In vitro human T-cell costimulation by CD28xVISTA BS2 potentiates LNCaP killing by a CD3xPSMA T-cell engager.

CD28xVISTA geometries BS2 and BS3 display efficient target binding.

Expression of VISTA on Kasumi-3 and LNCaP cells.

Reinvigoration of tumor-reactive T-cells using co-stimulatory bispecific antibodies (bsAbs) targeting CD28 or CD137 is emerging as a promising therapeutic strategy. Conditional, tumor-specific recruitment can offer a necessary layer of control and specificity. We developed pH-selective CD28xVISTA bsAbs to act specifically within the acidic tumor microenvironment (TME), aiming for enhanced T-cell-mediated cancer cell killing while minimizing systemic T-cell activation and Cytokine Release Syndrome (CRS) risk. CD28 agonism by CD28xVISTA bsAbs relies on pH-selective engagement of VISTA, a protein robustly expressed on myeloid cells highly prevalent in most solid tumors. This modality avoids engagement of tumor-associated antigens (TAAs) with the potential to provide highly tumor specific activity with minimal on-target/off-tumor side effects. We report the identification of a lead candidate with pH-dependent simultaneous engagement of both targets, and VISTA-dependent CD28 signaling in a reporter cell line. CD28xVISTA avidly bound VISTA-positive cells, and co-stimulation was shown in vitro by its ability to activate and expand T-cells and enhance T-cell mediated cancer cell killing in co-cultures of human PBMCs and cancer cells in the presence of a TAA-targeted anti-CD3 T-cell engager. Interestingly, our findings support both signaling in cis (between T-cell and cell displaying peptide-MHC complex) and in trans with stimulation occurring through CD28 clustering outside of the immune synapse. Our lead candidate displayed efficient tumor growth inhibition of human VISTA-expressing MC38 cells in a humanized CD28 syngeneic mouse model in combination with PD-1 blockade. Importantly, our CD28xVISTA bsAb showed no signs of superagonistic properties in several in vitro assays geared towards revealing induction of CRS. Our data supports clinical development in combination with anti-PD-1 or any TAA-targeted anti-CD3 T-cell engagers developed for solid tumors. ### Competing Interest Statement T.T., F.D.S., Z-G.J., Z.B., A.O., Y.K., K.M., V.S., A.M, and E.H.vdH are current or former employees of Sensei Biotherapeutics, Inc. The authors declare no other conflicts of interest.

Reinvigoration of tumor-reactive T cells using costimulatory bispecific antibodies (bsAb) targeting CD28 is emerging as a promising therapeutic strategy. Conditional, tumor-specific recruitment can offer a layer of control and specificity. We developed pH-selective CD28xV-domain Ig-containing suppressor of T-cell activation (VISTA) bsAbs to act specifically within the acidic tumor microenvironment, aiming for enhanced T cell-mediated cancer cell killing while minimizing systemic T-cell activation and cytokine release syndrome risk. CD28 agonism by our CD28xVISTA bsAbs relies on pH-selective engagement of VISTA, a protein robustly expressed on myeloid cells abundant in most solid tumors. Our lead candidate displayed pH-dependent engagement of VISTA and simultaneous binding to CD28, resulting in VISTA-dependent CD28 signaling in a reporter cell line. CD28xVISTA avidly binds VISTA+ cells, and costimulatory activity was shown in vitro by its ability to activate and expand T cells and enhance T cell-mediated cancer cell killing in cocultures of human peripheral blood mononuclear cells and cancer cells in the presence of a tumor-associated antigen-targeted anti-CD3 T-cell engager. This CD28xVISTA bsAb efficiently inhibited the growth of human VISTA-expressing MC38 tumors in a humanized CD28 syngeneic mouse model in combination with PD-1 blockade. Our findings support signaling both in cis (between T cell and target cell displaying peptide-MHC complex) and in trans, with stimulation occurring through CD28 clustering outside of the immune synapse. This CD28xVISTA bsAb showed no signs of superagonistic properties in several in vitro cytokine release syndrome assays. Thus, our data support clinical development for solid tumors in combination with anti-PD-1 or tumor-associated antigen-targeted anti-CD3 T-cell engagers.

