(W0930-06-35) Development of Rabbit Anti-Mertansine (DM1) Monoclonal Antibodies

Purpose: Antibody-drug conjugates (ADCs) are promising anti-cancer immuno-therapeutics. An Antibody–Drug Conjugate (ADC) typically comprises a monoclonal antibody covalently linked to a cytotoxic drug (also known as a payload) through a chemical linker. The
bioanalytical strategies for ADCs necessitate innovative analytical methods, encompassing both existing large- and small-molecule techniques. Maytansine and its synthetic derivatives, are widely investigated as the drug parts in ADCs.

Methods: In this project, we employed Yurogen’s SMABTM platform and have successfully developed a panel of rabbit monoclonal antibodies, that specifically bind to DM1

Results: Notably, these antibodies exhibit exceptional selectivity, with minimal cross-reactivity against the linker molecules. These
antibodies can be used in conducting pharmacokinetic studies for any ADC with DM1 as the payload

Conclusion: This innovative approach enables a novel, orthogonal and simplified approach for researchers to analyze ADC, accelerating the development and evaluation of these promising cancer therapeutics.

References: [1] Chu R, Gerstein J, Wu H, Huang H, Lou Y, Palmer R. Development of Rabbit Monoclonal Antibodies for Quantitation of Therapeutic Human Antibodies in Preclinical Non-Human Primate Studies. Monoclon Antib Immunodiagn Immunother. 2020 Oct;39(5):175-183. doi: 10.1089/mab.2020.0024. Epub 2020 Sep 30. PMID: 32996830.
[2] Lewis Phillips GD, Li G, Dugger DL, Crocker LM, Parsons KL, Mai E, Blättler WA, Lambert JM, Chari RV, Lutz RJ, Wong WL, Jacobson FS, Koeppen H, Schwall RH, Kenkare-Mitra SR, Spencer SD, Sliwkowski MX. Targeting HER2-positive breast cancer with trastuzumab-DM1, an antibody-cytotoxic drug conjugate. Cancer Res. 2008 Nov 15;68(22):9280-90. doi: 10.1158/0008-5472.CAN-08-1776. PMID: 19010901.


(A) Immunization strategy: rabbits received five doses of the KLH conjugated Lys-SMCC-DM1 on days 0, 7, 21, 42, 63 and a final boost dose of the molecule was given on days 84. Sera were collected on days 0, 49, 70 for immunoassay. (B)The serological IgG titer to Kadcyla® and BSA conjugated Lys-SMCC-DM1 for pre-immunization bleed (day 0) and post-immunization bleeds (days 28 and 56) were measured by ELISA assay.


Figure 4 (A) All three rabbit monoclonal antibodies showed specific binding to DM1-BSA and Kadcyla, but minimum or no binding to Trastuzumab, BSA-SMCC-peptide and total human IgG (negative control). (B) Competition ELISA of rabbit anti-DM1 antibodies further confirmed their specific binding to DM1 but not to SMCC linker. (C) All three rabbit anti-DM1 antibodies were able to pair with anti-trastuzumab monoclonal antibody for Kadcyla® measurement by sandwich ELISA assay.


Figure 5 (A) Kinetics of rabbit mAb 14D9, 10G10 and 11A9 to Lys-SMCC-DM1 was measured by surface plasmon resonance (SPR) technology (Biomolecular Interaction Analysis System, S-CLASS, Polariton Life Technologies Ltd.). Changes in resonance units (RU) over time are indicative of binding kinetics. (B) Summary of ka (equilibrium association constant), kd (dissociation constant) and KD for rabbit mAbs 14D9, 10G10 and 11A9.