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Updated: Dec 25, 2025
Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody
Published on: May 16, 2020
Affinity maturation, humanization, and co-crystallization of a rabbit anti-human ROR2 monoclonal antibody for
Rebecca S Goydel1, Justus Weber1, Haiyong Peng1
1Department of Immunology and Microbiology, The Scripps Research Institute, Jupiter, Florida 33458.
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.
Insights
Researchers developed a novel humanized antibody targeting ROR2, a protein overexpressed in many cancers but not healthy tissues. This antibody shows potent cancer cell killing ability, offering a promising new avenue for antibody-based cancer therapies.
Area of Science:
- Oncology
- Immunology
- Biotechnology
Background:
- Antibody therapeutics are crucial in cancer treatment but limited by the scarcity of cancer-specific antigens.
- Receptor tyrosine kinase-like orphan receptor 2 (ROR2) is highly expressed in various cancers but not in healthy adult tissues, making it a potential therapeutic target.
- ROR2's restricted expression pattern presents an opportunity for developing targeted antibody therapies.
Purpose of the Study:
- To develop and optimize a humanized monoclonal antibody (mAb) targeting the ROR2 antigen.
- To enhance the antibody's affinity and specificity for ROR2 through affinity maturation and humanization.
- To evaluate the therapeutic potential of the engineered antibody in ROR2-expressing cancer cells.
Main Methods:
- Affinity maturation of a rabbit mAb against ROR2 using HCDR3-focused mutagenesis and selection, guided by co-crystallography with the human ROR2 kringle domain.
- Humanization of the affinity-matured mAb via CDR grafting and framework optimization.
- Co-crystallization of the humanized mAb with the ROR2 kringle domain to confirm structural integrity.
- Conversion of the humanized mAb into a T cell-engaging bispecific antibody for functional assessment.
- Evaluation of the bispecific antibody's cytotoxicity against ROR2-expressing cancer cells.
Main Results:
- Successful affinity maturation and humanization of a rabbit anti-ROR2 mAb, yielding an antibody with high affinity and specificity for ROR2.
- The engineered humanized mAb maintained strong binding to ROR2.
- The derived T cell-engaging bispecific antibody demonstrated potent cytotoxicity against ROR2-expressing cancer cells.
- The antibody did not bind to ROR1 or other tested cell-surface antigens, confirming specificity.
Conclusions:
- A novel, affinity-matured, and humanized mAb targeting ROR2 has been successfully developed.
- This engineered antibody exhibits potent anti-cancer activity when formatted as a T cell-engaging bispecific antibody.
- The developed ROR2-targeting antibody represents a promising candidate for antibody-based therapies against ROR2-expressing hematologic and solid malignancies.
