Toxicology Studies In Monkeys Research Articles

Abstract P440: Discovery of clinical candidate GSC002219, a highly selective oral CYP11B2 inhibitor for hypertension

Background: Aldosterone is an important mediator of hypertension and its aberration could contribute to heart and kidney diseases. Aldosterone synthase inhibitors (ASI) attenuate the production of aldosterone directly and have been proposed as an alternative to mineralocorticoid receptor antagonists (MRA) for treatment of resistant hypertension and chronic renal disease. GSC002219 is a potent inhibitor of aldosterone synthase (CYP11B2) with high selectivity over the closely related enzyme CYP11B1. The aim of the study was to evaluate the effect of GSC002219 on aldosterone and blood pressure. Methods: The inhibitory effect of GSC002219 against CYP11B2 and CYP11B1 proteins was assessed in human renal leiomyoblastoma cells expressing recombinant human / cynomolgus CYP11B1or CYP11B2 enzymes, respectively. GSC002219 was further evaluated in Synacthen (ACTH) challenged cynomolgus monkey model for its effects against various related key biomarkers including cortisol, aldosterone and precursors. The effects of GSC002219 on systolic blood pressure (SBP) and diastolic blood pressure (DBP) were evaluated in stroke-prone spontaneously hypertensive rat (SHRSP) model. Results: GSC002219 potently inhibits CYP11B2 with an IC 50 of 27nM, while maintains a >150-fold selectivity over CYP11B1. In ACTH challenged cynomolgus monkey model, single oral doses of GSC002219 at 0.03 mg/kg and 0.1 mg/kg could suppress aldosterone production to 80% and 93%, respectively, comparing to vehicle-treated animals without any effect towards cortisol levels. In the rat SHRSP hypertension model, GSC002219 could effectively lower both SBP and DBP. GSC002219 has an excellent in vitro ADME and toxicology profile as well as a balanced physicochemical property. In 14-day rat and monkey toxicology studies, GSC002219 displayed good tolerance, excellent PK profile and a great safety window (>100 fold). Conclusions: Taken together, GSC002219 is a safe and potent inhibitor of CYP11B2 and efficacious in various disease models of hypertension. GSC002219 is under IND-enabling study and may enter the clinical development early 2025.

Selectivity and Safety of VIP943: A Novel CD123-Targeting Antibody-Drug Conjugate (ADC) Using a Proprietary Linker and Payload Class

Introduction ADCs afford high efficacy though often have shortcomings in their safety profile. Most ADC payloads are cytotoxic agents that can inhibit general processes of cellular homeostasis, including microtubule polymerization or DNA damage repair. While these are good targets for cytotoxicity, they have the drawback of being nonselective for dividing cancer cells. Here, we report the potent, on-target activity and selectivity of our novel CD123-targeting ADC VIP943, which uses a kinesin spindle protein (KSP; also known as KIF11 or Eg5) inhibitor as the payload (VIP716). The kinesin superfamily (KIF) is comprised of motor-proteins involved in various cellular mechanisms, including formation of the bipolar mitotic spindle during cell division. Targeting KSP selectively inhibits dividing cells, while sparing nondividing, differentiated cells thereby increasing the therapeutic index of VIP943. Methods VIP716 selectivity and potency: The motor domain of the human KSP and the other KIF family members were incubated with stabilized microtubules in a biochemical assay. The freshly prepared mixture was then incubated VIP716 and ispinesib, a clinical stage KSPi. ATPase activity was directly analyzed by measuring the formed inorganic phosphate. VIP943 selectivity: The binding of VIP943 to 6,019 human plasma membrane proteins expressed on HEK cells was evaluated (Retrogenix cell microarray). Cell cycle/death analysis: The effect of VIP943 on the 3 phases of the cell cycle (G0/G1- S- G2/M) was measured with DNA staining and flow cytometry in MOLM-13 and MV-4-11 cell lines. In addition, cell death was measured by flow cytometry after a 4 h incubation with VIP943 in MOLM-13 cells. Isotype ADC was used as a control. In vitro cytotoxicity: 56 hematologic cell lines were treated in vitro with VIP331 (cell permeable analog of VIP716). IC 50 results were matched with the DNA/RNA profile of relevant oncogenes to determine correlations. Nonhuman primate safety and toxicokinetics: VIP943 was intravenously administered to Cynomolgus monkeys at 1, 3, or 9 mg/kg once weekly for 4 weeks in a Good Laboratory Practice (GLP) toxicology study. Serial plasma samples were collected on Day 1 and Day 22. Total antibody from VIP943 (as a surrogate for VIP943) and VIP716 concentrations were measured by LC/MS/MS. Toxicokinetics (TK) were assessed using noncompartmental methods. Results In a biochemical assay, VIP716 (payload), achieved comparable potency to ispinesib with IC 50 values of 1.4 and 1.6 nM, respectively, for KSP. VIP716 did not inhibit any of the 11 other KIF family members up to 10 µM. In the Retrogenix assay, VIP943 (ADC) had no interaction with any of the analyzed human cell surface proteins except for CD123. Cell cycle analysis in AML cells showed a dose- and time-dependent increase in G2/M arrest with VIP943 treatment, but not with isotype control. An increase in cell death with VIP943 treatment was also observed after a 4-hour incubation. In a broad hematologic cell line panel, the median cytotoxicity IC 50 for VIP331 (cell permeable KSPi) was 2.49 nM. Of note, no genomic alterations correlated with sensitivity, suggesting VIP943 may be effective even in hard-to-treat hematologic malignancies. VIP943 was well tolerated in the GLP monkey toxicology study. No mortality occurred in any monkeys. No neutropenia, thrombocytopenia or anemia were observed. VIP943 and VIP716 exposures increased with increasing dose in a dose-proportional or greater than dose-proportional manner. No consistent differences in TK were observed with sex. Based on area under the concentration curve (AUC), no accumulation was observed. Payload to parent ratios were consistent with a low level of non-specific release of VIP716 from VIP943. Conclusion High on-target activity of VIP716, the payload of our new CD123-targeting ADC, VIP943, was demonstrated in a biochemical assay and selectivity against other KIF family members was confirmed. This activity translated into cell cycle arrest in the G2/M phase and subsequent cell death in a dose- and time-dependent manner. Additionally, toxicology in non-human primates and in vivo TK studies confirm safety, favorable drug exposures, and little non-specific release of the payload. Based on these data, evaluation of VIP943 in human clinical trials is warranted.

