Repeat-dose Toxicology Studies Research Articles

MYTX-011: a pH-dependent anti-cMET antibody-drug conjugate designed for enhanced payload delivery to cMET expressing tumor cells.

Advances in linker payload technology and target selection have been at the forefront of recent improvements in antibody-drug conjugate (ADC) design, leading to several approvals over the last decade. In contrast, the potential of novel ADC technologies to enhance payload delivery to tumors is relatively underexplored. We demonstrate that incorporation of pH-dependent binding in the antibody component of a cMET targeting ADC (MYTX-011) can overcome the requirement for high cMET expression on tumors, an innovation that has the potential to benefit a broader population of patients with lower cMET levels. MYTX-011 drove four-fold higher net internalization than a non-pH engineered parent ADC in non-small cell lung cancer (NSCLC) cells and showed increased cytotoxicity against a panel of cell lines from various solid tumors. A single dose of MYTX-011 showed at least three-fold higher efficacy than a benchmark ADC in mouse xenograft models of NSCLC ranging from low to high cMET expression. Moreover, MYTX-011 showed improved pharmacokinetics over parent and benchmark ADCs. In a repeat dose toxicology study, MYTX-011 exhibited a toxicity profile similar to other MMAE-based ADCs. These results highlight the potential of MYTX-011 for treating a broader range of NSCLC patients with cMET expression than other cMET targeting ADCs. A first in human study is ongoing to determine the safety, tolerability, and preliminary efficacy of MYTX-011 in patients with NSCLC (NCT05652868).

Preclinical characterization of a novel investigational monoclonal antibody CM313 with potent CD38-positive cell killing activity.

CM313 is currently under clinical investigation for treatments of multiple myeloma, systemic lupus erythematosus, and immune thrombocytopenia. We aimed to report the preclinical profile of the novel therapeutic anti-CD38 monoclonal antibody (mAb) CM313, with an emphasis on the difference with other CD38-targeting mAb. The binding of CM313 to CD38 recombinant protein across species was assessed using ELISA. The binding of CM313 to CD38-positive (CD38+) cells was detected using flow cytometry assays. CM313-induced complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and apoptosis on different CD38+ cells were assessed by LDH release assays or flow cytometry assays. The effect of CM313 on CD38 enzymatic activity was measured using fluorescence spectroscopy. CM313 immunotoxicity in human blood was assessed using flow cytometry assays, ELISA, and LDH release assays. Anti-tumor activity of CM313 was assessed in multiple mouse xenograft models. Safety profile of CM313 were evaluated in cynomolgus monkeys and human CD38 transgenic (B-hCD38) mice. There exist unique sequences at complementarity-determining regions (CDR) of CM313, which facilitates its affinity to CD38 is consistently higher across a spectrum of CD38+ cell lines than daratumumab. In vitro studies showed that CM313 induces comparable killing activity than daratumumab, including ADCC, CDC, ADCP, apoptosis induced by Fc-mediated cross-linking, and effectively inhibited the enzymatic activity of CD38. However, CM313 showed more potent CDC than isatuximab. In vivo, CM313 dose-dependently inhibited xenograft tumor growth, both as a monotherapy and in combination with dexamethasone or lenalidomide. Furthermore, CM313 was well tolerated with no drug-related clinical signs or off-target risks, as evidenced by 4-week repeat-dose toxicology studies in cynomolgus monkeys and B-hCD38 mice, with the later study showing no observed adverse effect level (NOAEL) of 300mg/kg once weekly. CM313 is a novel investigational humanized mAb with a distinct CDR sequence, showing comparable killing effects with daratumumab and stronger CDC activity than isatuximab, which supports its clinical development.

Abstract 1891: Preclinical characterization of ADRX-0706: A next-generation anti-Nectin-4 antibody-drug conjugate with improved therapeutic window

Abstract Nectin-4 is a well validated tumor target, which is highly expressed in several solid cancers such as urothelial, head-and-neck, breast, lung, pancreatic, ovarian, and cervical cancer, while expression in normal tissues is limited. A first-generation antibody-drug conjugate targeting Nectin-4, called enfortumab vedotin-ejfv (EV), was granted accelerated FDA approval in 2019 for urothelial cancers, and is currently approved for frontline therapy in cisplatin-ineligible patients with locally advanced or metastatic urothelial carcinoma, providing groundbreaking improvement in patient survival. However, clinical use of EV has also revealed safety-driven limitations, which result in reduced and/or interrupted drug exposures impacting efficacy. To overcome these limitations in therapeutic window and to maximize on the potential of Nectin-4 as a tumor target beyond urothelial cancers, Adcentrx has developed ADRX-0706, a Nectin-4-targeting next-generation ADC with a drug-antibody ratio of 8. ADRX-0706 is comprised of a novel fully human IgG1k monoclonal antibody linked to an optimized tubulin inhibitor payload AP052 through a proprietary cleavable linker. ADRX-0706 has strong binding affinity and selectively induces cytotoxicity in Nectin-4-expressing tumor cells. Importantly, ADRX-0706 is designed to have improved bystander effect, which allows tumor regression even in cases of heterogeneous Nectin-4 expression. This translates to superior in vivo efficacy of ADRX-0706 over EV in multiple mouse xenograft models of other indications beyond bladder cancer. In a mouse clinical trial of patient-derived cervical cancers with a range of Nectin-4 expression levels, ADRX-0706 reaches a 73% ORR. Since peripheral neuropathy is a main platform toxicity for this payload class, ADRX-0706 is assessed in an in vitro co-culture model to evaluate its neurotoxic potential. While EV displays a 75% decrease in neurite outgrowth, ADRX-0706 strikingly has no impact at concentrations up to 1mg/ml, suggesting a lower risk of peripheral neuropathy in humans for ADRX-0706. In preclinical repeat dose toxicology study, ADRX-0706 reaches an HNSTD of 18mg/kg in NHPs well above therapeutic exposures in mouse pharmacology studies, indicating a tremendous increase in the therapeutic index compared to EV. In summary, the highly selective and potent anti-tumor activity in multiple tumor types and wide preclinical therapeutic index of ADRX-0706 support clinical evaluation of this next generation anti-Nectin-4 ADC. ADRX-0706 is currently in a phase 1a/b study (NCT06036121). Citation Format: Andrew M. Hau, Maria Shahmoradgoli, Dong Jun Lee, Wes Sisson, Anna Wang, Peter P. Challita, Erin Nye, Mario M. Kuo, Alexis Mahloch, Monica Leung, Oscar Betancourt, Alexander Chu-Kung, Maojun Guo, Hui Li, Sabine Rottmann, Pia M. Challita-Eid. Preclinical characterization of ADRX-0706: A next-generation anti-Nectin-4 antibody-drug conjugate with improved therapeutic window [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 1891.

