Antibody-drug conjugates (ADCs) have become an important therapeutic modality for oncology, with three approved by the FDA and over 60 others in clinical trials. Despite the progress, improvements in ADC therapeutic index are desired. Peptide-based ADC linkers that are cleaved by lysosomal proteases have shown sufficient stability in serum and effective payload-release in targeted cells. If the linker can be preferentially hydrolyzed by tumor-specific proteases, safety margin may improve. However, the use of peptide-based linkers limits our ability to modulate protease specificity. Here we report the structure-guided discovery of novel, nonpeptidic ADC linkers. We show that a cyclobutane-1,1-dicarboxamide-containing linker is hydrolyzed predominantly by cathepsin B while the valine-citrulline dipeptide linker is not. ADCs bearing the nonpeptidic linker are as efficacious and stable in vivo as those with the dipeptide linker. Our results strongly support the application of the peptidomimetic linker and present new opportunities for improving the selectivity of ADCs.
Background: MUC16 is a transmembrane protein that is overexpressed by ovarian cancer. The role of MUC16 in the pathogenesis of ovarian cancer is unknown; however, MUC16 may facilitate binding of ovarian tumor cells to mesothelial cells lining the peritoneal cavity. Traditional antibody drug conjugates, with conjugation through inter-chain disulfides, are heterogeneous mixtures of drug antibody ratios (DAR) ranging from 0-8, resulting in complex pharmacokinetics (PK) and potentially unfavorable safety and efficacy. In contrast, THIOMABTM drug conjugates (TDCs) use novel technology that achieves site-directed drug conjugation yielding a homogeneous DAR. DMUC4064A is a cysteine-engineered TDC comprising a humanized anti-MUC16 IgG1 and 2 potent anti-mitotic monomethyl auristatin E (MMAE) molecules. Methods: The dose-escalation component of the Phase I study evaluated safety, tolerability, PK, pharmacodynamics, and early activity of DMUC4064A given Q3W to patients with platinum-resistant ovarian cancer. A standard 3+3 design was used to determine the maximum-tolerated dose. Archival tumor tissue was used to assess expression of MUC16 and other markers. Clinical activity was evaluated per RECIST v4.0 criteria. Results: Forty-four female patients, median age 63 (35-84), ECOG PS 0-1, received a median of 4 doses (range 1-12) of DMUC4064A at 1.0-5.6 mg/kg. At 5.6 mg/kg, one patient experienced three adverse events (AE) each qualifying as a DLT (colitis, hyperglycemia, and hypokalemia; all Grade 3). At 5.2 mg/kg, another patient experienced a DLT of Grade 5 septic shock. Grade ≥ 3 AE occurring in ≥ 5% of patients included hyponatremia (1 each at 1.8, 3.2, and 5.2 mg/kg; 2 at 2.4 mg/kg; 11% total), ascites (2 at 2.4 mg/kg and 1 at 3.2 mg/kg; 7% total) and hyperglycemia (3 at 5.6 mg/kg; 7% total), all unrelated except hyperglycemia. The most common (≥20%) related AEs for all dose levels were fatigue (34%), nausea (32%), diarrhea (23%) and abdominal pain (21%). Ocular toxicities (related, Grade ≥ 2) included keratitis (Grade 3, n=2), blurred vision (Grade 3, n=1; Grade 2, n=3), and dry eye (Grade 2, n=1). Total antibody and conjugated MMAE showed dose-dependent PK; neither were impacted by circulating CA125. Total antibody, conjugated and free MMAE accumulation was minimal. At doses ≥2.4 mg/kg, DMUC4064A had decreased clearance and achieved higher exposures versus MUC16 MMAE ADC. Confirmed responses (1 CR and 6 PRs) occurred at ≥ 3.2 mg/kg (n=30). Tumor MUC16 IHC scores were 2+/3+ in responders for whom data were available (n=5). Conclusions: These data are the first reported for an MMAE-containing TDC in a clinical trial (clinicaltrials.gov NCT02146313). DMUC4064A has an acceptable safety profile with improved stability compared to MUC16 MMAE ADC and shows evidence of anti-tumor activity, warranting further evaluation in ovarian cancer. Citation Format: Joyce F. Liu, Kathleen N. Moore, Judy S. Wang, Manish Patel, Michael J. Birrer, Erika Hamilton, Lisa Barroilhet, William M. Flanagan, Yulei Wang, Amit Garg, Xuyang Lu, Anjali Vaze, Dilip Amin, Doug Leipold, S Renee Commerford, Eric W. Humke, Harold A. Burris. Targeting MUC16 with the THIOMABTM-drug conjugate DMUC4064A in patients with platinum-resistant ovarian cancer: a Phase I escalation study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT009. doi:10.1158/1538-7445.AM2017-CT009
Anti-Ly6E-seco-CBI dimer antibody-drug conjugate (ADC) has been reported to form an adduct with alpha-1-microglobulin (A1M) in animal plasma, but with unknown impact on ADC PK and tissue distribution. In this study, we compared the PK and tissue distribution of anti-Ly6E ADC with unconjugated anti-Ly6E mAb in rodents and monkeys. For PK studies, animals received an intravenous (IV) administration of anti-Ly6E ADC or unconjugated anti-Ly6E mAb. Plasma samples were analyzed for total antibody (Tab) levels and A1M adduct formation. PK parameters were generated from dose-normalized plasma concentrations. Tissue distribution was determined in tumorbearing mice following a single IV dosing of radiolabeled ADC or mAb. Tissue radioactivity levels were analyzed using a gamma counter. The impact of A1M adduct formation on target cell binding was assessed in an in-vitro cell binding assay. The results show that ADC Tab clearance was slower than that of mAb in mice and rats, but faster than mAb in monkeys. Correspondingly, the formation of A1M adduct appeared to be faster and higher in mice followed by rats, but slowest in monkeys. While ADC trended to show an overall lower distribution to normal tissues, it had a strikingly reduced distribution to tumors in compared to mAb, likely due to A1M adduct formation interfering with target binding as demonstrated by the in-vitro cell binding assay. Together, these data demonstrate that anti-Ly6E ADC that forms A1M adduct had slower systemic clearance with strikingly reduced tumor distribution and highlight the importance of selecting an appropriate linker-drug for successful ADC development.