A sensitive LC-MS/MS assay to quantitate free payload Aur0101 from ADC PYX-201 in rat and monkey plasma.

Aim: Aur0101 is a cytotoxic and small-molecule microtubule depolymerizing agent, and is the payload conjugated to antibody-drug conjugatePYX-201. Developing and validating a sensitive bioanalytical method to quantitate Aur0101 was novel and crucial in preclinical PYX-201 studies. Materials & methods: Reference standard Aur0101 and its stable isotope labelled internal standard Aur0101-d8 were used in this LC-MS/MS method. Results: This sensitive assay was validated at alower limit of quantitation of15pg/ml and successfully applied to support preclinical rat and monkey toxicology studies. Preclinical plasma toxicokineticparameters were presented. Conclusion: A sensitive and robust LC-MS/MS assay was validated for Aur0101 in rat and monkey plasma.

Abstract 5667: CYT-303 FLEX-NKTM engager dose response efficacy mechanisms in HCC tumor model and safety in cynomolgus monkey toxicology studies support clinical evaluation in hepato-cellular carcinoma

Abstract BACKGROUND: CYT-303 is a multifunctional bispecific NK engager (NKE) targeting NK cell activating receptor NKp46 and tumor antigen Glypican-3 (GPC3) expressed in HCC (hepatocellular carcinoma). Cytovia’s proprietary FLEX-NKTM platform utilizes a novel FLEX-linker and human IgG1 back bone to allow for simultaneous binding to targeted cancer cells and NK cells. We evaluated CYT-303 dose response pharmacology efficacy mechanisms in HCC tumor models and conducted safety assessment studies in cynomolgus monkeys to support first in human clinical studies in HCC patients. METHODS: CYT-303 dose response pharmacology mechanistic studies were conducted in NSG-hIL-15 mice bearing Hep3B tumors and flow cytometry was used to assess bold and tumor NK cells. A 4-week CYT-303 repeat dose GLP toxicology study in cynomolgus monkeys at 6, 20 and 60 mg/kg dose was conducted following weekly intravenous infusions followed by a 6-week recovery period. CYT-303 toxicokinetic and anti-drug antibody (ADA) assessments were conducted using validated immunoassays. RESULTS: CYT-303 dose response in HCC tumor models showed increased tumor growth inhibition at the lower 2-3 mg/kg doses compared to higher 5-10 mg/kg doses consistent with the dose response previously reported in NK cell redirected cytotoxicity assays against HCC tumors. The increased anti- tumor efficacy observed at the lower doses were associated increased infiltration of PBNK’s to the tumor and corresponding reductions in PBNK’s in blood suggesting CYT-303 facilitates PBNK entry and retention in HCC tumors expressing GPC3 tumor antigen. CYT-303 safety assessment in the 4-week toxicology study in cynomolgous monkeys showed no treatment related toxicities or cytokine release and the NOAEL was determined to 60 mg/kg/week the highest dose administered in the study. No treatment related clinical signs, clinical pathology (hematology and clinical chemistry) and anatomic pathology (macroscopic, microscopic and organ weights) changes were observed. CYT-303 toxicokinetic analysis showed dose dependent increases in Cmax and AUC’s and no evidence for CYT-303 accumulation. CYT-303 anti-drug antibodies in monkeys were minimal and only observed in one low dose animal (1 out of 22) which also showed reduced CYT-303 exposures. Based on these pharmacology and toxicology results a pharmacologically active dose (PAD) based approach together with pharmacokinetic modeling would be utilized to estimate first in human dosing in HCC patients. CONCLUSIONS: CYT-303 efficacious doses identified in the HCC model together with safety in cynomolgus monkeys and planned human pharmacokinetic modeling studies support clinical evaluation of CYT-303 in first in human HCC clinical trials. Citation Format: Vishal Khairnar, Christine Waters, Solgalim Diaz, David De Franco, Jean Kadouche, Daniel Teper, Wei Li, Antonio Arulanandam. CYT-303 FLEX-NKTM engager dose response efficacy mechanisms in HCC tumor model and safety in cynomolgus monkey toxicology studies support clinical evaluation in hepato-cellular carcinoma. [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 5667.

Immunosuppression Profile of CFZ533 (Iscalimab), a Non-Depleting Anti-CD40 Antibody, and the Presence of Opportunistic Infections in a Rhesus Monkey Toxicology Study.

CFZ533 (iscalimab) is a nondepleting anti-CD40 antibody intended for inhibition of transplant organ rejection and treatment of autoimmune diseases. In a safety assessment in rhesus monkeys, CFZ533 was administered for 13 weeks up to 150 mg/kg/week subcutaneously. CFZ533 was shown previously to completely inhibit primary and secondary T-cell-dependent antibody responses. CD40 is expressed on B cells, antigen-presenting cells, and endothelial and epithelial cells, but is not expressed on T cells. Here, we demonstrate the complete suppression of germinal center formation in lymphoid organs. CFZ533 was well tolerated and did not cause any dose-limiting toxicity. However, the histological evaluation revealed increased numbers of CD4+ and CD8+ T cells in the T-cell-rich areas of lymph nodes enlarged in response to observed adenovirus and Cryptosporidium infections which suggest that T-cell immune function was unaffected. Background infections appear as the condition leading to unraveling the differential immunosuppressive effects by CFZ533. The presence of T cells at lymph nodes draining sites of infections corroborates the immunosuppressive mechanism, which is different from calcineurin-inhibiting drugs. Furthermore, CFZ533 did not show any hematological or microscopic evidence of thromboembolic events in rhesus monkeys, which were previously shown to respond with thromboembolism to treatment with anti-CD154 antibodies.