Abstract 2371: BGB-B167, a first-in-class 4-1BB/CEACAM5 bispecific antibody, exhibits potent in vitro and < for vivo antitumor activity and < for superior safety profile in preclinical models

Abstract 4-1BB (CD137) is a key costimulatory immunoreceptor and a promising therapeutic target in cancer. CEACAM5 (CEA) is a well-established tumor associated antigen overexpressed in many cancers, including colorectal, gastric, lung, pancreatic cancer, liver, breast and thyroid cancers. BGB-B167 is a novel immunoglobulin G (IgG)-based bispecific antibody targeting 4-1BB and CEA and is under clinical development for the treatment of advanced or metastatic solid tumors in humans. BGB-B167 binds to its target proteins with high specificity and affinity. Potent and CEA-dependent functional activities were demonstrated using peripheral blood mononuclear cell (PBMC)-based immune cell activation and cytotoxicity assays. In humanized 4-1BB knock-in mice bearing human CEA-expressing tumors, BGB-B167 exhibited potent, dose-associated single-agent efficacy as well as synergistic antitumor activity in combination with anti-PD-1 antibody. BGB-B167 was well tolerated in 1-month repeat-dose toxicology study in cynomolgus monkeys. Here, we describe the characterization of BGB-B167 with regard to preclinical proof-of-concept and basic drug-like properties. The combined dataset provides an overview on the design, mode of action, preclinical pharmacology and safety profile of BGB-B167. Citation Format: Zhuo Li, Liang Qu, Xin Chen, Liu Xue, Jie Li, Qi Liu, Jian Sun, Hanzi Sun, Yun Chen, Yuanyuan Xie, Wanyi Wang, Lin Zhu, Penghao Wang, Xiaosui Zhou, Hongjia Hou, Jie Chen, Xinyi Liu, Yilu Zhang, Ning Liu, Xinyi Liang, Shuo Zhang, Xiaoyan Tang, Jing Song, Tong Zhang, Xiaomin Song, Xuesong Liu, Kang Li, Chichi Huang. BGB-B167, a first-in-class 4-1BB/CEACAM5 bispecific antibody, exhibits potent in vitro and < for vivo antitumor activity and < for superior safety profile in preclinical models [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 2371.

Abstract LB320: Discovery and characterization of QTX3034, a potent, selective, and orally bioavailable allosteric KRAS inhibitor

Abstract The RAS family of proto-oncogenes are the most frequently mutated genes in cancer, in which mutations in KRAS account for approximately 25% of all human cancers. RAS oncogenes impair the ability of RAS to convert from its active GTP-bound form into its inactive GDP-bound state leading to the sustained activation of the MAPK signaling pathway and ultimately driving tumorigenesis. Until recently, KRAS was regarded as undruggable, but newly approved covalent inhibitors of KRASG12C have demonstrated clinical benefit in patients harboring this mutation. However, patients with KRASG12C mutation only represent approximately 10% of the KRAS-mutated or amplified cancer population, and multiple RAS-mediated resistance mechanisms have emerged with these covalent compounds. Here we show that QTX3034 is a potent, highly selective, and orally bioavailable mutant KRAS inhibitor. Using the most common KRAS mutant, KRASG12D, QTX3034 demonstrated picomolar binding affinity (0.6 nM) to the inactive form of KRASG12D and inhibited SOS1/2 mediated nucleotide exchange with picomolar potency (0.2 nM). In a NanoBiT cellular target engagement assay, QTX3034 treatment disrupted KRASG12D:RAF1 protein-protein interaction in a concentration-dependent manner with an EC50 of 17 nM in HEK293T cells. Western blot analyses and CTG proliferation assays illustrated that QTX3034 treatment inhibited downstream ERK phosphorylation and cell proliferation in KRASG12D -driven cancer cell lines. Similar inhibitory effects were observed across KRASG12D and KRASG12V mutant cancer cell lines, albeit less potent against the latter. Notably, QTX3034 produced no effect against NRAS or BRAF mutant cells. In vivo, QTX3034 achieved sustained systemic exposure levels required for efficacy following single oral administration in mice, indicating potential durable inhibition of KRAS signaling in vivo. In a KRASG12D -driven xenograft model, oral administration of QTX3034 twice daily (BID) demonstrated dose-dependent anti-tumor efficacy and achieved tumor regression in 100% of tumors. In a KRASG12V -driven xenograft model, oral administration of QTX3034 showed significant tumor growth inhibition, consistent with its activity against KRASG12V in vitro. QTX3034 was well-tolerated in rodent, repeat dose toxicology studies and exhibited significant exposure in the brain. QTX3034 has good solubility, permeability, and moderate protein binding in human plasma. The systemic clearance in rats and dogs was moderate to high, respectively, with good oral bioavailability. QTX3034 exhibited weak CYP inhibition (direct and time-dependent) with a favorable in vitro off-target selectivity/safety profile. Taken together, this preclinical characterization supports the advancement of QTX3034 into IND-enabling studies for KRAS mutant cancers. Citation Format: Yang W. Zhang, Dave Rominger, Elizabeth Donohue Vo, Jillian M. Silva, Yang J. Zhang, Greg Lee, John Micozzi, Ben Reid, Brooke McDonough, Audrey Hospital, Juan I. Luengo, Cameron Pitt, Hong Lin. Discovery and characterization of QTX3034, a potent, selective, and orally bioavailable allosteric KRAS inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB320.

