Josh Snyder

Development of ASG-15ME, a Novel Antibody–Drug Conjugate Targeting SLITRK6, a New Urothelial Cancer Biomarker

SLITRK6 is a member of the SLITRK family of neuronal transmembrane proteins that was discovered as a bladder tumor antigen using suppressive subtractive hybridization. Extensive immunohistochemistry showed SLITRK6 to be expressed in multiple epithelial tumors, including bladder, lung, and breast cancer as well as in glioblastoma. To explore the possibility of using SLITRK6 as a target for an antibody–drug conjugate (ADC), we generated a panel of fully human mAbs specific for SLITRK6. ADCs showed potent in vitro and in vivo cytotoxic activity after conjugation to Monomethyl Auristatin E or Monomethyl Auristatin F. The most potent ADC, ASG-15ME, was selected as the development candidate and given the product name AGS15E. ASG-15ME is currently in phase I clinical trials for the treatment of metastatic urothelial cancer. This is the first report that SLITRK6 is a novel antigen in bladder cancer and also the first report of the development of ASG-15ME for the treatment of metastatic bladder cancer. Mol Cancer Ther; 15(6); 1301–10. ©2016 AACR.

Inhibition of Megakaryocyte Differentiation by Antibody–Drug Conjugates (ADCs) is Mediated by Macropinocytosis: Implications for ADC-induced Thrombocytopenia

Thrombocytopenia is a common adverse event in cancer patients treated with antibody–drug conjugates (ADC), including AGS-16C3F, an ADC targeting ENPP3 (ectonucleotide pyrophosphatase/phosphodiesterase-3) and trastuzumab emtansine (T-DM1). This study aims to elucidate the mechanism of action of ADC-induced thrombocytopenia. ENPP3 expression in platelets and megakaryocytes (MK) was investigated and shown to be negative. The direct effect of AGS-16C3F on platelets was evaluated using platelet rich plasma following the expression of platelet activation markers. Effects of AGS-16C3F, T-DM1, and control ADCs on maturing megakaryocytes were evaluated in an in vitro system in which human hematopoietic stem cells (HSC) were differentiated into MKs. AGS-16C3F, like T-DM1, did not affect platelets directly, but inhibited MK differentiation by the activity of Cys-mcMMAF, its active metabolite. FcγRIIA did not appear to play an important role in ADC cytotoxicity to differentiating MKs. AGS-16C3F, cytotoxic to MKs, did not bind to FcγRIIA on MKs. Blocking the interaction of T-DM1 with FcγRIIA did not prevent the inhibition of MK differentiation and IgG1-mcMMAF was not as cytotoxic to MKs despite binding to FcγRIIA. Several lines of evidence suggest that internalization of AGS-16C3F into MKs is mediated by macropinocytosis. Macropinocytosis activity of differentiating HSCs correlated with cell sensitivity to AGS-16C3F. AGS-16C3F was colocalized with a macropinocytosis marker, dextran-Texas Red in differentiating MKs. Ethyl isopropyl amiloride (EIPA), a macropinocytosis inhibitor, blocked internalization of dextran-Texas Red and AGS-16C3F. These data support the notion that inhibition of MK differentiation via macropinocytosis-mediated internalization plays a role in ADC-induced thrombocytopenia. Mol Cancer Ther; 16(9); 1877–86. ©2017 AACR. See related article by Zhao et al., p. 1866

Modulation of Macropinocytosis-Mediated Internalization Decreases Ocular Toxicity of Antibody–Drug Conjugates

AGS-16C3F is an antibody-drug conjugate (ADC) against ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3) containing the mcMMAF linker-payload currently in development for treatment of metastatic renal cell carcinoma. AGS-16C3F and other ADCs have been reported to cause ocular toxicity in patients by unknown mechanisms. To investigate this toxicity, we developed an in vitro assay using human corneal epithelial cells (HCEC) and show that HCECs internalized AGS-16C3F and other ADCs by macropinocytosis, causing inhibition of cell proliferation. We observed the same mechanism for target-independent internalization of AGS-16C3F in fibroblasts and human umbilical vein endothelial cells (HUVEC). Macropinocytosis-mediated intake of macromolecules is facilitated by the presence of positive charges or hydrophobic residues on the surface of the macromolecule. Modification of AGS-16C3F, either by attachment of poly-glutamate peptides, mutation of residue K16 to D on AGS-16C3F [AGS-16C3F(K16D)], or decreasing the overall hydrophobicity via attachment of polyethylene glycol moieties, significantly reduced cytotoxicity against HCECs and other primary cells. Rabbits treated with AGS-16C3F showed significant ocular toxicity, whereas those treated with AGS-16C3F(K16D) presented with less severe and delayed toxicities. Both molecules displayed similar antitumor activity in a mouse xenograft model. These findings establish a mechanism of action for target-independent toxicities of AGS-16C3F and ADCs in general, and provide methods to ameliorate these toxicities.Significance: These findings reveal a mechanism for nonreceptor-mediated toxicities of antibody drug conjugates and potential solutions to alleviate these toxicities. Cancer Res; 78(8); 2115-26. ©2018 AACR.