Nonclinical toxicology development of a novel antibody antibiotic conjugate for treating invasive Staphylococcus Aureus infections

Invasive Staphylococcus aureus (S. aureus) infections are a leading cause of death and not effectively treated with prolonged standard of care antibiotics. A novel THIOMAB™ antibody antibiotic conjugate (TAC) was developed that uses a bacterial-wall specific antibody to deliver the antibiotic (dmDNA31, a rifamycin analogue) to bacteria to minimize toxicities typically seen with prolonged use of traditional antibiotics. The TAC nonclinical toxicology package included repeat dose rat and cynomolgus monkey toxicology studies for 8 weekly intravenous (IV) doses, a 7-day daily repeat dose IV toxicology study of dmDNA31 and an assessment of genotoxicity, cardiovascular toxicity, neurotoxicity and sperm parameters. TAC and dmDNA31 were well tolerated in rats and monkeys, and there was no evidence of genotoxicity, cardiovascular toxicity or neurotoxicity. Non-adverse findings were observed and included blue discoloration in skin, blood, etc. due to the blue color of dmDNA31, increased globulin due to the high doses of antibodies, and abnormal sperm morphology of small heads in male rats with no histopathology correlate in testis. This is an example of antibody-mediated delivery of an antibiotic that has the potential to offer a more effective way of eradicating infection while providing a better safety profile compared to traditional antibiotics.

Preclinical Assessment of a MUC12-Targeted BiTE (Bispecific T-cell Engager) Molecule

MUC12 is a transmembrane mucin that is highly expressed in >50% of primary and metastatic colorectal tumors. MUC12 is also expressed by normal epithelial cells of the colon and small intestine. Although MUC12 localization in normal epithelial cells is restricted to the apical membrane, expression in tumors is depolarized and shows broad membrane localization. The differential localization of MUC12 in tumor cells as compared with normal cells makes it a potential therapeutic target. Here, we evaluated targeting of MUC12 with a BiTE (bispecific T-cell engager) molecule. We generated a panel of proof-of-concept half-life extended (HLE) BiTE molecules that bind MUC12 on tumor cells and CD3 on T cells. We prioritized one molecule based on in vitro activity for further characterization in vivo In vitro, the MUC12 HLE BiTE molecule mediated T-cell-redirected lysis of MUC12-expressing cells with half-maximal lysis of 4.4 ± 0.9 to 117 ± 78 pmol/L. In an exploratory cynomolgus monkey toxicology study, the MUC12 HLE BiTE molecule administered at 200 μg/kg with a step dose to 1,000 μg/kg was tolerated with minimal clinical observations. However, higher doses were not tolerated, and there was evidence of damage in the gastrointestinal tract, suggesting dose levels projected to be required for antitumor activity may be associated with on-target toxicity. Together, these data demonstrate that the apically restricted expression of MUC12 in normal tissues is accessible to BiTE molecule target engagement and highlight the difficult challenge of identifying tumor-selective antigens for solid tumor T-cell engagers.

A Biparatopic Antibody-Drug Conjugate to Treat MET-Expressing Cancers, Including Those that Are Unresponsive to MET Pathway Blockade.

Lung cancers harboring mesenchymal-to-epithelial transition factor (MET) genetic alterations, such as exon 14 skipping mutations or high-level gene amplification, respond well to MET-selective tyrosine kinase inhibitors (TKI). However, these agents benefit a relatively small group of patients (4%-5% of lung cancers), and acquired resistance limits response durability. An antibody-drug conjugate (ADC) targeting MET might enable effective treatment of MET-overexpressing tumors (approximately 25% of lung cancers) that do not respond to MET targeted therapies. Using a protease-cleavable linker, we conjugated a biparatopic METxMET antibody to a maytansinoid payload to generate a MET ADC (METxMET-M114). METxMET-M114 promotes substantial and durable tumor regression in xenografts with moderate to high MET expression, including models that exhibit innate or acquired resistance to MET blockers. Positron emission tomography (PET) studies show that tumor uptake of radiolabeled METxMET antibody correlates with MET expression levels and METxMET-M114 efficacy. In a cynomolgus monkey toxicology study, METxMET-M114 was well tolerated at a dose that provides circulating drug concentrations that are sufficient for maximal antitumor activity in mouse models. Our findings suggest that METxMET-M114, which takes advantage of the unique trafficking properties of our METxMET antibody, is a promising candidate for the treatment of MET-overexpressing tumors, with the potential to address some of the limitations faced by the MET function blockers currently in clinical use.

Abstract LB175: APX601, a Potent TNFR2 Antagonist as a Novel and Promising Approach to Reverse Tumor Immune Suppression

Abstract A key barrier to effective immunotherapy for cancer is the immunosuppressive tumor microenvironment (TME) characterized by infiltrating regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSC). While depletion of immune-suppressive cells is a promising cancer immunotherapy strategy, current approaches are ineffective due to lack of specificity and safety concerns. Tumor Necrosis Factor Receptor 2 (TNFR2) is emerging as a novel, selective target to overcome immunosuppression in the TME. Here we present APX601 as a potent Treg and myeloid suppressive cell inhibitor. APX601 is a humanized IgG1 antibody to TNFR2 generated using APXiMAB™, Apexigen's proprietary antibody platform. APX601 selectively binds human and monkey TNFR2, blocks the binding of TNFR2 to TNF-α and depletes TNFR2-expressing Treg and tumor cells in vitro. In addition to depleting suppressive cells, APX601 can also reverse MDSC and Tregs mediated immune suppression. In human TNFR2 knock-in syngeneic mouse tumor models CT26 and MC38, treatment with APX601 results in significant tumor growth inhibition both as a single agent and in combination with anti-PD-1. Thus, APX601 is a potent antagonist antibody against TNFR2 with the ability to reverse immune suppression in TME and inhibit tumor growth. APX601 was evaluated in a repeat dose range finding toxicology study in monkeys and demonstrated a good safety profile when administered up to 100mg/kg. These data strongly support the development of APX601 as a novel therapeutic approach for reversing tumor immune suppression and for the treatment solid tumors. Citation Format: Sushma Krishnan, Ryan Alvarado, George Huang, Xiaodong Yang, Erin L. Filbert. APX601, a Potent TNFR2 Antagonist as a Novel and Promising Approach to Reverse Tumor Immune Suppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB175.