Abstract 465: The preclinical characterization of TST003, a first-in-class mAb targeting gremlin-1 overexpressed by cancer-associated fibroblasts and tumor cells

Abstract Cancer associated fibroblast (CAF) is a rich source of factors for immune suppression and promotes cancer progression and metastasis via epithelial to mesenchymal transition (EMT). It is well known that tumors with mesenchymal phenotype have significantly poor prognosis and reduced response to checkpoint inhibitors. Targeting CAF becomes an increasingly attractive cancer therapeutic approach. Gremlin-1 is a member of the TGFβ superfamily, expressed by CAFs and tumor cells, and known to play a key role in EMT transition, cancer cell proliferation and stroma maintenance. Gremlin-1 overexpression in CAF or tumor cells often correlates with poor clinical prognosis in multiple cancers including prostate, pancreatic, gastric cancer etc. Here we describe the characterization of TST003 (14E3), a novel humanized IgG1 monoclonal antibody targeting Gremlin-1. TST003 bound to human Gremlin-1 with high affinity and high selectivity, and blocked Gremlin-1 binding to BMP2/4 with an EC50 of 3.68 nM to BMP2 and 4.53 nM to BMP4 respectively. TST003 could dose-dependently reverse Gremlin-1 inhibition of the BMP4-mediated Smad1/5/9 phosphorylation in tumor cells as measured by Western blot. In an ex vivo assay using prostate cancer patient-derived organoids (PDOs), TST003 inhibited the growth of 9 out of 12 human PDOs significantly. TST003 also demonstrated its single agent tumor growth inhibition (TGI>50%) at 10 mg/kg in a human prostate cancer PC3 xenograft model on castrated male nude mice and a human colorectal cancer patient derived xenograft (PDX) mouse model in the presence of human PBMC. Both tumor models expressed Gremlin-1 as detected by IHC analysis. In addition, a significantly better anti-tumor activity was observed when TST003 combined with an anti-VEGFR2 antibody in this CRC PDX (MSS, Kras G12D, PD-L1 negative) model. The safety profiles of TST003 were characterized in the single and repeated dose toxicology studies integrated with safety pharmacology, local tolerance, and in vitro hemolysis study, TCR study, and cytokine release study. TST003 was well tolerated in cynomolgus monkeys without noteworthy abnormalities following a single dose or repeated doses. In summary, TST003 is a first-in-class therapeutic mAb targeting Gremlin-1 overexpressed by CAFs and tumor cells. Our preclinical characterization results provided the rationale for on-going clinical evaluation of TST003 in patients with advanced solid tumors with high unmet medical need either as monotherapy or in combination with SoC. Citation Format: Di Sun, Huanhuan Guo, Chaping Cheng, Hongjun Li, Xinlai Yao, Shuang Lu, Jie Ding, Shenjie Zhang, Shijie Zhou, Steven Yu, Yi Gu, Xueming Qian. The preclinical characterization of TST003, a first-in-class mAb targeting gremlin-1 overexpressed by cancer-associated fibroblasts and tumor cells [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 465.

Abstract 1882: SCR-9171, a MUC17-targeted bispecific T cell engager molecule for gastrointestinal cancer

Abstract Background: Although advances treatments have significantly improved the survival of patients with gastrointestinal cancer in recent decades, there is still an unmet medical need for more effective treatments. A promising strategy is the use of bispecific antibodies that recruit T cells to kill cancer cells by simultaneously binding to CD3 on T cells and tumor associated antigens. CD3-based T cell engagers are highly potent therapeutic molecules with T cell cytotoxicity activities. But high CRS risk and T cell exhaustion has been observed for high affinity CD3-based T cell engagers in clinicals. Effective and safe use of these therapeutics depends on selective targeting of cancer cells and optimal CD3 affinity. MUC17, a member of the mucin family, is a transmembrane glycoprotein only expressed on the apical membrane of normal gastrointestinal mucosal epithelial cells. MUC17 is overexpressed in 23%-52% of patients with gastric cancer. Compared to the adjacent normal tissues, the expression of MUC17 protein is consistently higher in gastric cancer tissues, suggesting that MUC17 is an attractive therapeutic target for gastric cancer. Method & Results: We created MUC17/CD3 T cell engaging bispecific antibodies by starting with a nanobody humanized from camel and a low affinity CD3 antibody. Here we report the design and the promising preclinical activity of SCR-9171 molecule in vitro and in vivo. SCR-9171 induced T cell activation and had potent cytotoxicity of MUC17 positive tumor cells in vitro. However, the cytokine release was much lower than the positive control, which established with a high affinity CD3 antibody. Importantly, in mouse models with reconstituted human immune cells, SCR-9171 showed strong antitumor efficacy against MUC17-positive tumors through the activation of T cells. Tolerability and pharmacokinetics of MUC17/CD3 bispecific antibody are being assessed in cynomolgus monkey. The results demonstrated that SCR-9171 was well tolerated in a one-month repeat-dose toxicology study at 3mg/kg with no associated clinical signs or changes in body weight. Conclusion: These findings suggest that MUC17/CD3 bispecific antibody is a potential therapeutic molecule for MUC17-positive solid tumor. Citation Format: Guangcun Cheng, Yayuan Fu, Hua Zheng, ng Li, Xiaolei He, Xi Jiao, Shuhui Luan, Shuai Wang, Lei Jiang, Wenjing Li, Yun Zhang, Lei Liu, Renhong Tang. SCR-9171, a MUC17-targeted bispecific T cell engager molecule for gastrointestinal cancer [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 1882.

A CD79b Targeting ADC with Superior Anti-Tumor Activity and Safety Resulting in Significantly Improved Therapeutic Index (TI): Safe and Efficacious CD79b ADC