Tumor Growth Inhibition Summary and Statistics

pH selective CD28xVISTApH-sens BS2 bsAb inhibits MC38-hVISTA tumor growth in hCD28 KI mice in combination with anti-PD-1.

Full blood cell characterization from ex vivo human whole blood ID.Flow assay.

VISTA, an inhibitory myeloid-T-cell checkpoint, holds promise as a target for cancer immunotherapy. However, its effective targeting has been impeded by issues such as rapid clearance and cytokine release syndrome observed with previous VISTA antibodies. Here we demonstrate that SNS-101, a newly developed pH-selective VISTA antibody, addresses these challenges. Structural and biochemical analyses confirmed the pH-selectivity and unique epitope targeted by SNS-101. These properties confer favorable pharmacokinetic and safety profiles on SNS-101. In syngeneic tumor models utilizing human VISTA knock-in mice, SNS-101 shows in vivo efficacy when combined with a PD-1 inhibitor, modulates cytokine and chemokine signaling, and alters the tumor microenvironment. In summary, SNS-101, currently in Phase I clinical trials, emerges as a promising therapeutic biologic for a wide range of patients whose cancer is refractory to current immunotherapy regimens. VISTA is a pH-dependent inhibitory checkpoint for T-cells that is abundant on myeloid lineage cells and antagonists of VISTA may successfully reinvigorate anti-tumour immunity. Here, the authors show that the antibody SNS-101, which is currently being investigated in humans in a clinical trial, is characterized by pH-sensitivity that endows it with favorable pharmacokinetic and safety profiles, and enhanced therapeutic effect when combined with PD-1 checkpoint inhibitors.

ObjectivesDespite their efficacy, some immunotherapies have been shown to induce immune-related adverse events, including the potentially life-threatening cytokine release syndrome (CRS), calling for reliable and translational preclinical models to predict potential safety issues and investigate their rescue. Here, we tested the reliability of humanized BRGSF mice for the assessment of therapeutics-induced CRS features in preclinical settings.MethodsBRGSF mice reconstituted with human umbilical cord blood CD34+ cells (BRGSF-CBC) were injected with anti-CD3 antibody (OKT3), anti-CD3/CD19 bispecific T-cell engager Blinatumomab, or VISTA-targeting antibody. Human myeloid and dendritic cells’ contribution was investigated in hFlt3L-boosted BRGSF-CBC mice. OKT3 treatment was also tested in human PBMC-reconstituted BRGSF mice (BRGSF-PBMC). Cytokine release, immune cell distribution, and clinical signs were followed.ResultsOKT3 injection in BRGSF-CBC mice induced hallmark features of CRS, specifically inflammatory cytokines release, modifications of immune cell distribution and activation, body weight loss, and temperature drop. hFlt3L-boosted BRGSF-CBC mice displayed enhanced CRS features, revealing a significant role of myeloid and dendritic cells in this process. Clinical CRS-managing treatment Infliximab efficiently attenuated OKT3-induced toxicity. Comparison of OKT3 treatment’s effect on BRGSF-CBC and BRGSF-PBMC mice showed broadened CRS features in BRGSF-CBC mice. CRS-associated features were also observed in hFlt3L-boosted BRGSF-CBC mice upon treatment with other T-cell or myeloid-targeting compounds.ConclusionsThese data show that BRGSF-CBC mice represent a relevant model for the preclinical assessment of CRS and CRS-managing therapies. They also confirm a significant role of myeloid and dendritic cells in CRS development and exhibit the versatility of this model for therapeutics-induced safety assessment.