Preclinical Safety Assessment and Toxicokinetics of Apitegromab, an Antibody Targeting Proforms of Myostatin for the Treatment of Muscle-Atrophying Disease.

Myostatin is a negative regulator of skeletal muscle and has become a therapeutic target for muscle atrophying disorders. Although previous inhibitors of myostatin offered promising preclinical data, these therapies demonstrated a lack of specificity toward myostatin signaling and have shown limited success in the clinic. Apitegromab is a fully human, monoclonal antibody that binds to human promyostatin and latent myostatin with a high degree of specificity, without binding mature myostatin and other closely related growth factors. To support the clinical development of apitegromab, we present data from a comprehensive preclinical assessment of its pharmacology, pharmacokinetics, and safety across multiple species. In vitro studies confirmed the ability of apitegromab to inhibit the activation of promyostatin. Toxicology studies in monkeys for 4 weeks and in adult rats for up to 26 weeks showed that weekly intravenous administration of apitegromab achieved sustained serum exposure and target engagement and was well-tolerated, with no treatment-related adverse findings at the highest doses tested of up to 100 mg/kg and 300 mg/kg in monkeys and rats, respectively. Additionally, results from an 8-week juvenile rat study showed no adverse effects on any endpoint, including neurodevelopmental, motor, and reproductive outcomes at 300 mg/kg administered weekly IV. In summary, the nonclinical pharmacology, pharmacokinetic, and toxicology data demonstrate that apitegromab is a selective inhibitor of proforms of myostatin that does not exhibit toxicities observed with other myostatin pathway inhibitors. These data support the conduct of ongoing clinical studies of apitegromab in adult and pediatric patients with spinal muscular atrophy (SMA).

Characterization of robust immune responses to a bispecific antibody, a novel class of antibody therapeutics.

Background: Anti-A/B is a bispecific monoclonal antibody thatblocks activities of soluble targets A and B. Robust immune responses were observed in a multiple-dose cynomolgus monkey toxicology study, negatively impacting the toxicokinetics/pharmacodynamics profile of anti-A/B in some animals. This was unexpected as similar findings were not observed in the two previously studied parental molecules. Methodology & Results: This paper discusses our characterization strategy for evaluating the immunogenic domain(s) of anti-A/B and our mitigation plan to monitor immunogenicity in the first-in-human clinical study. The characterization results from the cynomolgus monkey and Phase I studies are discussed. Conclusion: The characterization strategy discussed informed understanding of immunogenicity results and clinical impact, which can be broadly applied to other molecules with multiple-binding domains.

1283. New Generation Antiviral Conjugate (AVC): Stable, Safe, and Single

BackgroundCD377 is a novel antiviral Fc-conjugate (AVC) development candidate for influenza prevention and treatment, comprising multiple copies of a novel potent small-molecule antiviral and the Fc domain of human IgG1. CD377 was designed as a stable, long-acting molecule for treatment and prevention of influenza A and B. Studies were conducted to characterize CD377 stability/pharmacokinetics (PK), single-dose efficacy in influenza models, and safety/toxicology.MethodsPK in the mouse (1-100 mg/kg), rat (5-50 mg/kg), ferret (3 mg/kg), and monkey (5-20 mg/kg) were studied by sampling plasma over a 1-2 week interval. Plasma levels of intact molecule and total Fc were measured by neuraminidase (NA)-capture and Fc-capture with Fc-detection ELISA, respectively. Two-week safety/toxicology (bodyweight, coagulation, clinical signs, chemistries, hematology, cytokines, urinalsis, histopathology) was evaluated in monkeys (5-20 mg/kg on days 1 and 8). Prophylaxis efficacy was studied in a lethal influenza mouse model using a single dose of CD377 (0.3–3 mg/kg) 28 days prior to intranasal (IN) challenge with 3x the LD95 of A/California/07/2009 (H1N1)pdm, A/Hong Kong/1/68 (H3N2), or B/Malaysia (Victoria lineage). Treatment efficacy was studied in a similar mouse model using a single dose of CD377 (0.3–3 mg/kg) administered 2 hours after IN challenge with A/CA/12/2012 (H1N1)pdm.ResultsPlasma concentrations measured by Fc-capture/Fc-detection and NA-capture/Fc-detection were comparable, indicating that CD377 remained intact in vivo. In species tested, CD377 t1/2 was 3–10 days. Dose proportional increases in exposure were observed, notably from 1–100 mg/kg in mouse. High bioavailability (77%) was observed after subcutaneous (SC) or intramuscular (IM) administration. A single SC dose of 1 mg/kg administered 28 days prior to infection provided 100% protection against H1N1, B, and H3N2 subtypes in mouse (Fig. 1). Treatment efficacy was observed with a single 0.3 mg/kg IM dose. The 2-week monkey toxicology study showed no adverse effects.Figure 1. Efficacy (Survival and Body Weight) of CD377 in a 28-Day Prevention Model Against Influenza H1N1, H3N2, and B Subtypes in Mouse (IN infection challenge on Day t=0 and CD377 dosed t–28 days). ConclusionThe stability and safety of CD377, together with its long half-life and efficacy with a single dose, support the potential of CD377 as a long-acting, novel AVC for the prevention and treatment of influenza.DisclosuresVoon Ong, PhD, Cidara Therapeutics, Inc. (Employee, Shareholder) James Levin, PhD, Cidara Therapeutics (Shareholder) Allen Borchardt, PhD, Cidara Therapeutics (Employee) Thomas P. Brady, PhD Chemistry, Cidara Therapeutics (Employee) Thanh Lam, PhD, Cidara Therapeutics (Shareholder) Alain Noncovich, PhD, Cidara Therapeutics (Shareholder) Joanne Fortier, BSc, Cidara Therapeutics (Employee, Shareholder) Karin Amundson, B.S., Cidara Therapeutics (Shareholder) Jeffrey B. Locke, PhD, Cidara Therapeutics, Inc. (Employee, Shareholder) Amanda Almaguer, Bachelors, Cidara Therapeutics, Inc. (Employee, Shareholder) Nicholas Dedeic, n/a, Cidara Therapeutics (Employee) Grayson Hough, MS - Chemistry, Cidara Therapeutics (Employee) Jason Cole, PhD, Cidara Therapeutics (Shareholder) Simon Döhrmann, PhD, Cidara Therapeutics (Shareholder) Rajvir Grewal, n/a, Cidara Therapeutics, Inc. (Employee, Shareholder) Elizabeth Abelovski, B.S., Cidara Therapeutics (Shareholder) James M. Balkovec, PhD, Cidara Therapeutics (Consultant, Shareholder) Ken Bartizal, PhD, Cidara Therapeutics, Inc. (Consultant, Shareholder) Les Tari, PhD, Cidara Therapeutics (Shareholder)