The Araris' site-specific and 1-step enzymatic linker conjugation technology aims at generating safe and highly potent ADCs without the need for antibody engineering prior to linker-payload conjugation. We developed a very stable anti-CD79b-MMAE ADC (ARC-02) with this technology showing a higher therapeutic index (TI) compared to the approved ADC polatuzumab-vedotin (Polivy®) in different preclinical models. Therefore, ARC-02 is considered a promising next generation treatment alternative for patients suffering from Non-Hodgkin lymphoma (NHL). Using native polatuzumab as the targeting antibody and monomethyl auristatin E (MMAE) as payload, we generated within 24 hours the highly homogeneous and pure ARC-02 development candidate with a well-defined drug-to-antibody ratio (DAR) of 1.9 and with high purity (monomer content > 98%). ARC-02 is highly stable under stressed conditions at elevated temperatures and maintains the FcyR/FcRn-binding properties of the parental mAb. In-vitro ARC-02 demonstrated potent cytotoxicity in four different NHL cell lines tested so far with similar potency (EC50 values) compared to the approved Polivy® ADC despite of the lower toxin load of ARC-02 (DAR of 1.9) compared to Polivy® (DAR of 3.5). ARC-02 clearly differentiates from Polivy® by extraordinary stability in mouse, cynomolgus monkey and human sera exemplified by the absence of payload deconjugation or linker cleavage under physiological conditions (at 37°C) and long-term incubation (>2 weeks). Exceptional stability could also be confirmed in vivo in pharmacokinetic studies in mice, rats and cynomolgus monkeys demonstrating an antibody-like exposure profile comparable to the unmodified polatuzumab parental antibody without premature toxin deconjugation in the circulation resulting in improved pharmacokinetics of ARC-02 compared to Polivy® (half-life in mice 10 days for ARC-02 vs 5 days for Polivy®). Despite the excellent stability of ARC-02 in vitro and in vivo, the MMAE toxin is still efficiently released from ARC-02 in lysosomal human Cathepsin B cleavage or human liver-lysosome release assays which is providing the foundation for the observed robust anti-tumor activity in vivo: ARC-02 was compared head-to-head to Polivy® in two different CD79b-expressing established tumor models in vivo (Granta-519 and Ramos). Single dose treatment of ARC-02 at very low payload doses induced effective and long-lasting anti-tumor responses with complete tumor eradication achieved at about half of the Polivy® payload dose in both NHL established tumor models. Based on these in vivo efficacy experiments, the minimal effective dose (MED) of ARC-02 that is still inducing eradication of established tumors is determined to be as low as 10 μg/kg based on the MMAE payload dose or 1 mg/kg based on the total ADC dose. Finally, the highest non-severely toxic dose (HNSTD) of ARC-02 in repeat dose toxicology studies was determined to be ≥30 mg/kg and ≥24 mg/kg in rats and cynomolgus monkeys, respectively, which is substantially higher than the HNSTD reported for Polivy® (i.e. 10 mg/kg in the rat and 3 mg/kg in the cynomolgus monkey as described in the Polivy® Biologic License Application). Therefore, the tolerability in rodents and non-human primates is at least 3 and 8 times higher when compared to historical safety data of Polivy®. Using the HNSTD derived from repeat dose non-human primate toxicity study (24 mg/kg) and the MED calculated from single dose rodent efficacy studies (1 mg/kg) an impressive TI of 24 can be calculated for ARC-02 which is substantially higher than the TI of Polivy®. These encouraging results obtained so far indicate that ARC-02 a) has very favorable biophysical properties, b) shows a consistent and highly defined DAR, c) is highly stable in vitro and in vivo, d) is highly potent and efficacious in multiple tumor models and e) has improved tolerability in rodents and non-human primates and f) shows an impressive improvement in TI and ultimately warrant further development of ARC-02.

Evaluation of Common Gamma Chain Cytokine Signaling Blockade with REGN7257, an Interleukin 2 Receptor Gamma (IL2RG) Monoclonal Antibody, on Immune Cell Populations in Monkey and Human

The common γ chain cytokine receptor (γc; IL2RG) family of cytokines includes interleukin 2 (IL2), IL4, IL7, IL9, IL15, and IL21. This set of cytokines exhibits broad pleiotropic actions on both the innate and adaptive immune system with each cytokine sharing the IL2RG chain as part of its signaling receptor complex. Mutations in the IL2RG gene result in X-linked severe combined immunodeficiency (XSCID) in humans, whereby patients present with dramatically diminished numbers of T-cells and NK-cells, and dysfunctional B-cells. Given the crucial role for γc cytokines in the development and function of lymphocytes, modulating their activities may offer therapeutic potential in a range of immune-mediated diseases. To understand the effects of γc cytokine blockade on immune cells including T-cell subsets, we utilized REGN7257, a fully human IL2RG monoclonal antibody that inhibits γc cytokine-induced signaling, and tested its ability to suppress immune cell populations and their functions in cynomolgus monkey and human. Mixed lymphocyte reaction assays were performed to look at the impact of γc cytokine signaling blockade on activation/proliferation of human T-cells. To further analyze the contribution of γc cytokines to T-cell differentiation and function, we performed in vitro transcriptomic studies on human peripheral blood mononuclear cells stimulated with anti-CD3/CD28 beads. In addition, we assessed the effects of γc cytokine signaling blockade on both human T-cell and NK-cell effector functions in target cell killing assays. Finally, we evaluated the effects of γc cytokine signaling blockade with REGN7257 on immune cell populations and their activation/proliferation status in cynomolgus monkeys by flow cytometry. γc cytokine signaling blockade with REGN7257 demonstrated potent inhibition of allogeneic responses in mixed human lymphocyte reaction assays, by preventing T-cell activation and proliferation. Two-tailed gene set enrichment analysis identified 4 distinct patterns of significantly enriched gene sets that were impacted by γc cytokine signaling upon CD3/CD28-induced activation of human T-cells: gene sets involved in inflammatory responses, differentiation and proliferation, activation and immune responses, as well as those related to adhesion and migration. These signatures were blocked with REGN7257 treatment. In addition, REGN7257 potently blocked activation (i.e. IFNG production) and cytotoxic activity of both human T-cells and NK-cells in in vitro target cell killing assays. Finally, the impact of γc cytokine signaling blockade on lymphocyte populations in vivo were investigated in cynomolgus monkeys in a single dose pharmacokinetic/pharmacodynamic study and a repeat-dose toxicology study. Inhibition of γc cytokine signaling with REGN7257 in cynomolgus monkeys reduced circulating T-cells and NK-cells, without impacting B-cells, granulocytes, platelets or red blood cells. γc cytokine signaling blockade led to a reduction in both peripheral CD4+ and CD8+ T-cell counts with effector memory T-cells being the most impacted T-cell population studied. Similarly to that observed in human T-cell MLR assays, both activated and proliferating T-cells were reduced in monkeys dosed with REGN7257. Taken together, these pharmacological observations highlight the major role for γc cytokine signaling in maintenance of lymphocyte populations (i.e. NK-cells and T-cells), but not other immune cell populations, such as granulocytes, platelets and red blood cells. Furthermore, our data highlight the importance of γc cytokines in driving functions of cynomolgus monkeys and human effector NK and T-cells, opening a potential new route for the management of immune-mediated diseases.