Abstract Introduction: Tumor-specific recruitment of co-stimulatory bispecific antibodies (bsAbs) has emerged as a promising therapeutic strategy. Here, we investigated pH-selective CD28xVISTA bsAbs to act selectively within the acidic tumor microenvironment (TME). Our CD28xVISTA bsAbs are designed for tripartite “trans-activation” of CD28 in the TME, aiming for enhanced T-cell-mediated cancer cell killing while minimizing systemic T-cell activation and Cytokine Release Syndrome (CRS) risk. Experimental Procedures: We evaluated various CD28xVISTA bsAbs, focusing on a prototype 1+2 format with monovalent CD28 binding, bivalent VISTA binding and Fc receptor interaction null mutations (BS2). BS2 was tested for T-cell trans-activation using Jurkat-IL-2-luciferase reporter cells in the presence of HEK293 cells expressing membrane bound OKT3-scFv and CHO cells expressing human VISTA. BS2’s potency was further evaluated in an xCelligence-based human T-cell killing assay that dynamically monitors growth of LNCaP prostate cancer cells co-cultured with VISTA+ Kasumi-3 cells, alone or in combination with a CD3xPSMA bispecific T-cell engager. T-cell activation and proliferation were also measured using flow cytometry with CD3, CD4, CD8 and CD25 markers. Additionally, we modified BS2 to introduce pH-selective VISTA binding, limiting its activity to the TME, and tested this version in a syngeneic mouse model in combination with anti-murine PD-1 (anti-mPD-1). Finally, cytokine release from human PBMCs, co-cultured with human umbilical vein endothelial cells (HUVECs) and treated with the pH-selective BS2 (or TGN1412 as a positive control), was measured using a bead-based multiplex immune assay. Results: Our data show that the combination of anti-mPD-1 and our prototype CD28xVISTA bsAb (BS2) with pH-selective VISTA binding significantly inhibited tumor growth in vivo. In vitro, CD28xVISTA co-stimulation in trans by BS2 potentiated the activity of a CD3xPSMA bispecific T-cell engager in our human T-cell killing assay. Cytokine release assessments demonstrated negligible induction of inflammatory cytokines, indicating a favorable safety profile for this antibody. Conclusion: Our study demonstrates the feasibility of cis and trans CD28 co-stimulation using a CD28xVISTA bsAb (BS2). This approach, which bypasses the need for tumor-associated antigens (TAAs) required by other CD28xTAA bispecifics in development, suggests a potentially safer alternative for T-cell engagement and stimulation. Moreover, our novel myeloid-directed TME-selective co-stimulation strategy with a CD28xVISTA bsAb may broaden the potential for T-cell engagement approaches in solid tumors by enabling rational combinations with existing CD3xTAA T-cell engagers without competing for the same target. Citation Format: Thomas Thisted, Zhi-Gang Jiang, Zuzana Biesova, Adejumoke Onumajuru, Yuliya Kleschenko, Kanam Malhotra, Vikas Saxena, Arnab Mukherjee, F. Donelson Smith, Edward H. van der Horst. Conditionally active CD28xVISTA bispecific antibodies induce myeloid-driven tumor-specific T-cell co-stimulation for improved cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5294.

