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 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 similarantitumor 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. These findings reveal a mechanism for nonreceptor-mediated toxicities of antibody drug conjugates and potential solutions to alleviate these toxicities. .

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 antibodydrug 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.

Antibody drug conjugates offer a targeted cancer treatment for the delivery of potent cytotoxic drugs. Derivatives of the natural product dolastatin 10 containing pyridines and other basic amines were examined with the objective of determining if a more hydrophilic auristatin derivative would be potent enough for use as part of an ADC. This may be advantageous if a less hydrophobic drug makes a better ADC. A pyridine derivative, monomethyl auristatin PYE, showed the greatest potency when tested in vivo. While only a modest tumor growth inhibition was observed when the HCC1954 human breast cancer xenografts were treated with"non-cleavable" linker ADCs, tumor regression was seen when treated with an enzymatically degradable "cleavable" linker ADC when conjugated to trastuzumab. Based on these studies, monomethyl auristatin PYE shows promise for use as an ADC payload.

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 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..

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).