NON-CLINICAL SAFETY EVALUATION OF A NOVEL THERAPEUTIC VACCINE FOR PEANUT ALLERGY

<h3>Introduction</h3> Peanut allergy is the most common cause of fatal food-associated anaphylaxis. VLP Peanut is a novel virus-like particle (VLP)-based vaccine being developed for the treatment of peanut allergy. Prior to first in human trials, its safety was assessed in a repeat dose toxicology study. <h3>Methods</h3> VLP Peanut was administered with or without the adjuvant 1.95% MicroCrystalline Tyrosine, subcutaneously, to Wistar Han rats three times daily (TID) once every 4 weeks for 12 weeks as described (Fig 1). A maximum dose level of 3.6 mg was evaluated (anticipated maximum dose in humans). Assessment of toxicity included clinical, post-dose, and dose site observations; body weights; food consumption; ophthalmic observations; and clinical and anatomic pathology. <h3>Results</h3> Minor changes in bodyweight gains, food consumption and clinical pathology were noted in animals administered VLP Peanut ±MCT and minor, non-adverse, increases in spleen and liver weights were noted in some female animals. These changes were reversible and not adverse. Animals administered VLP Peanut ± MCT had occasional dose site observations of very slight or slight edema, more frequently noted for animals administered MCT. Macroscopic observations at subcutaneous injection site/s were noted for all test article-treated groups, which correlated with microscopic observations of inflammation and neutrophilic reactions at the subcutaneous injection site/s. Dose site observations were considered adverse but were fully reversed after a 4week recovery. <h3>Conclusion</h3> VLP Peanut administered to rats, was systemically well tolerated, with only minor, non-adverse clinical changes. Slight dose site observations were fully-reversible and did not impact on the overall health of the animals.

Abstract 1077: Stability and safety evaluation of ELU001, a targeted C’Dot drug conjugate for the potential treatment of folate receptor alpha-overexpressing cancers

Abstract While several antibody-drug conjugates (ADCs) have shown efficacy in the treatment of solid tumors ADCs remain limited in their therapeutic efficacy due to long circulatory half-life and limited tumor penetration. Ultra-small (sub-10nm) C’Dot-drug-conjugates (CDCs) directly address this, exhibiting deep penetration and retention in solid tumor models, with limited systemic exposure following rapid renal clearance. CDCs can also achieve greater potency by delivering over 10x more payload and by binding to tumor cells with a higher avidity allowing effective killing of tumors with broader range of moderate to high expression of target antigens. ELU001 is a CDC that targets folate receptor alpha (FRα) overexpressing tumors via folic acid moieties on its surface and delivers an average of 21 exatecan topoisomerase-1 inhibitor molecules as its payload. FRα is overexpressed on a variety of tumors including ovarian, endometrial, triple negative breast and non-small cell lung, but is minimally expressed on normal tissues making it an attractive tumor-associated antigen for targeted drug delivery. ELU001 is highly stable in plasma and elicits antitumor efficacy in a variety of cell line and PDX-derived tumor models both in vitro and in vivo. In 15-day repeat dose toxicology and toxicokinetic studies performed in Wistar Han rats and Beagle dogs, ELU001 was well tolerated at up to 0.87 mg/kg/day in rats and 0.174 mg/kg/day in dogs based upon conjugated exatecan concentration when administered on a QWx3 schedule via a 1-hour infusion. Observed dose-related toxicities for both species were limited to the bone marrow and GI tract - the same organs as those observed when free payload (exatecan) was administered suggesting that the delivery of exatecan conjugated to the CDC did not broaden the tissue toxicity profile. Observed toxicities were recovered or substantially reduced by the end of a two-week recovery period. No drug-related hepatic, renal, pulmonary or ocular toxicities were observed and there were no drug-related deaths in the repeat dose toxicity study. TK parameters, estimated in the 15-day GLP studies revealed similar plasma exposure values in males and females for ELU001, Total Exatecan (conjugated and released) and Released Exatecan for rats and dogs, respectively. ELU001 exhibited an average circulatory half-life ranging from approximately 15 to 20 hrs in rats and 24 to 29 hrs in dogs with no accumulation of ELU001, Total Exatecan or Free Exatecan observed from day 1 to day 15. Based upon AUC0-last (hr*ng/ml) released payload levels in the circulation were less than approximately 0.3% and 0.1% of the total payload levels in the rat and the dog respectively. No ELU001 anti-drug antibodies were induced in either species. In summary, ELU001 has a favorable nonclinical safety/TK profile and is currently under evaluation in a clinical safety study - NCT05001282. Citation Format: Gregory P. Adams, Kai Ma, Feng Chen, Marion Scocca, Aranapakam Venkatesan, Cathy Reddick, Mary Hilgart, Thomas Gardinier, Fei Wu, Melik Turker, Peiming Chen, Tin Khor, Vaibhav Patel, Eliel Bayever. Stability and safety evaluation of ELU001, a targeted C’Dot drug conjugate for the potential treatment of folate receptor alpha-overexpressing cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1077.

Abstract ND03: AMX-818: Next generation conditionally active, masked T-cell engager for the treatment of HER2-expressing solid tumors