Abstract CT245: NEON-1: A first-in-human phase I open-label study of ALPN-202, a conditional CD28 costimulator and dual checkpoint inhibitor, in advanced malignancies

Abstract Background: Checkpoint inhibitors (CPI) targeting the PD-(L)1 and CTLA-4 pathways have demonstrated significant clinical benefit in many cancers, but the majority of patients unfortunately still fail to demonstrate objective responses and/or develop resistance, at least in part due to insufficient activation and/or excessive exhaustion of tumor-specific T cells. Strong preclinical rationale has emerged for combining CPIs with T cell costimulatory agonists, but such approaches have not yet translated into significantly improved clinical benefit. The majority of these approaches, though, have not targeted CD28, a well-known, critical T cell costimulatory pathway. ALPN-202 is a variant CD80 vIgD-Fc fusion that mediates PD-L1-dependent CD28 costimulation and inhibits the PD-L1 and CTLA-4 checkpoints. As monotherapy, ALPN-202 has demonstrated superiority to CPI-only therapies in preclinical tumor models, while demonstrating a favorable safety profile in rat and monkey toxicology studies. It is in development for the treatment of multiple advanced malignancies. Methods: This is a cohort-based, open-label dose escalation and expansion study in adults with advanced solid tumors or lymphoma (NCT04186637). The objectives of this study are to evaluate the safety, efficacy, pharmacokinetics (PK), anti-drug antibodies (ADA), and pharmacodynamics (PD) of ALPN-202. Subjects who are refractory or resistant to standard therapy (including approved CPIs), or without available standard or curative therapy are eligible. ALPN-202 will be administered by IV infusion. Dose escalation will initially use single-subject cohorts as an accelerated titration strategy to minimize the number of patients that will receive dose levels with low likelihood to elicit clinically meaningful PD activity. At dose levels predicted to elicit clinically meaningful PD activity, a standard 3+3 strategy will be used in two arms studying different dose schedules–Q1W and Q3W. Thereafter, an expansion cohort(s) will enroll subjects at a selected dose(s) and selected indication(s). Outcomes include safety assessments such as adverse events and dose-limiting toxicities. Tumor responses will be categorized by RECIST v1.1 for solid tumors, and by Lugano Classification for lymphoma. Biomarkers will include tumor measurements of PD-L1, CD28, CD80 and CD86 to explore the possible relationship between baseline expression and outcomes. Other PD will include target saturation, inflammatory cytokines and immunophenotyping of circulating leukocytes. Citation Format: Jing Yang, Gary Means, Almudena Tercero, Kristi Manjarrez, Jan Hillson, Stanford L. Peng. NEON-1: A first-in-human phase I open-label study of ALPN-202, a conditional CD28 costimulator and dual checkpoint inhibitor, in advanced malignancies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT245.

Nonclinical Safety Assessment of AMG 553, an Investigational Chimeric Antigen Receptor T-Cell Therapy for the Treatment of Acute Myeloid Leukemia.

Feline McDonough Sarcoma-like tyrosine kinase 3 (FLT3), a tyrosine-protein kinase involved in hematopoiesis, is detectable on the cell surface of approximately 80% of leukemia isolates from adult patients with acute myeloid leukemia (AML). AMG 553 is an investigational chimeric antigen receptor (CAR) T-cell immunotherapy for the treatment of AML. FLT3 expression analysis and in vitro and in vivo studies were leveraged to evaluate the nonclinical safety of AMG 553. Cynomolgus monkeys administered autologous anti-FLT3 CAR T cells demonstrated no evidence of CAR T-cell-mediated toxicity, expansion, or persistence, likely due to restricted cell surface FLT3 protein expression in healthy animals. This highlights the limited value of such in vivo studies for safety assessment of the CAR T-cell modality when directed against a target with restricted expression. To complement these studies and directly evaluate the potential toxicities of eliciting T-cell-mediated cytotoxicity against cells with surface expression of FLT3 protein in vivo, data from cynomolgus monkey toxicology studies with 2 bispecific T-cell engager molecules targeting FLT3 were leveraged; findings were consistent with the targeted killing of bone marrow cells expressing cell surface FLT3. Potential AMG 553-induced cytotoxicity was assessed against a wide range of normal human primary cells and cell lines; cytotoxicity was observed against FLT3-positive AML cell lines and a percentage of primary bone marrow CD34+ cells. In conclusion, the nonclinical safety data suggest that AMG 553 can target FLT3 protein on AML cells, whereas only affecting a percentage of normal hematopoietic stem and progenitor cells, supporting clinical development.