Background CD39 expression is upregulated in the tumor microenvironment (TME), which is characterized by high levels of extracellular ATP (eATP) and low pH. CD39 catalyzes the rate-limiting degradation step of this immunostimulatory ATP, leading to a rise in immunosuppressive adenosine (ADO). CD39 displays broad expression on endothelial cells and macrophages, which represents a significant peripheral sink for CD39 targeting antibodies. Our strategy aims to circumvent this problem through delivery of pH-selective anti-CD39 blocking antibodies that will achieve a high target occupancy in the tumor, maintaining eATP and inhibiting ADO generation to enhance anti-tumor immunity. Methods Anti-CD39 antibodies were generated through a yeast-based screening platform and characterized for efficacy of blocking CD39 activity. Cell lines (HEK293 stably overexpressing CD39, parental HEK293, CD39+ SK-MEL28, and CD39high ARH77) or soluble CD39 protein were incubated with candidate antibodies. CD39 enzymatic activity was determined using CellTiter Glo 2.0 assay (Promega) and EnzCheck Phosphate Assay (Thermo Fisher). Results Monovalent affinity of 83 candidate antibodies was measured; KDs ranged from 1 × 10−9 - 5 × 10−7M under both physiological (pH 7.4) and acidic (pH 6.0) conditions. All 83 antibodies bound CD39-overexpressing HEK293 cells at pH 6.0; 76 bound CD39-overexpressing HEK293 cells at pH 7.4. We identified 30 antibodies (100 nM) that block CD39 activity on HEK293-CD39 cells (threshold: > 5% inhibition) under either pH 7.4 or pH 6.0 conditions. 26 antibodies inhibit CD39 activity at pH 7.4, whereas 22 antibodies block CD39 at pH 6.0. Among these 30 antibodies, ten (at 200 nM) inhibit CD39 activity on SK-MEL28 cells (moderate CD39 expression) at both pH 7.4 and pH 6.0; 5 of these preferentially inhibit CD39 at pH 6.0 (threshold: > 7.5% inhibition). The 30 antibodies selected above were tested on ARH77 cells (high CD39 expression). Among them, 16 antibodies inhibited CD39 enzymatic activity beyond 10% at pH 6.0 (maximal inhibition: 43%) and > 6.5% at pH 7.4 (maximal inhibition: 35%). All 16 suppressed soluble CD39 activity under both pH conditions. A set of 8 antibodies demonstrating higher efficacy of CD39 inhibition on both cell lines at pH 6.0 versus pH 7.4 were prioritized for lead optimization. Summary and conclusions A panel of 83 antibodies were identified and characterized for inhibition of CD39 enzymatic activity at neutral and acidic pH. The 8 most promising antibodies in terms of pH-selective CD39 binding and inhibition are currently undergoing lead optimization. This pH-dependent TME targeting strategy may alleviate undesirable properties of on-target/off-tumor binding such as target-mediated drug disposition (that results in suboptimal pharmacokinetic properties) and effectively bolster the specific blocking of CD39 in the local tumor environs. Citation Format: Aiping Bai, Elise Renahan, Kanam Malhotra, Thomas Thisted, F. Donelson Smith, Robert H Pierce, Edward H van der Horst. Identification of conditionally active antibodies that selectively block CD39 activity in the acidic tumor microenvironment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4645.

Immune checkpoints and other immunoregulatory targets can be difficult to precisely target due to expression on non-tumor immune cells critical to maintaining immune homeostasis in healthy tissues. On-target/off-tumor binding of therapeutics results in significant pharmacokinetic and pharmacodynamic problems. Target-mediated drug disposition (TMDD) significantly limits effective intratumoral drug levels and adversely affects anti-tumor efficacy. Target engagement outside the tumor environment may lead to severe immune-related adverse events (irAEs), resulting in a narrowing of the therapeutic window, sub-optimal dosing, or cessation of drug development altogether. Overcoming these challenges has become tractable through recent advances in antibody engineering and screening approaches. Here, we review the discovery and development of conditionally active antibodies with minimal binding to target at physiologic pH but high-affinity target binding at the low pH of the tumor microenvironment by focusing on the discovery and improved properties of pH-dependent mAbs targeting two T cell checkpoints, VISTA and CTLA-4.

