Multiplex LC-MS/MS Assays for Clinical Bioanalysis of MEDI4276, an Antibody-Drug Conjugate of Tubulysin Analogue Attached via Cleavable Linker to a Biparatopic Humanized Antibody against HER-2

Abstract

Bioanalysis of complex biotherapeutics, such as antibody-drug conjugates (ADCs), is challenging and requires multiple assays to describe their pharmacokinetic (PK) profiles. To enable exposure-safety and exposure-efficacy analyses, as well as to understand the metabolism of ADC therapeutics, three bioanalytical methods are typically employed: Total Antibody, Antibody Conjugated Toxin or Total ADC and Unconjugated Toxin. MEDI4276 is an ADC comprised of biparatopic humanized antibody attached via a protease-cleavable peptide-based maleimidocaproyl linker to a tubulysin toxin (AZ13599185) with an approximate average drug-antibody ratio of 4. The conjugated payload of MEDI4276 can undergo ester hydrolysis to produce the conjugated payload AZ13687308, leading to the formation of MEDI1498 (de-acetylated MEDI4276). In this report, we describe the development, validation and application of three novel multiplex bioanalytical methods. The first ligand-binding liquid chromatography coupled with tandem mass spectrometry (LBA-LC-MS/MS) method was developed and validated for simultaneous measurement of total antibody and total ADC (antibody-conjugated AZ13599185) from MEDI4276. The second LBA-LC-MS/MS assay quantified total ADC (antibody-conjugated AZ13687308) from MEDI1498. The third multiplex LC-MS/MS assay was used for simultaneous quantification of unconjugated AZ13599185 and AZ13687308. Additional stability experiments confirmed that quantification of the released warhead in the presence of high concentrations of MEDI4276 was acceptable. Subsequently, the assays were employed in support of a first-in-human clinical trial (NCT02576548).

Crosstalk between HER2 and PD-1/PD-L1 in Breast Cancer: From Clinical Applications to Mathematical Models

“…For instance, hertuzumab-based ADC (RC48) shows improved efficacy compared to trastuzumab, lapatinib, and T-DM1 in the resistant BT474/L1.9 xenograft model [42]. MEDI4276 (trastuzumab scFv with AZ13599185, a tubulysin payload), PF-06804103 (anti-Trop2 Aur0101), A166 (undisclosed payload), ALT-P7 (HM2-monomethyl auristatin E), ARX 788 (monoclonal antibody with monomethyl auristatin E), DHES0185A (monoclonal antibody with benzodiazepine monoamide), and SYD 985 (trastuzumab duocarmazine with seco-DUBA) are other ADCs under investigation for HER2 + BC [42,[56][57][58][59][60][61][62]. With respect to the promising performance in Phase I and II clinical trials for the treatment of HER2 + metastatic BC, in December 2019, the FDA granted accelerated approval for trastuzumab deruxtecan (DS-8201) [12,[63][64][65].…”

“…With respect to the promising performance in Phase I and II clinical trials for the treatment of HER2 + metastatic BC, in December 2019, the FDA granted accelerated approval for trastuzumab deruxtecan (DS-8201) [12,[63][64][65]. When it comes to the desired properties of drugs, it is important to have optimal stability properties while the drug moves through the human plasma, along with efficient target-specific drug release [42,[56][57][58][59][60][61][62]. Out of the above mentioned ADCs, preclinical experiments on animal models are reported only for MEDI4276, RC48, ARX 788, DS-8201, and SYD 985, and hence more in vitro and in vivo experiments are imperative in this area [59,61,62,66].…”

“…When it comes to the desired properties of drugs, it is important to have optimal stability properties while the drug moves through the human plasma, along with efficient target-specific drug release [42,[56][57][58][59][60][61][62]. Out of the above mentioned ADCs, preclinical experiments on animal models are reported only for MEDI4276, RC48, ARX 788, DS-8201, and SYD 985, and hence more in vitro and in vivo experiments are imperative in this area [59,61,62,66]. Similarly, mathematical models that depict the dynamics of these novel drugs, as well as many of FDA-approved anti-HER2 agents are yet to be devised.…”

Expeditious Generation of Biparatopic Common Light Chain Antibodies via Chicken Immunization and Yeast Display Screening

“…To date, common light chain antibodies have been generated for various bispecific applications (18)(19)(20)(21)(22)(23)(24). Nonetheless, they are mostly based on other formats, such as scFvs or camelid singledomain antibodies (VHH) (8)(9)(10)(25)(26)(27)(28)(29). Even though the generation of such molecules is straightforward, they do not exhibit an IgG-like architecture.…”

Anti-ErbB2 immunotherapeutics: struggling to make better antibodies for cancer therapy

“…MEDI4276 is a bispecific antibody comprising the single-chain variable fragment (scFv) of trastuzumab, fused to the heavy chains of the second anti-ErbB2 antibody 39S (which binds to an independent ErbB2 domain), and conjugated to tubulysin. 48 A Phase 1/2 study (NCT02576548) of MEDI4276 in patients with select ErbB2-expressing advanced solid tumors has been completed.…”