Immune complex disease in a chronic monkey study with a humanised, therapeutic antibody against CCL20 is associated with complement-containing drug aggregates.

Despite the potential for the chemokine class as therapeutic targets in immune mediated disease, success has been limited. Many chemokines can bind to multiple receptors and many receptors have multiple ligands, with few exceptions. One of those exceptions is CCL20, which exclusively pairs to CCR6 and is associated with several immunologic conditions, thus providing a promising therapeutic target. Following successful evaluation in a single dose, first time in human clinical study, GSK3050002-a humanized IgG1 monoclonal antibody against human CCL20-was evaluated in a 26-week cynomolgus monkey toxicology study. A high incidence of unexpected vascular and organ inflammation was observed microscopically, leading to the decision to halt clinical development. Here we report a dose-responsive increase in the incidence and severity of inflammation in multiple organs from monkeys receiving 30 and 300 mg/kg/week by either subcutaneous or intravenous injection. Histomorphological changes resembled an immune complex-mediated pathology, which is often due to formation of anti-drug antibodies in monkeys receiving a human protein therapeutic and thus not predictive of clinical outcome. However, the presentation was atypical in that there was a clear dose response with a very high incidence of inflammation with a low incidence of ADA that did not correlate well individually. Additionally, the immunohistologic presentation was atypical in that the severity and distribution of tissue inflammation was greater than the numbers of associated immune complexes (i.e., granular deposits). An extensive ex vivo analysis of large molecular weight protein complexes in monkey serum from this study and in human serum samples demonstrated a time-dependent aggregation of GSK3050002, that was not predicted by in vitro assays. The aggregates also contained complement components. These findings support the hypothesis that immune complexes of drug aggregates, not necessarily including anti-drug antibodies, can fix complement, accumulate over time, and trigger immune complex disease. A situation which may have increased clinical relevance than typical anti-drug antibody-associated immune complex disease in monkeys administered human antibody proteins.

Abstract 540: AO-176, a normal cell sparing humanized anti-CD47 antibody

Abstract AO-176 is a next generation humanized anti-CD47 IgG2 that binds human and cynomolgus monkey CD47 equivalently. AO-176, like other CD47 antibodies, blocks the interaction of CD47 with SIRPα, inducing phagocytosis of tumor cells by activated macrophages. Unique to AO-176 is its ability to directly kill tumor cells via programmed cell death type III and immunogenic cell death in a cell autonomous (non-ADCC) manner. AO-176 also exhibits preferential binding to tumor vs. normal cells, a notable characteristic, as CD47 is expressed on many normal cells including red blood cells (RBC), platelets, T cells and endothelial cells. While other CD47 antibodies induce hematological toxicities such as anemia and thrombocytopenia in both primate models and in patients, AO-176 negligibly binds RBCs and platelets and even at high doses, minimally impacts hematology in cynomolgus monkey toxicology studies. AO-176 is currently being evaluated in phase 1 clinical trials for the treatment of solid tumors. In recent head to head experiments conducted with other CD47 antibodies, AO-176 bound all normal cells tested to a significantly lower degree. For example, unlike anti-CD47 antibodies such as Hu-5F9-G4, we observed negligible and minimal ex vivo binding to healthy human RBCs and platelets respectively and significantly lower binding of AO-176 to other hematologic and non-hematologic cells such as T cells and endothelial cells. Although AO-176 binds cynomolgus monkey RBCs to a slightly greater degree than human RBCs ex vivo, AO-176 versus comparative published Hu5F9-G4 findings has demonstrated dramatically reduced receptor occupancy when evaluated in monkey toxicology studies. We have also developed a clinic-ready receptor occupancy assay to measure AO-176 binding to circulating PBMCs in patients treated with AO-176 and to demonstrate minimal binding to CD47 on normal human cells. We have begun to evaluate mechanisms that may underlie the normal cell sparing effects of AO-176. Published studies have demonstrated that in addition to CD47 protein expression level, clustering and mobility of CD47 at the cell surface may impact binding to ligands, downstream signaling and subsequent cellular responses such as apoptosis. For example, avidity for SIRPα and clearance by macrophages is modulated by CD47 clustering that can be modulated by protein glycosylation, association with lipid rafts, cytoskeleton, integrins or other cis-acting factors. Here, we compare the distribution of CD47 on tumor and RBCs to evaluate receptor association with other cellular membrane components that may impact distribution on the cell surface. In summary, AO-176 is a next generation anti-CD47 antibody that, aside from induction of phagocytosis, possesses additional attributes that include direct tumor cell killing and preferential binding to tumor versus normal cells. These attributes of AO-176 differentiate it from other CD47 axis targeting agents currently in clinical evaluation. Citation Format: Prabir Chakraborty, Myriam N. Bouchlaka, Benjamin J. Capoccia, Ronald R. Hiebsch, Michael J. Donio, Robyn J. Puro, Vicki Sung, Daniel S. Pereira. AO-176, a normal cell sparing humanized anti-CD47 antibody [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 540.

Discovery of A Novel EGFR-Targeting Antibody-Drug Conjugate, SHR-A1307, for the Treatment of Solid Tumors Resistant or Refractory to Anti-EGFR Therapies.