BackgroundTumor associated macrophages (TAMs) are important regulators of immunosuppression in the tumor microenvironment (TME) and are often associated with poor clinical responses in patients. VSIG4 (V-Set and Immunoglobulin domain containing 4) is a negative checkpoint regulator (NCR) and its expression has been well established on both tissue resident macrophages and TAM populations.1-3 However, the inhibitory molecular mechanism of VSIG4 remains largely unknown1 2 and many therapeutic strategies targeting TAMs in general have been limited either by a lack of TME-specificity or high target abundance leading to target-mediated drug disposition (TMDD).4 5 The development of immunomodulatory antibodies that selectively target antigens in acidic environments, such as the TME, has the potential to increase tumor exposure and reduce toxicity.6 We have characterized endogenous expression patterns of VSIG4 and observed an absence of expression on peripheral immune cell populations but a significant induction on differentiated primary macrophages under conditions similar to the TME. Additionally, we have performed a proteomics screen for potentially novel T-cell receptor(s) that interact with VSIG4.MethodsEndogenous expression patterns of VSIG4 were characterized in primary human immune cell populations including whole blood, peripheral blood mononuclear cells (PBMCs), and polarized macrophage populations. The induction of VSIG4 expression in polarized macrophage populations was demonstrated by both flow cytometry and RNA expression analysis. A Ligand Receptor Capture-Trifunctional Chemoproteomic Reagents (LRC-TriCEPS)-based proteomics strategy was used to identify receptors on primary human T-cells that interact with recombinant VSIG4 protein.7ResultsOur results show a robust upregulation of VSIG4 expression in polarized human macrophage populations. Additionally, we established multiple functional assays demonstrating VSIG4-mediated suppression of primary human T-cells. Finally, the LRC-TriCEPS-based proteomics screen yielded novel candidate receptors that interact with VSIG4.ConclusionsVSIG4 inhibits human T-cell activation in multiple assay formats and through numerous functional measurements. A group of T-cell receptors was found to be involved in novel interactions with VSIG4, and their potential roles in VSIG4-mediated regulation are currently being validated. Collectively, our work will lead to an enhanced mechanistic understanding of how VSIG4 suppresses T-cell activation and provide a strategy and tools for discovery of therapeutic relevant anti-VSIG4 antibodies.ReferencesLiao et al. Laboratory Investigation. 2014; 94: 706–715. Zang et al. J Clin Invest. 2006; 116: 2590–2593. Helmy et al. Cell. 2006; 124: 915–927. Ries et al. Cancer Cell. 2014; 25: 846–859. Graversen et al. Mol Ther. 2012; 20: 1550–1558. Johnston et al. Nature. 2019; 574: 565–570. Frei et al. Nat Biotechnol. 2012; 30: 997–1001.