Abstract T Cell Engagers (TCEs) are a highly potent, cancer killing modality that redirect T cell cytotoxicity against tumors that express a selected tumor-associated antigen, or TAA, bypassing the requirements for T cell recognition of tumor specific MHC/peptides. In addition to their induction of direct cytotoxicity, their potency is amplified by cytokine driven actions downstream of T cell activation that enhance the anti-tumor immune response. Because of this two-fold mechanism of action, TCEs have demonstrated clinical activity in patients with “cold” tumors that are not responsive to other immune therapies such as checkpoint inhibitors. While a razor-thin therapeutic index (TI) has been observed in hematologic malignancies for TCEs like blinatumomab, and similarly with CAR-Ts, developing solid tumor T-cell-directed therapies has been hampered by on-target off-tumor toxicities including cytokine release syndrome (CRS), other toxicities, and other challenges related to immunogenicity. Attempts to circumvent CRS include step-up dosing, prophylactic steroids, complex molecular designs, have had limited success. Unmasked TCEs targeting HER2 have been unable to achieve a therapeutic index in the clinic due to dose limiting CRS toxicities at doses below 1 ug/kg. To address this challenge, we have engineered conditionally-activated T cell engagers or XPATs (XTENylated Protease Activated T Cell Engagers), that exploit the intrinsically high protease activity in tumors relative to healthy tissues. XPATs utilize our universal protease-releasable masking technology (Pro-XTEN) to provide preferential unmasking and activation in tumors, thereby mitigating off-tumor toxicities. Several key features of the technology have clinical validation including half-life extension, protease-driven unmasking and low immunogenicity, as over 200 patients have been treated with XTENylated products. AMX-818 is a conditionally active, masked T cell engager targeting HER2. AMX-818 is comprised of a tandem scFv core with a HER2-binding domain and a proprietary low affinity CD3 binding domain with each domain masked by a protease-releasable XTEN polymer that works by spatial/steric shielding of the active domains. AMX-818 is designed to overcome the on-target, off-tumor toxicities associated with existing TCEs by mitigating toxicity against healthy tissues that express the target antigen. Extensive in vitro and in vivo preclinical testing demonstrate the potential for AMX-818 to drive anti-tumor activity similar to an unmasked TCE, yet significantly expand the safety margin in NHPs. In vitro, proteolytically-unmasked AMX-818 demonstrates potent cytotoxicity against tumor lines with EC50s in the single-digit pM range. AMX-818 reduces target-directed T cell cytotoxicity and T cell activation by up to 4 orders of magnitude, while singly-masked variants of AMX-818 show intermediate activity relative to unmasked HER2 T cell engager. Only minimal complete unmasking of AMX-818 is required to generate potent cytotoxicity. In the established HER2-high (BT-474) and HER2-low (HT-55) xenograft models, AMX-818 induced protease-dependent tumor regressions comparable to the unmasked (active) TCE while remaining stable in circulation. In vivo, preferential cleavage of XPATs were demonstrated in patient-derived xenograft tumors relative to healthy organs (average of 24% unmasked TCE was observed in tumors). In NHPs, AMX-818 demonstrated a wide safety margin (MTD of AMX-818 = 42mg/kg vs MTD of fully unmasked HER2 TCE = 0.2mg/kg/day), supported by its protease stability in circulation and a maximum tolerated Cmax that was &amp;gt;400-fold higher than that of its active form (unmasked HER2 TCE). No CRS or systemic T cell activation was observed even at 50 mg/kg, supportive of minimal CRS risk for XPATs, whereas the unmasked form induced lethal CRS at &amp;gt; 0.3 mg/kg/day. Only 1-3% of the singly-cleaved XPAT metabolites were detected in plasma from NHPs treated with high doses of AMX-818 (25 &amp; 42 mg/kg), while the fully unmasked TCE form was undetectable. In vitro studies in which AMX-818 was incubated for 7-days at 37°C in plasma from healthy and diseased NHPs and humans, (conditions which over-represent the accumulation of metabolites in vivo) demonstrate that AMX-818 is stable in plasma with comparable amounts of fully unmasked TCE generated between species. The similar rates of cleavage between species and the strong safety margin observed with AMX-818 in NHPs predict limited systemic accumulation of active TCE in human patients. In a repeat-dose GLP toxicology study, the highest dose evaluated (6 mg/kg) was the NOAEL, suggesting that HER2-XPAT has a wide TI based on predicted human PK and predicted efficacious exposures. The collective data demonstrate a significant decrease in toxicity with AMX-818 compared to its potent, fully unmasked TCE counterpart. Together with the data supporting stability of AMX-818 in plasma and healthy tissue and its preferential cleavage in tumors, the nonclinical data package supports further evaluation in a planned clinical trial. AMX-818 represents a promising solution to widen the TI for TCE activity in HER2-high and HER2-low expressing tumors. Citation Format: Bryan Irving. AMX-818: Next generation conditionally active, masked T-cell engager for the treatment of HER2-expressing solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr ND03.

Abstract 2910: A CD79b targeting ADC with superior anti-tumor activity and therapeutic index

Abstract The Araris’ site-specific and 1-step linker conjugation technology aims at generating safe and highly potent ADCs without the need for antibody engineering prior to linker-payload conjugation. We developed a very stable anti-CD79b-MMAE ADC with this technology showing a higher therapeutic index compared to polatuzumab-vedotin in preclinical models. Our ADC may represent a safe and efficacious alternative for the treatment of patients with diffuse-large B-cell lymphoma (DLBCL).Using native polatuzumab (non-engineered, same antibody sequence as present in approved polatuzumab-vedotin) as the targeting antibody and monomethyl auristatin E (MMAE) as payload, we generated within 24hours highly homogeneous and pure ADCs with a well-defined drug-to-antibody ratio (DAR) of 1.9, with a &amp;gt 98% monomer content. The ADC is highly stable under stressed conditions at elevated temperatures and maintains the FcyR/FcRn-binding properties of the parental mAb. In in-vitro assays our ADC demonstrated potent cytotoxicity in four tested cell-lines, similar to the approved polatuzumab-vedotin (Polivy®). Moreover, our anti-CD79b ADC (ARADC) is highly stable in mouse, cynomolgus and human sera exemplified by the absence of payload deconjugation or linker cleavage. Though highly stable, the ADC is still efficiently released by lysosomal human Cathepsin B cleavage or human liver-lysosome enzymes. Most importantly, the resulting ADC is extremely stable in circulation as shown in pharmacokinetic studies in mice and rats demonstrating an antibody-like exposure profile comparable to the unmodified polatuzumab antibody and twice as long as the approved polatuzumab-vedotin (half-life 10d vs 5d).Most importantly, the in vivo efficacy of ARADC (DAR 1.9) was compared with approved polatuzumab-vedotin (DAR 3.5) in two CD79b-expressing tumor models: Granta-519 and Ramos. ARADC provided equal tumor growth inhibition and survival at about half the payload dose relative to polatuzumab-vedotin in both models. At approximately equal payload doses, ARADC treatment led to greater antitumor effects and a considerably longer survival advantage over polatuzumab-vedotin in both models. Finally, the highest non-severely toxic dose (HNSTD) of ARADC was determined at 30mg/kg in a 4-week repeat dose toxicology study in rats. This observation, together with the high anti-tumor potency at low dose - the minimal effective dose (MED), results in an overall 4-6-fold increased therapeutic index (TI).These encouraging results obtained so far indicate that ARADC a) has very favorable biophysical properties, b) shows a clearly defined drug-to-antibody ratio, c) is highly stable in vitro and in vivo, d) is highly potent and efficacious in multiple tumor models and e) showed an improvement in TI by a factor of 4-6 and ultimately warrant further development of ARADC. Citation Format: Isabella Attinger-Toller, Philipp Probst, Romain Bertrand, Ramona Stark, Roger Santimaria, Emma Renard, Rachael Fay, Dragan Grabulovski, Bernd Schlereth, Philipp René Spycher. A CD79b targeting ADC with superior anti-tumor activity and therapeutic index [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2910.