Although inhibiting EGFR-mediated signaling proved to be effective in treating certain types of cancers, a quickly evolved mechanism that either restores the EGFR signaling or activates an alternative pathway for driving the proliferation and survival of malignant cells limits the efficacy and utility of the approach via suppressing the EGFR functionality. Given the fact that overexpression of EGFR is commonly seen in many cancers, an EGFR-targeting antibody-drug conjugate (ADC) can selectively kill cancer cells independently of blocking EGFR-mediated signaling. Herein, we describe SHR-A1307, a novel anti-EGFR ADC, generated from an anti-EGFR antibody with prolonged half-life, and conjugated with a proprietary toxin payload that has increased index of EGFR targeting-dependent versus EGFR targeting-independent cytotoxicity. SHR-A1307 demonstrated strong and sustained antitumor activities in EGFR-positive tumors harboring different oncogenic mutations on EGFR, KRAS, or PIK3CA. Antitumor efficacy of SHR-A1307 correlated with EGFR expression levels in vitro and in vivo, regardless of the mutation status of EGFR signaling mediators and a resultant resistance to EGFR signaling inhibitors. Cynomolgus monkey toxicology study showed that SHR-A1307 is well tolerated with a wide therapeutic index. SHR-A1307 is a promising therapeutic option for EGFR-expressing cancers, including those resistant or refractory to the EGFR pathway inhibitors.

Variability of Spleen and Mesenteric Lymph Node in Control Cynomolgus Monkeys ( Macaca fascicularis) from Nonclinical Safety Studies: A Retrospective Assessment.

We assessed the variability of spleen and mesenteric lymph node (MLN) microscopic observations and the correlations of these observations with other study data from 478 control cynomolgus monkeys from 53 routine nonclinical safety studies. Spleen weight parameters (absolute and relative to body or brain weights) were highly variable both within a control group on an individual study (up to 5.11-fold) and among animals with the same light microscopic observation. Grades for microscopic observations were also highly variable. The most frequent microscopic observations for spleen were changes in the size and number of germinal centers (58%), acidophilic (hyaline) material in lymphoid follicles (52%), and compound lymphoid follicles (20%). The most frequent microscopic observations in the MLN were eosinophil infiltrates (90%), changes in size and number of germinal centers (42%), and brown pigment (21%). The only meaningful relationships ( r2 > 0.3) were positive correlations between reticuloendothelial hyperplasia and malarial pigment in the spleen and between each of these observations and spleen weight parameters. We conclude that determination of test article-related effects on the immune system in routine monkey toxicology studies requires careful consideration and a weight-of-evidence approach due to the low numbers of animals/group, the inherent variability in spleen and MLN parameters, and the infrequent correlation among immune system-related end points.

Investigating Potential Mechanism(s) By Which ASO-Based Drugs Cause Thrombocytopenia

IntroductionAntisense oligonucleotides (ASOs) are a new class of single-stranded DNA based drugs that hold great therapeutic promise for their disease modifying potential in a wide range of genetic diseases. Preclinical toxicology studies in monkeys, as well as late stage clinical trials in humans, have upon repeated dosing, reported events of ASO sequence-specific lowering of platelet counts (mild to severe thrombocytopenia) (Henry et al. Nucleic acid therapeutics 2017). The underlying cause of this platelet decrease is still unclear in humans. We have investigated if the thrombocytopenia associated with ASOs is due to either impaired platelet production and/or destruction of platelets (clearance) due to increased platelet reactivity (activation/aggregation status). Preliminary data from mouse derived fetal liver megakaryocytes suggest that pro-platelet production does not seem to be reduced by ASOs and hence in the current study we hypothesized that the ASO-induced thrombocytopenia is due to increased clearance of platelets from the circulation.MethodsIn the current study we explored how ASOs affect platelet aggregation in platelet rich plasma (PRP) and platelet-leukocyte aggregates in whole blood (WB) obtained from healthy volunteers after informed consent. PRP or WB was treated with a clinically relevant concentration of ASO (5µM) corresponding to expected maximum plasma concentration levels, or a 20x-supra-therapeutic concentration (100µM). Four ASOs were tested: two CpG-rich phosphorothioate deoxyoligonucleotide (PS ODN) sequences: 818290 and 120704, and two non-CpG 2'-MOE containing sequences: 104838 and 501861. 818290 was included as a positive control since it has been shown to cause direct platelet activation (Flierl et al. JEM 2015). 104838 have been reported to cause moderate, dose dependent drops in platelet counts in monkeys and humans, with platelet sequestration in the liver and spleen (Narayanan PK, et al. Toxicol Sci. 2018). 501861 has triggered sporadic severe thrombocytopenia in select monkeys. ASO treated PRP was analyzed for platelet aggregation using 96-well optimul aggregometry (Lordkipanidzé et al. Blood 2014) in the presence of vehicle (PBS) or 6 concentrations of thrombin receptor activating peptide-6 (0.08-80µM,TRAP6). In a separate experiment, PRP was incubated with ASOs plus the spleen tyrosine kinase (Syk) inhibitor PRT-060318 (10µM). ASO treated WB was incubated with fluorescently labelled CD41/61 antibody to label platelets and a leukocyte-specific antibody CD14, and platelet-leukocyte aggregates were analyzed by FACS according to (Gerrits et al. Curr. Protoc. 2016).ResultsThe two non-CpG rich 2'-MOE ASO sequences 104838 and 501861 did not affect platelet aggregation at either concentration (5µM + 100µM) (Figure 1 A+B). Whilst the two CpG-rich PS ODN ASOs (818290 and 120704) triggered spontaneous platelet aggregation in PRP at 100µM (Figure 1 C+D), that was normalized by co-incubating these ASOs with a Syk inhibitor (Figure 2). 5µM of ASO treatment triggered a significant increase in platelet-leukocyte aggregates in WB for all the ASOs tested (Figure 3).ConclusionWe have shown that the two CpG-rich PS ODN ASOs (818290 and 120704) triggered spontaneous platelet aggregation in PRP at 100µM. This effect was inhibited by a Syk inhibitor. 818290 has previously been identified to activate platelets through a Syk-dependent, GPVI receptor mediated mechanism (Flierl et al. JEM 2015). Here, we report for the first time that the aggregatory effects of 120704 have been identified to be Syk dependent as well, possibly through a similar interaction with platelet GPVI receptors.We have also presented novel data that therapeutically relevant concentrations of all the ASOs tested cause an increase in platelet-leukocyte aggregates in WB. Based on these data we highlight the importance of screening ASOs in multi-cellular assays, not just PRP, since there was no effect of the ASOs 104838 or 501861 on platelet aggregation. Enhanced formation of platelet-leukocyte aggregates could be one contributing factor for increased platelet clearance, explaining ASO-induced thrombocytopenia. Further investigation into the ASO-induced interactions between platelets and immune cells are warranted. Defining the mechanisms by which ASO-based drugs cause low platelet count may yield strategies to manage this drug-induced thrombocytopenia in patients. [Display omitted] DisclosuresThon:Platelet Biogenesis: Employment, Equity Ownership, Other: Co-founder, Patents & Royalties. Henry:Ionis Pharmaceuticals: Employment. Narayanan:Ionis Pharmaceuticals: Employment. Italiano:Platelet Biogenesis: Equity Ownership, Other: Co-founder, Patents & Royalties.