Background VISTA (V-domain Ig suppressor of T-cell activation) is a negative checkpoint regulator (NCR), highly expressed on myeloid cells.1 PSGL-1 on T-cells has been identified as a novel NCR that limits survival and promotes T-cell exhaustion.2 Recently, VISTA was reported to bind PSGL-1 and suppress T-cell activity exclusively under acidic conditions (~pH 6 in lymph nodes or the tumor microenvironment).3, 4 Although VISTA inhibition demonstrated excellent therapeutic combinability with other modalities targeting NCRs (e.g. CTLA-4, PD-1/PD-L1),5 clinical development of anti-VISTA antibodies has been challenging due to: 1) high clearance via target-mediated drug disposition (TMDD) by VISTA+ neutrophils and monocytes at physiologic pH; and 2) cellular activation and cytokine release syndrome (CRS) at sub-therapeutic doses by engagement of VISTA in the blood.6 We developed SNS-101, a human monoclonal IgG1 antibody specific for the protonated, active form of VISTA, which is designed to disrupt the immunosuppressive VISTA:PSGL-1 interaction, avoid TMDD and mitigate potential CRS. Methods The binding potential of SNS-101 to VISTA+ cells was determined in human and non-human primate (NHP) whole-blood by flow cytometry. The effect of SNS-101 on human monocytes and T-cells was evaluated in vivo in human CD34+ cord blood cell reconstituted BRGSF mice, which develop both human lymphoid and myeloid compartments. The pharmacokinetic (PK) profile was assessed in NHPs. Anti-tumor efficacy was assessed in VISTA-KI mice implanted with the syngeneic tumor model, MC38, and tumor-infiltrating T-cells were analyzed by flow cytometry. Results SNS-101 did not bind to human or NHP VISTA+ monocytes, neutrophils and natural killer cells. In humanized BRGSF mice, SNS-101 induced significant expansion of CD4 and CD8 central memory (CCR7+CD45RA-), and naïve (CCR7+CD45RA+) CD8 T-cells, respectively, but had no significant impact on monocyte activation. PK studies in NHPs showed linear elimination kinetics. Conversely, a non-pH-sensitive antibody bound VISTA+immune cells, induced monocyte activation followed by a decrease in cell numbers and was rapidly cleared in NHPs. Anti-tumor efficacy studies in MC-38 demonstrate that SNS-101 enhanced anti-PD-1 response and dose-dependently increased tumor-infiltrating CD8 T-cells. Conclusions Our results demonstrate that SNS-101 exhibits linear elimination kinetics in NHPs, overcoming TMDD-induced PK limitations observed with other anti-VISTA antibodies. Importantly, SNS-101 induced expansion of naïve and memory T-cell phenotypes in vivo without activation or depletion of monocytes, differentiating it from non-pH-selective VISTA antibodies. In the MC-38 syngeneic tumor model, SNS-101 demonstrated significant enhancement of anti-tumor effects in combination with anti-PD-1 antibodies through an increase in CD8+ T-cells. References Yuan L, Tatineni J, Mahoney KM, et al. VISTA: a mediator of quiescence and a promising target in cancer immunotherapy. Trends Immunol. 2021; 42:209–227. Tinoco R, Carrette F, Barraza ML, et al. PSGL-1 is an immune checkpoint regulator that promotes T cell exhaustion. Immunity. 2016; 44:1190–1203. Johnston RJ, Su LJ, Pinckney J, et al. VISTA is an acidic pH-selective ligand for PSGL-1. Nature 2019; 574:565–570. Wu H, Estrella V, Beatty M, et al. T-cells produce acidic niches in lymph nodes to suppress their own effector functions. Nat Commun. 2020; 11:4113. Gao J, Ward JF, Pettaway CA, et al. VISTA is an inhibitory immune checkpoint that is increased after ipilimumab therapy in patients with prostate cancer. Nat. Med. 2017; 23:551–555. Curis Corporate Presentation Jan 2022 [http://investors.curis.com/events-and-presentations?item=100]

e14504 Background: Active cancer immunotherapeutics frequently give rise to immune-mediated adverse events (AEs), including cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICANS). On-target, off-tumor activation of myeloid lineage cells (e.g. monocytes) has been implicated as a factor in the generation of these immune-mediated AEs. VISTA (V-domain Ig suppressor of T-cell activation) is an immune checkpoint, which is highly expressed on myeloid cells, and binds PSGL-1 on T-cells, but only when ‘activated’ by protonation at low pH (̃pH 6). Inhibition of the VISTA:PSGL-1 interaction in the acidic tumor microenvironment has been shown to be efficacious in multiple syngeneic murine tumor models. However, antibodies binding to VISTA at physiological pH 7.4 have significant potential to induce dose-limiting toxicities such as CRS and/or ICANS and be prematurely eliminated from circulation through targeted-mediated drug disposition (TMDD), reducing the likelihood of reaching efficacious drug occupancy levels. Among several non-pH-selective antibodies in clinical development, JNJ-61610588 (now CI-8993) induced dose-limiting on-target CRS at subtherapeutic dose levels and exhibited TMDD. To mitigate potential CRS and prevent TMDD, we developed SNS-101, a highly selective monoclonal IgG1 antibody for “active” VISTA, which inhibits the critical interaction with PSGL-1. Methods: Using flow cytometry at physiological pH, we compared SNS-101 with clinical stage non-pH-selective anti-VISTA antibodies and examined binding to VISTA-positive cell populations in human PBMCs as well as in human CD34+ cord blood cells reconstituted BRGSF-HIS mice (humanized BRGSF-HIS mice), which develop both human lymphoid and myeloid compartments. Furthermore, we performed in vitro and in vivo CRS studies in a HUVEC/PBMC co-culture system and humanized BRGSF-HIS mice, respectively. Results: Under physiological pH, non-pH-selective antibodies bound to monocytes, neutrophils and NK cells whereas SNS-101 did not exhibit any significant interactions. CRS assays indicate that the magnitude of induced cytokine levels was significantly higher with non-pH-selective antibodies compared to SNS-101. Conclusions: We assessed the binding profile of SNS-101 vs. non-pH-selective VISTA antibodies. Our results demonstrate that SNS-101 does not bind to VISTA-positive cells in circulating blood. In addition, in vitro and in vivo CRS studies suggest that SNS-101 has a lower risk of inducing CRS compared to non-pH selective anti-VISTA antibodies, alleviating liabilities previously associated with anti-VISTA antibodies. IND-enabling studies, including pharmacokinetic and toxicology studies, are ongoing.

