Affinity maturation, humanization, and co-crystallization of a rabbit anti-human ROR2 monoclonal antibody for therapeutic applications

Abstract

Antibodies are widely used as cancer therapeutics, but their current use is limited by the low number of antigens restricted to cancer cells. A receptor tyrosine kinase, receptor tyrosine kinase-like orphan receptor 2 (ROR2), is normally expressed only during embryogenesis and is tightly down-regulated in postnatal healthy tissues. However, it is up-regulated in a diverse set of hematologic and solid malignancies, thus ROR2 represents a candidate antigen for antibody-based cancer therapy. Here we describe the affinity maturation and humanization of a rabbit mAb that binds human and mouse ROR2 but not human ROR1 or other human cell-surface antigens. Co-crystallization of the parental rabbit mAb in complex with the human ROR2 kringle domain (hROR2-Kr) guided affinity maturation by heavy-chain complementarity-determining region 3 (HCDR3)-focused mutagenesis and selection. The affinity-matured rabbit mAb was then humanized by complementarity-determining region (CDR) grafting and framework fine tuning and again co-crystallized with hROR2-Kr. We show that the affinity-matured and humanized mAb retains strong affinity and specificity to ROR2 and, following conversion to a T cell–engaging bispecific antibody, has potent cytotoxicity toward ROR2-expressing cells. We anticipate that this humanized affinity-matured mAb will find application for antibody-based cancer therapy of ROR2-expressing neoplasms.

The WNT/ROR Pathway in Cancer: From Signaling to Therapeutic Intervention

“…Subsequently, they developed the mAb into a CAR T cell format, which demonstrated a high specificity towards ROR2. Moreover, XBR2-401 was used as a base for designing a biAb (ROR2 x CD3), showing specificity for ROR2 in vitro [ 199 ]. Further investigation concerning ROR2-targeting ADCs are also in progress [ 200 ].…”

WNT11 is a novel ligand for ROR2 in human breast cancer

“…Interestingly, while the deletion of the Ig and CRD domain of ROR2 decreased its invasion-promoting effect, an additional reduction was observed upon deletion of the Kringle domain. As the Kringle domain has been shown to contain a lysine-binding site that could potentially mediate receptor-receptor interactions 61 , this observation might point to the involvement of other receptors in the WNT11/ROR2 complex.…”

Effective rational humanization of a PASylated anti-galectin-3 Fab for the sensitive PET imaging of thyroid cancer in vivo

“…Whereas the concept of CDR grafting between Igs from different species was originally devised on the basis of structural considerations 13 , 14 , common humanization procedures mostly rely on a homology assessment at the level of the amino acid sequence till today 16 , 43 . This is somewhat surprising particularly in cases where three-dimensional structural information for the donor antibody actually is available 44 – 46 . In the early endeavors, acceptor framework sequences from structurally characterized antibodies or Bence Jones proteins were chosen 13 , 14 , 47 ; this was followed by the use of human consensus Ig sequences 20 , including the famous example of the αHER2 antibody trastuzumab 15 .…”

“…In the early endeavors, acceptor framework sequences from structurally characterized antibodies or Bence Jones proteins were chosen 13 , 14 , 47 ; this was followed by the use of human consensus Ig sequences 20 , including the famous example of the αHER2 antibody trastuzumab 15 . Today, however, frequently expressed germline Ig sequences from the human genome serve as preferred templates with presumably low immunogenicity 43 , 46 , 48 .…”