Humanization of a recombinant monoclonal antibody to produce a therapeutic HER dimerization inhibitor, pertuzumab

Abstract

Dimerization is essential for activity of human epidermal growth factor receptors (HER1/EGFR, HER2/ErbB2, HER3/ErbB3, and ErbB4) and mediates intracellular signaling events leading to cancer cell proliferation, survival, and resistance to therapy. HER2 is the preferred dimerization partner. Activation of HER signaling pathways may be blocked by inhibition of dimer formation using a monoclonal antibody (MAb) directed against the dimerization domain of HER2. The murine MAb 2C4 that specifically binds the HER2 dimerization domain was cloned as a chimeric antibody, humanized using a computer-generated model to guide framework substitutions, and variants were tested as Fabs. Pharmacokinetics and toxicology were evaluated in rodents and cynomolgus monkeys. Cloning the variable domains of MAb 2C4 into a vector containing human kappa and CH1 domains allowed construction of a mouse-human chimeric Fab. DNA sequencing of the chimeric clone permitted identification of CDR residues. The full-length IgG1 of variant F-10 was equivalent in binding to chimeric IgG1 and was designated pertuzumab (rhuMAb 2C4; Omnitarg). Pertuzumab pharmacokinetics was best described by a two-compartment model with a distribution phase of <1 day, terminal half-life of approximately 10 days, and volume of distribution of approximately 40 mL/kg that approximates serum volume. With the exception of diarrhea, pertuzumab was generally well tolerated in cynomolgus monkeys. Pertuzumab, a recombinant humanized IgG1 MAb, is the first of a new class of agents known as HER dimerization inhibitors. Inhibition of HER dimerization may be an effective anticancer strategy in tumors with either normal or elevated expression of HER2.

Fig. 1

Amino acid sequences of murine 2C4, humanized 2C4 (Fab-10) (pertuzumab), and human consensus sequences of L chain subgroup kappa I (humκI) and H chain subgroup III (hum III). CDR loops are enclosed in brackets, and differences between sequences are denoted by an asterisk (*). Residue numbering is according to Kabat et al. [26]. a VL domain. b VH domain

Fig. 2

Humanized 2C4 IgG1 (pertuzumab) expressed from either 293 cells (filled square) or CHO cells (filled circle) binds HER2-ECD equally well compared with the 2C4 chimeric full-length IgG1 antibody (filled triangle)

Fig. 3

Interspecies scaling used to predict human clearance. The predicted CL of pertuzumab in humans by allometric scaling is approximately 330 mL/day, which is equivalent to 4.76 mL/day/kg for a 70 kg individual

Fig. 4

X-ray structure of humanized 2C4 Fab (pertuzumab) [48] (PDB accession number 1L71). The CDR loops of the light (L1-3) and heavy chains (H1-3) are indicated. Side chains of VH residues that were investigated or changed during humanization are shown (refer to Table 1 for details for specific changes and their effect on binding relative to the chimera)