Use of nicotine-specific monoclonal antibodies (mAbs) to sequester and reduce nicotine distribution to brain has been proposed as a therapeutic approach to treat nicotine addiction (the basis of tobacco use disorder). A series of monoclonal antibodies with high affinity for nicotine (nic•mAbs) was isolated from B-cells of vaccinated smokers. Genes encoding 32 unique nicotine binding antibodies were cloned, and the mAbs expressed and tested by surface plasmon resonance to determine their affinity for S-(–)-nicotine. The highest affinity nic•mAbs had binding affinity constants ( K D ) between 5 and 67 nM. The 4 highest affinity nic•mAbs were selected to undergo additional secondary screening for antigen-specificity, protein properties (including aggregation and stability), and functional in vivo studies to evaluate their capacity for reducing nicotine distribution to brain in rats. The 2 most potent nic•mAbs in single-dose nicotine pharmacokinetic experiments were further tested in a dose-response in vivo study. The most potent lead, ATI-1013, was selected as the lead candidate based on the results of these studies. Pretreatment with 40 and 80 mg/kg ATI-1013 reduced brain nicotine levels by 56 and 95%, respectively, in a repeated nicotine dosing experiment simulating very heavy smoking. Nicotine self-administration was also significantly reduced in rats treated with ATI-1013. A pilot rat 30-day repeat-dose toxicology study (4x200mg/kg ATI-1013) in the presence of nicotine indicated no drug-related safety concerns. These data provide evidence that ATI-1013 could be a potential therapy for the treatment of nicotine addiction.

BackgroundImmunotherapies, especially immune checkpoint inhibitors, have become a cornerstone of cancer treatment. Remarkable clinical responses have been observed blocking the programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) axis across a spectrum of indications. However, innate and/or acquired resistance to anti-PD-1 blockade remains a major challenge. V-domain Ig suppressor of T-cell activation (VISTA) is a B7-family member, which promotes T-cell and myeloid quiescence and represents a promising target, particularly in combination with anti-PD-1/PD-L1 treatment. Recently, the interaction of VISTA with its receptor PSGL-1 was demonstrated to be significantly enhanced by the acidic tumor microenvironment (TME). As VISTA is highly expressed on myeloid cells, including those in the blood, antibodies binding VISTA at physiological pH 7.4 could result in rapid elimination from circulation through targeted-mediated drug disposition, making efficacious drug occupancy levels difficult to reach and potentially narrowing the therapeutic window. An antibody engineered to selectively bind and block VISTA at low pH in the TME may therefore be an ideal drug candidate.MethodsIn this study, fully human anti-VISTA antibodies were generated through pH-selective enrichment strategies of a yeast-based display library comprising highly diverse synthetic immune repertoires. The ‘parental’ antibodies have been extensively characterized using in vitro flow-cytometry, surface-plasmon resonance (SPR) and PSGL-1/VISTA inhibition assays in primary human CD4 and CD8 T-cells at pH 6.0 and pH 7.4. Eight parental antibodies were identified and tested for combinatorial efficacy with anti-PD-1 in vivo in human VISTA knock-in mice inoculated with syngeneic MC-38 tumors. These antibodies underwent further optimization for enhanced binding affinity at pH 6.0 and decreased binding at pH 7.4. ‘Progeny’ antibody ranking was based on the same in vitro and in vivo characterization as parental antibodies.ResultsEighty four parental antibodies were initially discovered. Flow-cytometry and SPR analysis revealed candidates displaying pH-dependent binding to endogenously expressed native VISTA on cells, and a PSGL-1/VISTA inhibition assay at pH 6.0 was run to identify and rank potent interface blockers. Eight candidate antibodies were tested in an in vivo intervention study in combination with anti-murine PD-1 demonstrating varied combinatorial efficacy with a subset leading to superior tumor rejection. Characterization of optimized progeny antibodies led to identification of anti-VISTA antibody SNS-101.ConclusionsEnrichment of highly diverse antibody libraries led to the identification of a pH-selective inhibitory anti-VISTA antibody SNS-101, which exerts excellent combinability with anti-PD-1 leading to superior anti-tumor activity in a mouse model.