Metabolism of an Oxime-Linked Antibody Drug Conjugate, AGS62P1, and Characterization of Its Identified Metabolite

AGS62P1 is an antibody drug conjugate (ADC) composed of a human IgG1κ monoclonal antibody against FLT3 (FMS-like tyrosine kinase 3) with a p-acetyl phenylalanine (pAF) residue inserted at position 124 of each heavy chain linked to the proprietary microtubule disrupting agent AGL-0182-30 via an alkoxyamine linker that forms an oxime upon conjugation to the antibody. AGS62P1 is currently in Phase I human clinical trials for acute myelogenous leukemia (AML). The identified primary metabolite of an oxime-linked ADC is presented for the first time. AGS62P1 metabolism was assessed in xenograft tumor-bearing mice and rats treated with the ADC using liquid chromatography and mass spectrometry-based methods described herein. In this study, we identified the metabolite of AGS62P1 as pAF-AGL-0185-30, which contains a fragment resulting from the catabolism of the antibody component of the ADC and hydrolysis of the terminal amide portion of the linker-payload. We demonstrated that the metabolite of AGS62P1 is tolerated in rats above 1.5 mg/kg and above 0.334 mg/kg in cynomolgus monkeys when given as a single dose. Furthermore, we established in vitro that pAF-AGL-0185-30 does not significantly inhibit hERG or cytochrome P450 family enzymes (CYPs).

Preclinical Evaluation of an Anti-Nectin-4 ImmunoPET Reagent in Tumor-Bearing Mice and Biodistribution Studies in Cynomolgus Monkeys

PurposeNectin-4 is selectively overexpressed in a variety of cancers and is currently under clinical investigation as a therapeutic target. A monoclonal antibody against nectin-4 (AGS-22M6) was evaluated as an Immuno-positron emission tomography (ImmunoPET) reagent. Its ability to assay nectin-4 expression as well as detect nectin-4 positive tumors in the liver and bone was evaluated using mouse models.ProceduresThe biodistribution of [89Zr]AGS-22M6 was evaluated in mice bearing tumors with varying levels of nectin-4 expression. An isogenic breast cancer tumor line was used to model metastatic liver and bone disease in mice. The biodistribution of [18F]AGS-22M6 in cynomolgus monkeys was evaluated.ResultsA positive correlation was demonstrated between tumor nectin-4 expression and [89Zr]AGS-22M6 uptake. Tumors in the liver and bone were detected and differentiated based on nectin-4 expression. [18F]AGS-22M6 showed limited uptake in cynomolgus monkey tissues.Conclusions[89Zr]AGS-22M6 is a promising ImmunoPET reagent that can assay nectin-4 expression in both primary and metastatic lesions.

Abstract 3853: Potential mechanisms for thrombocytopenia and neutropenia induced by antibody-drug conjugates

Thrombocytopenia and neutropenia are common adverse events in cancer patients treated with antibody-drug conjugates (ADCs). We used human hematopoietic stem cells (HSC) in vitro and various ADCs to investigate the potential mechanisms of thrombocytopenia and neutropenia. Two ADCs, AGS-16C3F and T-DM1, were selected for the investigation of megakaryocytes (MKs) as they both induced thrombocytopenia in patients. AGS-16C3F is an ENPP3-targeting antibody conjugated to microtubule-disrupting agent MMAF (licensed from Seattle Genetics) via a non-cleavable linker (mcMMAF) tested in a phase 1 trial in kidney cancer patients. T-DM1 (KADCYLA) is an anti-HER2 antibody conjugated to the maytansine derivative DM1, approved for the treatment of advanced Her-2 positive breast cancer. HSCs were differentiated to MKs and percentage of CD41 positivity from FACS analysis was used to measure the effect of ADCs on MK differentiation. Results showed that while MKs do not express ENPP3 or Her-2, both AGS-16C3F (IC50 = 40nM) and T-DM1 inhibited MK differentiation. MKs express FcγRIIA to which T-DM1 (IgG1) and, to lesser degree, AGS-16C3F (IgG2) can bind; however, blockage of FcγRIIA failed to rescue MKs from cytotoxicity induced by either T-DM1 or AGS-16C3F suggesting that binding to FcγRIIA is not critical for MK cytotoxicity. AGS-16C3F is internalized into MKs via macropinocytosis and causes significant disorganization of tubulin structure as observed by confocal microscopy. HSCs were also differentiated to neutrophils which were characterized by the percentage of CD66b positivity in FACS analysis. Several antibodies to targets absent in neutrophils were conjugated to both MMAE and MMAF and were tested for cytotoxicity during neutrophil differentiation. Our results showed that ADCs containing MMAE via a cleavable linker were more cytotoxic to neutrophil differentiation than the same ADC containing MMAF via a non-cleavable linker, in agreement with clinical results. Different from MKs, results showed that neutrophils have low macropinocytosis activity. Secretion of cathepsins increased during neutrophil differentiation, and a membrane impermeable cathepsin inhibitor blocked ADC cytotoxicity to neutrophils, suggesting that extracellular protease cleavage is critical for neutrophil cytotoxicity in vitro. Neutrophils express high levels of FcγRIIA and saturation of this receptor by control IgG inhibited ADC binding to neutrophils and at least partially rescued neutrophils from ADC cytotoxicity, suggesting that FcγRIIA can play a role in neutropenia induced by ADCs, particularly with IgG1 isotype. In conclusion, applying these in vitro data to clinical experience suggests that thrombocytopenia may be mediated by macropinocytosis of ADC in MK and neutropenia by secreted proteases and interactions with their FcγRs. Understanding these toxicities may help design new ADCs with improved toxicity profile. Citation Format: Hui Zhao, Sara Gulesserian, Sathish K. Ganesan, Zhilan Zeng, Jimmy Ou, Veronica Robles, Josh Snyder, David R. Stover, Fernando Donate. Potential mechanisms for thrombocytopenia and neutropenia induced by antibody-drug conjugates. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3853.