For antibody-drug conjugates (ADCs) that carry a cytotoxic drug, doses that can be administered in preclinical studies are typically limited by tolerability, leading to a narrow dose range that can be tested. For molecules with non-linear pharmacokinetics (PK), this limited dose range may be insufficient to fully characterize the PK of the ADC and limits translation to humans. Mathematical PK models are frequently used for molecule selection during preclinical drug development and for translational predictions to guide clinical study design. Here, we present a practical approach that uses limited PK and receptor occupancy (RO) data of the corresponding unconjugated antibody to predict ADC PK when conjugation does not alter the non-specific clearance or the antibody-target interaction. We used a 2-compartment model incorporating non-specific and specific (target mediated) clearances, where the latter is a function of RO, to describe the PK of anti-CD33 ADC with dose-limiting neutropenia in cynomolgus monkeys. We tested our model by comparing PK predictions based on the unconjugated antibody to observed ADC PK data that was not utilized for model development. Prospective prediction of human PK was performed by incorporating in vitro binding affinity differences between species for varying levels of CD33 target expression. Additionally, this approach was used to predict human PK of other previously tested anti-CD33 molecules with published clinical data. The findings showed that, for a cytotoxic ADC with non-linear PK and limited preclinical PK data, incorporating RO in the PK model and using data from the corresponding unconjugated antibody at higher doses allowed the identification of parameters to characterize monkey PK and enabled human PK predictions.
Key defining attributes of an antibody-drug conjugate (ADC) include the choice of targeting antibody, linker, and the drug-to-antibody ratio (DAR). The choice of DAR, within the constraints of acceptable physicochemical properties for the given platform, is a function of balancing delivery of sufficient payload to targeted cells with the ability to achieve sustained in vivo exposures. Previous reports have described lower DAR mc-VC-MMAE conjugates, DAR = 1-2, that demonstrated higher in vivo exposure and lower clearance when compared to higher DAR (e.g. 4-8) counterparts. In theory, high DAR conjugates may be especially desirable when targeting low antigen expressing tumors or when lower potency payloads are used, as each binding and internalization event results in greater payload delivery. Here we report a systematic exploration of DAR across a much wider range than has been previously reported, by combining THIOMAB® protein engineering technology with the Dolasynthen platform. Homogeneous, site-specific ADCs spanning a discrete range of DARs – 2, 4, 6, 12, and 18 – were made by conjugation of Trastuzumab IgG1 THIOMAB constructs with 1, 2, or 3 engineered cysteines to monomeric or trimeric Dolasynthen. The cytotoxicity of the resulting well-defined ADCs was assessed in vitro in cell lines with high or low expression of HER2 antigen. Pharmacokinetic data for all test articles in mice were generated in tumor bearing mice. In high HER2 expressing cell lines, in vitro cytotoxicity by payload was comparable across DARs. In a lower HER2 expressing system, the higher DAR ADCs performed better. In vivo, our data demonstrated comparable pharmacokinetics for the Dolasynthen conjugates across all DARs. These results illustrate the utility of a DAR ranging platform, such as Dolasynthen when evaluating ADCs as it enables the interrogation of a range of antibody and payload dosing regimens. Citation Format: Kalli C. Catcott, Susan Clardy, Jack Sadowsky, Rebecca K. Rowntree, Naniye Malli Centibas, Ling Xu, Andy Polson, Kenneth Avocetien, Tyler Carter, Mark Nazzaro, Dokyong "DK" Kim, Thomas H. Pillow, Neelie Zacharias, Cong Wu, Jeffrey Zurita, Elizabeth Ditty, Stephen Bradley, Alex Uttard, Bingfan Du, William S. Sawyer, Doug Leipold, Gail Lewis Phillips, LiuLiang Qin, Kelly Slocum, Geoffrey Del Rosario, Ginny Li, Shang-Fan Yu, David Lee, Radha Iyengar, Marc Damelin, Dorin Toader, Timothy B. Lowinger. Site-specific Dolasynthen ADCs demonstrate consistent exposure across a wide range of drug-to-antibody ratios [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P167.
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