Site-Specific Polymer Attachment to HR2 Peptide Fusion Inhibitors against HIV-1 Decreases Binding Association Rates and Dissociation Rates Rather Than Binding Affinity.

A popular strategy for overcoming the limited plasma half-life of peptide heptad repeat 2 (HR2) fusion inhibitors against HIV-1 is conjugation with biocompatible polymers such as poly(ethylene glycol) (PEG). However, despite improved resistance to proteolysis and reduced renal elimination, covalent attachment of polymers often causes a loss in therapeutic potency. In this study, we investigated the molecular origins of the loss in potency upon conjugation of linear, midfunctional, and hyperbranched PEG-like polymers to peptides that inhibit HIV-1-host cell membrane fusion. Fluorescence binding assays revealed that polymer conjugation imparted mass transport limitations that manifested as coexistent slower association and dissociation rates from the gp41 target on HIV-1. Furthermore, reduced association kinetics rather than affinity disruption was responsible for the loss in antiviral potency. Finally, the binding assays indicated that the unmodified HR2-derived peptide demonstrated diffusion-limited binding. The observed high potency of the unmodified peptide in HIV-1 inhibition assays was therefore attributed to rapid peptide conformational changes upon binding to the gp41 prehairpin structure. This study emphasizes that the view in which polymer ligation to therapeutic peptides inadvertently leads to loss in potency due to a loss in binding affinity requires scientific verification on a case-by-case basis and that high peptide potency may be due to rapid target-binding events.

A Flexible Multiplatform Bioanalytical Strategy for Measurement of Total Circulating Shed Target Receptors: Application to Soluble B Cell Maturation Antigen Levels in the Presence of a Bispecific Antibody Drug.

B cell maturation antigen (BCMA) is a membrane-bound receptor that is overexpressed on multiple myeloma cells and can be targeted with biotherapeutics. Soluble shed forms of membrane-associated receptors in circulation can act as a drug sink, especially when it is present in high molar ratio compared to drug concentration, potentially derailing the intended pharmacological mechanism and impacting pharmacokinetic (PK) measurements and efficacious dose predictions. In this study, we present a bioanalytical strategy for assessing dynamic levels of total soluble BCMA before and during treatment with a bispecific antibody targeting BCMA and CD3. Implementation of a ligand binding assay was not successful due to extensive bispecific antibody interference. Instead, we explored two types of immunoaffinity (IA) liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays, one at the protein level and one at the surrogate peptide level. Ultimately, the protein-level IA-LC-MS/MS method was optimized for use in a cynomolgus monkey PK/pharmacodynamic study. In addition, we demonstrated that the method was easily adapted for use with human samples in preparation for translation to the clinic. This work demonstrates the benefit of flexibility and agility in bioanalytical method development in early drug development. Multiplatform suitability assessments enable rapid, resource-sparing identification and qualification of clinically translatable assays. We recommend early adoption of this strategy to provide enough time for critical reagent development and assay validation for analysis of shed targets.

Optimal design, anti-tumour efficacy and tolerability of anti-CXCR4 antibody drug conjugates.

Antibody-drug conjugates (ADCs) are promising therapies for haematological cancers. Historically, their therapeutic benefit is due to ADC targeting of lineage-restricted antigens. The C-X-C motif chemokine receptor 4 (CXCR4) is attractive for targeted therapy of haematological cancers, given its expression in multiple tumour types and role in cancer "homing" to bone marrow. However, CXCR4 is also expressed in haematopoietic cells and other normal tissues, raising safety challenges to the development of anti-CXCR4 ADCs for cancer treatment. Here, we designed the first anti-CXCR4 ADC with favourable therapeutic index, effective in xenografts of haematopoietic cancers resistant to standard of care and anti-CXCR4 antibodies. We screened multiple ADC configurations, by varying type of linker-payload, drug-to-antibody ratio (DAR), affinity and Fc format. The optimal ADC bears a non-cleavable linker, auristatin as payload at DAR = 4 and a low affinity antibody with effector-reduced Fc. Contrary to other drugs targeting CXCR4, anti-CXCR4 ADCs effectively eliminated cancer cells as monotherapy, while minimizing leucocytosis. The optimal ADC selectively eliminated CXCR4+ cancer cells in solid tumours, but showed limited toxicity to normal CXCR4+ tissues, sparing haematopoietic stem cells and progenitors. Our work provides proof-of-concept that through empirical ADC design, it is possible to target proteins with broad normal tissue expression.