In vitro and in vivo evaluation of cysteine and site specific conjugated herceptin antibody-drug conjugates

PLoS One. 2014 Jan 14;9(1):e83865. doi: 10.1371/journal.pone.0083865. eCollection 2014.

Abstract

Antibody drug conjugates (ADCs) are monoclonal antibodies designed to deliver a cytotoxic drug selectively to antigen expressing cells. Several components of an ADC including the selection of the antibody, the linker, the cytotoxic drug payload and the site of attachment used to attach the drug to the antibody are critical to the activity and development of the ADC. The cytotoxic drugs or payloads used to make ADCs are typically conjugated to the antibody through cysteine or lysine residues. This results in ADCs that have a heterogeneous number of drugs per antibody. The number of drugs per antibody commonly referred to as the drug to antibody ratio (DAR), can vary between 0 and 8 drugs for a IgG1 antibody. Antibodies with 0 drugs are ineffective and compete with the ADC for binding to the antigen expressing cells. Antibodies with 8 drugs per antibody have reduced in vivo stability, which may contribute to non target related toxicities. In these studies we incorporated a non-natural amino acid, para acetyl phenylalanine, at two unique sites within an antibody against Her2/neu. We covalently attached a cytotoxic drug to these sites to form an ADC which contains two drugs per antibody. We report the results from the first direct preclinical comparison of a site specific non-natural amino acid anti-Her2 ADC and a cysteine conjugated anti-Her2 ADC. We report that the site specific non-natural amino acid anti-Her2 ADCs have superior in vitro serum stability and preclinical toxicology profile in rats as compared to the cysteine conjugated anti-Her2 ADCs. We also demonstrate that the site specific non-natural amino acid anti-Her2 ADCs maintain their in vitro potency and in vivo efficacy against Her2 expressing human tumor cell lines. Our data suggests that site specific non-natural amino acid ADCs may have a superior therapeutic window than cysteine conjugated ADCs.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Monoclonal, Humanized / chemistry*
  • Antineoplastic Agents / chemistry*
  • Binding Sites
  • Cell Line, Tumor
  • Cysteine / chemistry*
  • Drug Stability
  • Female
  • Gene Expression Regulation, Neoplastic / drug effects
  • Humans
  • Immunoconjugates / blood
  • Immunoconjugates / chemistry*
  • Immunoconjugates / pharmacokinetics
  • Immunoconjugates / pharmacology
  • Male
  • Mice
  • Rats
  • Receptor, ErbB-2 / metabolism
  • Serum Albumin / metabolism
  • Substrate Specificity
  • Trastuzumab
  • Xenograft Model Antitumor Assays

Substances

  • Antibodies, Monoclonal, Humanized
  • Antineoplastic Agents
  • Immunoconjugates
  • Serum Albumin
  • ERBB2 protein, human
  • Receptor, ErbB-2
  • Cysteine
  • Trastuzumab

Grant support

All studies were funded by Agensys, Inc and/or Ambrx, Inc. There were no private or government funding agencies used to fund these studies. All studies were conceived and performed by Agensys or Ambrx employees, with the exception of the rat toxicology studies, which were designed by Agensys and Ambrx employees and paid for by Agensys, Inc. The data analysis, preparation of the manuscript and the decision to publish were made by Agensys, Inc and Ambrx, Inc.