently transinfect adjacent human liver cells. Virus capture and transinfection require internalization of the SIGN-HCV pseudovirus complex. In vivo, L-SIGN is largely expressed on endothelial cells in liver sinusoids, whereas DC-SIGN is expressed on dendritic cells. Capture of circulating HCV particles by these SIGN+ cells may facilitate virus infection of proximal hepatocytes and lymphocyte subpopulations and may be essential for the establishment of persistent infection.

Hepatitis C virus (HCV) envelope glycoproteins E1/E2 can pseudotype retroviral particles and efficiently mediate entry into target cells. Using this experimental system, we determined HCV tropism for different cell types. Only primary hepatocytes and one hepatoma cell line were susceptible to HCV pseudovirus entry, which could be inhibited by sera from HCV-infected individuals. Furthermore, expression of the putative HCV receptor CD81 on nonpermissive human hepatic but not murine cells enabled HCV pseudovirus entry. Importantly, inhibition of viral entry by an anti-CD81 mAb occurred at a step following HCV attachment to target cells. Our results indicate that CD81 functions as a post-attachment entry coreceptor and that other cellular factors act in concert with CD81 to mediate HCV binding and entry into hepatocytes.

Hepatitis C virus (HCV) infects nearly 3% of the population of the world and is a major cause of liver disease. However, the mechanism whereby the virus targets the liver for infection remains unknown, because none of the putative cellular receptors for HCV are both expressed specifically in the liver and capable of binding HCV envelope glycoproteins. Liver/lymph node-specific intercellular adhesion molecule-3-grabbing integrin (L-SIGN) is a calcium-dependent lectin expressed on endothelial cells of liver and lymph nodes. Dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), a homologous molecule expressed on dendritic cells, binds HIV and promotes infection. By using a virus-binding assay, we demonstrate that L-SIGN and DC-SIGN specifically bind naturally occurring HCV present in the sera of infected individuals. Further studies demonstrate that binding is mediated by the HCV envelope glycoprotein E2 and is blocked by specific inhibitors, including mannan, calcium chelators, and Abs to the lectin domain of the SIGN molecules. Thus, L-SIGN represents a liver-specific receptor for HCV, and L-SIGN and DC-SIGN may play important roles in HCV infection and immunity.