A Randomized, Double-Blind, Placebo-Controlled Phase 1 Study of the Pharmacokinetics and Pharmacodynamics of REGN3918, a Human Antibody Against Complement Factor C5, in Healthy Volunteers

INTRODUCTION: Blockade of complement factor C5 has demonstrated benefit in Paroxysmal Nocturnal Hemoglobinuria (PNH), atypical Hemolytic Uremic Syndrome and Generalized Myasthenia Gravis. We developed a human IgG4P antibody, REGN3918, that binds with high affinity to wild-type and variant (R885H/C) human C5. REGN3918 was well-tolerated in monkey toxicology studies with up to 26 weeks of dosing at up to 100 mg/kg/week. This finding was supportive of conducting this first-in-human study of REGN3918 in healthy volunteers.OBJECTIVE: The primary objective of this ongoing study is to evaluate the safety and tolerability of single ascending intravenous (IV) and subcutaneous (SC) doses and a multiple dose regimen consisting of an IV loading dose plus multiple weekly SC doses of REGN3918 administered in healthy volunteers. The secondary objectives of the study are to assess the pharmacokinetic (PK) and pharmacodynamic (PD) profile of REGN3918.METHODS: 56 subjects were randomized to 4 sequential ascending IV dose cohorts plus 2 sequential ascending SC cohorts followed by 1 multiple dose cohort (consisting of an IV loading dose and weekly SC doses). Each cohort consisted of 8 subjects randomized to receive REGN3918 or placebo (6 active: 2 placebo). An adaptive design was implemented to allow for dose level and dosing interval adjustment utilizing in-study PK and PD measures. The PD profile of REGN3918 was assessed utilizing a sheep red blood cell complement activity assay (CH50 assay) as well as serum concentrations of total C5. REGN3918 was administered as follows:•Cohort 1: 1 mg/kg IV, single dose•Cohort 2a: 3 mg/kg IV, single dose•Cohort 2b: 300 mg SC, single dose•Cohort 3a: 10 mg/kg IV, single dose•Cohort 3b: 600 mg SC, single dose•Cohort 4: 30 mg/kg IV, single dose•Cohort 5: Loading dose of 15 mg/kg IV followed by 4 repeat SC doses of 400 mg administered once weekly for four weeks.RESULTS: REGN3918 was found to be well tolerated in single doses of up to 30 mg/kg IV and 600 mg SC. The multiple dose Cohort 5 has completed dosing in all subjects and is currently in safety follow-up. Thus far, there has been one SAE, salpingitis in a subject with an intra-uterine contraceptive device. The SAE occurred in a Cohort 5 subject after completion of dosing and has since resolved. REGN3918 exhibited dose-dependent increases in exposure in serum, with a trend toward prolonged serum concentrations at IV doses ≥10 mg/kg. Following SC administration, concentrations of REGN3918 in serum peaked at 4 to 8 days post dose and bioavailability was estimated as approximately 70%. REGN3918 exposure led to dose-dependent inhibition of CH50. In all 4 IV dosing cohorts, suppression of hemolysis was observed at 15 min post-injection. Complete suppression of hemolysis was achieved with ≥ 3mg/kg dosing. At 30 mg/kg, complete suppression of hemolysis was maintained for >6 weeks, consistent with observed prolonged REGN3918 concentrations following this dose. In the 2 SC cohorts, peak suppression of hemolysis was observed 3-7 days post dosing, again consistent with observed peak concentrations of REGN3918 in serum. In the multiple dose cohort 5, complete suppression of CH50 was observed over the four-week dosing period.CONCLUSIONS: REGN3918 was well tolerated and resulted in dose-dependent inhibition of hemolytic activity in normal healthy volunteers. A single IV infusion of R3918, at 30 mg/kg, blocked hemolytic activity completely for >6 weeks. Complete inhibition of hemolytic activity was maintained over a 4-week dosing period by a weekly SC regimen following an IV loading dose. DisclosuresWeyne:Regeneron Pharmaceuticals: Employment, Equity Ownership. Ni:Regeneron Pharmaceuticals: Employment, Equity Ownership. DelGizzi:Regeneron Pharmaceuticals: Employment, Equity Ownership. Godin:Regeneron Pharmaceuticals: Employment. Morton:Regeneron Pharmaceuticals: Employment, Equity Ownership. Prasad:Regeneron Pharmaceuticals: Employment, Equity Ownership. Rankin:Regeneron Pharmaceuticals: Employment, Equity Ownership. Simek-Lemos:Regeneron Pharmaceuticals: Employment, Equity Ownership. Wang:Regeneron Pharmaceuticals: Employment, Equity Ownership. Rippley:Regeneron Pharmaceuticals: Employment, Equity Ownership. Harari:Regeneron Pharmaceuticals, Inc.: Employment.