Aspartate β-hydroxylase (ASPH) is a unique cancer target that is expressed exclusively on the surface of cancer cells. It is a type II transmembrane protein predominantly expressed during embryogenesis where it promotes cell migration for organ development, but has very low expression in healthy adult tissue, and is localized to the intracellular compartment of the endoplasmic reticulum. However, re-expression and translocation to the tumor cell surface has been detected in more than 20 different types of cancers, with expression levels inversely correlated with disease prognosis. Promising phase I clinical trial results have been achieved with our ASPH-targeted vaccine, SNS-301, in the treatment of biochemical recurrence of prostate cancer (reported previously: Nordquist, et al. (2018) J Clin Oncol. 36, suppl; abstr e15166; Nordquist, et al. Annals of Oncology, 29, suppl_8, 1 October 2018, mdy279.404). This study demonstrated reductions in disease-specific biomarkers (i.e. PSA) which correlated to the breaking of immune self-tolerance as indicated by stimulation of both cellular and humoral ASPH-specific immune responses. Importantly the vaccine was well tolerated with minimal numbers of adverse events or off-target effects. Thus, ASPH represents a promising target for immunotherapeutic approaches to human cancers. Here we present studies that we have initiated towards the development of ASPH-targeted CAR (chimeric antigen receptor)-T cell therapies. A series of ASPH-targeting chimeric antigen receptor constructs have been produced which combine various ASPH-specific scFv domains and either the 4-1BB or CD28 co-stimulatory domains. The ASPH-specific scFv explored include several scFv that we previously isolated from a fully human non-immune scFv library displayed on the surface of yeast and targeted at different epitopes within the extracellular domain of ASPH as well as several affinity matured variations of these scFv (Yeung, et al. (2007) Human Antibodies 16, 163-76). All constructs were incorporated into a lentiviral vector which was used to transfect human CD4+ and CD8+ T-cells. CAR expression and affinities were determined by flow cytometry using a fluorescently labeled antigen. Cell killing efficacy and cytokine responses of the resulting CAR-T cells were observed in co-culture experiments with a series of ASPH-expressing human cancer cell lines allowing for the ranking of the CAR constructs in terms of overall extent of immune stimulation ex vivo. It is our expectation that the unique features of the ASPH tumor specific antigen will help overcome one of the major hurdles in T-cell therapy: the identification of antigens that permit effective targeting of tumors in the absence of non-tolerable and/or off-target toxicities to essential tissues and organs. Experiments to further characterize ASPH-targeted CAR-T cells are ongoing with the goal of moving these promising therapeutics into clinic. Citation Format: Thomas Thisted, Kanam Malhotra, Michael S. Lebowitz, Hossein Ghanbari. CAR-T cell therapies targeting aspartyl β-hydroxylase (ASPH) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2306.