Abstract 5202: AMG 794, a Claudin 6-targeted half-life extended (HLE) bispecific T cell engager (BITE®) molecule for non-small cell lung cancer and epithelial ovarian cancer

Abstract AMG 794 is a half-life extended BiTE® immune therapy targeting the oncofetal antigen Claudin 6 (CLDN6). AMG 794 redirects T cells to kill CLDN6-expressing tumor cells and is being developed for the treatment of non-small cell lung cancer (NSCLC) and epithelial ovarian cancer (EOC). CLDN6 is a compelling tumor antigen that is expressed during embryonic and fetal development, transcriptionally silenced in adult tissues, and re-expressed on the surface of NSCLC and EOC cells. By immunohistochemistry, CLDN6 staining of the cell membrane was observed in 27% of non-squamous NSCLC (n = 63) and 69% of EOC (n = 92) samples, the majority of which were of the high-grade serous ovarian cancer subtype. Expression of CLDN6 protein was not detected in most normal adult tissues, with rare CLDN6 immunostaining limited to individual cells in the pituitary, pancreas, small intestine, kidney, and female reproductive organs. AMG 794 is a fully human BiTE® molecule that binds both human and cynomolgus monkey CLDN6 and CD3. AMG 794 binds human CLDN6 and CD3 with equilibrium dissociation constant (KD) of 13 nM and 36 nM, respectively. In vitro, AMG 794 redirects human T cells to kill CLDN6-expressing cancer cells with a half-maximal lysis concentration (EC50) of 2.6 ± 1.1 pM to 127.4 ± 53.4 pM. Consistent with the mechanism of action of BiTE® immune therapy, AMG 794 induces T cell activation and transient production of cytokines in co-cultures of T cells and CLDN6-expressing tumor cells. Remarkably, AMG 794 binding and cytotoxic activity is selective for CLDN6 over other claudin family proteins, despite high homology in the extracellular loops with CLDN9. Weekly dosing of AMG 794 significantly inhibited the growth of established lung and ovarian xenograft tumors in immunocompromised mice injected with human T cells. Anti-tumor activity was associated with an increase in tumor-infiltrating T cells. AMG 794 was well tolerated in a one-month repeat-dose toxicology study in cynomolgus monkey, with evidence for target engagement. The potent, selective activity of AMG 794 for CLDN6-expressing NSCLC and EOC cells, together with an acceptable nonclinical safety profile, supported the advancement of AMG 794 into clinical development. A first-in-human study to explore the safety, tolerability, pharmacokinetics, and anti-tumor activity of AMG 794 in patients with CLDN6-positive advanced/metastatic non-squamous NSCLC or EOC will be enrolling patients in March 2022. Citation Format: Elizabeth Pham, Anja Henn, Beate Sable, Joachim Wahl, Kip Conner, Katja Matthes, Shivani Gupta, Rodolfo Yabut, Famke Aeffner, Kristin Lewis Wilson, Jonas Anlahr, Christoph Dahlhoff, Vijay Kale, Matthias Friedrich, Tobias Raum, Peter Kufer, Angela Coxon, Sabine Stienen, Julie M. Bailis. AMG 794, a Claudin 6-targeted half-life extended (HLE) bispecific T cell engager (BITE®) molecule for non-small cell lung cancer and epithelial ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5202.

Characterization of an Anti-CD70 Half-Life Extended Bispecific T-Cell Engager (HLE-BiTE) and Associated On-Target Toxicity in Cynomolgus Monkeys.

Bispecific T-cell engager (BiTE) molecules have great potential to treat cancer. Nevertheless, dependent on the targeted tumor antigen, the mechanism of action that drives efficacy may also contribute to on-target/off-tumor toxicities. In this study, we characterize an anti-CD70 half-life extended BiTE molecule (termed N6P) which targets CD70, a TNF family protein detected in several cancers. First, the therapeutic potential of N6P was demonstrated using in vitro cytotoxicity assays and an orthotopic xenograft mouse study resulting in potent killing of CD70+ cancer cells. Next, in vitro characterization demonstrated specificity for CD70 and equipotent activity against human and cynomolgus monkey CD70+ cells. To understand the potential for on-target toxicity, a tissue expression analysis was performed and indicated CD70 is primarily restricted to lymphocytes in normal healthy tissues and cells. Therefore, no on-target toxicity was expected to be associated with N6P. However, in a repeat-dose toxicology study using cynomolgus monkeys, adverse N6P-mediated inflammation was identified in multiple tissues frequently involving the mesothelium and epithelium. Follow-up immunohistochemistry analysis revealed CD70 expression in mesothelial and epithelial cells in some tissues with N6P-mediated injury, but not in control tissues or those without injury. Collectively, the data indicate that for some target antigens such as CD70, BiTE molecules may exhibit activity in tissues with very low antigen expression or the antigen may be upregulated under stress enabling molecule activity. This work illustrates how a thorough understanding of expression and upregulation is needed to fully address putative liabilities associated with on-target/off-tumor activity of CD3 bispecific molecules.

MO557: Lanthanum Dioxycarbonate is Safe in Rats

Abstract BACKGROUND AND AIMS Over 7 million patients suffer from end-stage kidney disease (ESKD) worldwide.[1] Due to low kidney function, patients often become hyperphosphatemic, which leads to negative outcomes[2] including increased risk of death.[3] Patients usually rely on dietary restriction and phosphate(P) binders to address hyperphosphatemia. However, current P binders often do not achieve desired P levels[4] and have a high pill burden due to high quantity of large pills.[5,6] Having a therapeutic option using the same quantity of elemental lanthanum as existing therapies (and therefore similar efficacy) but with a potentially smaller and easy-to-administer pills would enhance patient quality of life (QoL). Lanthanum dioxycarbonate, RENAZORB(LDC), is a novel nanotechnology product that combines lanthanum, which has the highest binding capacity versus other P binders,[7] with a potentially smaller pill size that is swallowed rather than chewed to treat hyperphosphatemia in ESRD patients. However, its safety results have not yet been published. We present the results from a toxicity study evaluating safety of lanthanum dioxycarbonate in rats. METHOD A repeat-dose toxicology study was conducted to evaluate the potential toxicological profile of LDC. Rats were administered oral doses of LDC at 200, 600 or 2000 mg/kg/day by gavage for approximately 6 weeks. Blood samples for TK analysis were obtained from cohorts of 3 rats/sex/group/time pre-dose and at 1, 2, 4, 8, 12, 24 and 48 h after Day 1 and 38 dosing. RESULTS LDC was well tolerated at all test doses. The only test article-related effects noted at the high dose of 2000 mg/kg in both sexes were decreased mean cell hemoglobin (MCH) and mean cell hemoglobin concentration (MCHC), and increased urine pH, and at 600 mg/kg increased urine pH in female rats. The increased urinary pH was considered not of toxicologic relevance, and the decreases in MCH and MCHC were considered to be small enough to not adversely affect the animals’ health. The no-observed-adverse-effect level was 2000 mg/kg. CONCLUSION All doses of lanthanum dioxycarbonate were well tolerated and found safe in rats. These results suggest lanthanum dioxycarbonate may be a suitable candidate for further development. Potential benefits of LDC may include reduced pill burden and ease of dose administration (smaller easy-to-swallow versus chewable), which collectively has potential to enhance patient adherence, improve treatment outcomes and enhance QoL.

Investigational New Drug Enabling Nonclinical Safety Assessment of the Iminosugar UV-4, a Broad-Spectrum Host-Targeted Antiviral Agent.

The iminosugar UV-4 is a broad-spectrum antiviral drug candidate with activity in vitro and in vivo against multiple, diverse viruses. The toxicological profile of UV-4, dosed as the hydrochloride salt, was evaluated in single-dose and repeat-dose oral toxicity studies in mice, rats, dogs, and non-human primates (NHP). No moribundity or deaths were associated with the drug up to the maximum tolerated dose. No treatment-related adverse effects were observed following single oral doses in dogs, rats, and mice up to 250, 400, 1000mg/kg, respectively, and in NHP up to 180mg/kg administered three times daily for 10days. UV-4-related findings were generally seen at higher doses after 7- or 14-day exposure. The most common clinical pathology findings (increase in aspartate aminotransferase and decreased platelet count) were consistently found across species and each appeared dose related. The kidney, mesenteric lymph nodes, stomach including gastrointestinal tract, and thymus were identified as target organs in mice, rats, and dogs. In 14-day repeat-dose toxicology studies in mice and dogs conducted in compliance with Good Laboratory Practice regulations, the dog was considered to be the most sensitive species to UV-4 exposure based on the treatment-related adverse effects noted in the identified target organs. The results of these studies demonstrate the safety profile of UV-4 hydrochloride and supported the selection of starting and maximal doses for a single ascending dose first-in-human clinical study.

Attenuating nicotine's effects with high affinity human anti-nicotine monoclonal antibodies.

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 (KD) 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.

Phosphatidylinositol 3-Kinase δ-Specific Inhibitor-Induced Changes in the Ovary and Testis in the Sprague Dawley Rat and Cynomolgus Monkey.

Phosphatidylinositol 3-kinase (PI3K) δ is a lipid kinase primarily found in leukocytes, which regulates important cell functions. AMG2519493 was a PI3K δ-specific inhibitor in development for treatment of various inflammatory diseases. AMG2519493-related changes in the male and/or female reproductive organs were observed in the 1- and 3-month oral repeat dose toxicology studies in the rat and cynomolgus monkey. Hemorrhagic corpora lutea cysts and increased incidence of corpora lutea cysts without hemorrhage were observed in the ovaries at supra pharmacological doses in the rat. A decrease in seminiferous germ cells in the testis, indicative of spermatogenesis maturation arrest, was observed in both the rat and cynomolgus monkey. Although the characteristics were comparable, the drug systemic exposures associated with the testicular changes were very different between the 2 species. In the rat, the testicular change was only observed at supra pharmacologic exposure. Isotype assessment of PI3K signaling in rat spermatogonia in vitro indicated a role for PI3K β, but not δ, in the c Kit/PI3K/protein kinase B signaling pathway. Therefore, changes in both the ovary and testis of the rat were considered due to off target effect as they only occurred at suprapharmacologic exposure. In contrast, the testicular changes in the cynomolgus monkey (decrease in seminiferous germ cells) occurred at very low doses associated with PI3K δ-specific inhibition, indicating that the PI3K δ isoform may be important in spermatogenesis maturation in the cynomolgus monkey. Our results suggest species-related differences in PI3K isoform-specific control on reproductive organs.

Application of a newly-developed cynomolgus macaque BiTE-mediated cytotoxic T-lymphocyte activity assay to various immunomodulatory agents in vitro

The immunotoxic potential of drug candidates is assessed through the examination of results from a variety of in vitro and in vivo immunophenotyping and functional study endpoints in pre-clinical studies. CD8+ cytotoxic T-lymphocyte (CTL) activity impairment by immunosuppressive agents is recognized to be a potentiating factor for decreased antiviral defense and increased cancer risk. A bi-specific T-cell engager (BiTE®)-mediated CTL activity assay that applies to ex vivo experimentation in non-human primates in the context of toxicology studies was successfully developed and applied in cynomolgus monkey regulatory studies. While an ex vivo analysis conducted in the context of repeat-dose toxicology studies focuses on the long-term impact on CTL function, an in vitro assay with the same experimental design captures acute effects in the presence of the test article. Here, the in vitro assay was applied to a list of drugs with known clinical immunomodulatory impact to understand the applicability of the assay. The results showed this assay was sensitive to a wide range of immunosuppressants directly targeting cell-intrinsic signaling pathways in activated CTL. However, agents executing immuno-modulation through inhibiting cytokines/cytokine receptors, co-stimulatory molecules, and cell adhesion and migration pathways did not impair the CTL activity in this short-term in vitro culture. In addition, anti-PD-1/PD-L1 immune checkpoint blockers enhanced the CTL activity. Taken together, the results here demonstrate that in concordance with their mechanism of action, the in vitro BiTE®-mediated CTL assay is applicable and sensitive to immunomodulatory agents acting via a variety of mechanisms.