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Published on: December 19, 2020
Minimizing immunogenicity of the SDR-grafted humanized antibody CC49 by genetic manipulation of the framework
Noreen R Gonzales1, Eduardo A Padlan, Roberto De Pascalis
1Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, Room 8B09, 10 Center Drive, Building 10, Bethesda, MD 20892-1750, USA.
Abstract:
The murine mAb CC49 specifically recognizes a tumor-associated glycoprotein (TAG)-72, which is expressed on the majority of human carcinomas. This Ab has potential applications in the diagnosis and treatment of human carcinomas. However, patients receiving murine CC49 generate human anti-murine Ab (HAMA) responses, preventing repeated administration of the Ab for effective treatment. To minimize the HAMA response, two versions of humanized CC49 (HuCC49) were developed: (a) HuCC49 and (b) HuCC49V10 (V10). HuCC49 was developed by grafting the CC49 CDRs, while V10 was generated by grafting only the specificity determining residues (SDRs) of the CC49 onto the frameworks of the human Abs. During the generation of both HuCC49 and V10, a few murine framework residues that were believed to be essential for the integrity of the Ag-binding site were retained. However, the indispensability of these residues for the Ag-binding activity of CC49 has not been experimentally validated. In this study, an array of V10 variants were generated by replacing, by site-specific mutagenesis, the murine framework residues that were retained in the humanized Ab with their counterparts in the human templates. The variants were tested for their (a) Ag-binding activity and (b) reactivity to sera from patients who were previously administered murine CC49 in a clinical trial. One such variant, V59, compared to the parental V10, shows a significant decrease in its reactivity to the anti-variable region Abs present in the patients' sera, while it binds to the TAG-72 Ag with a slightly higher affinity. Variant 59, which is expected to be minimally immunogenic because of its low sera reactivity, is a potentially useful clinical reagent against human carcinomas. In this study, we show for the first time that experimental validation rather than reliance on the protein data bank (PDB) should be the criterion for the indispensability of framework residues for the humanization of any murine Ab to retain its Ag-binding property and reduce its immunogenicity in patients.
Insights
Humanizing murine antibodies like CC49 for cancer therapy reduces immune responses. This study developed a variant, V59, with lower immunogenicity and maintained TAG-72 binding, offering a promising tool for carcinoma treatment.
Area of Science:
- Immunology and Cancer Therapeutics
- Monoclonal Antibody Engineering
Background:
- Murine monoclonal antibody (mAb) CC49 targets tumor-associated glycoprotein (TAG)-72, present on most human carcinomas, with diagnostic and therapeutic potential.
- Human anti-murine antibody (HAMA) responses limit repeated administration of murine CC49.
- Humanized versions (HuCC49, HuCC49V10) were created by grafting CC49's specificity-determining residues (SDRs) onto human antibody frameworks, retaining some murine residues.
Purpose of the Study:
- To experimentally validate the necessity of retained murine framework residues in humanized CC49 (HuCC49V10) for antigen binding and immunogenicity.
- To generate and characterize variants of HuCC49V10 to reduce human anti-murine antibody (HAMA) responses while maintaining TAG-72 binding.
Main Methods:
- Site-specific mutagenesis was used to replace retained murine framework residues in HuCC49V10 with human counterparts, creating variants.
- Variants were tested for binding affinity to TAG-72 antigen.
- Reactivity of variants to antibodies in sera from patients previously treated with murine CC49 was assessed.
Main Results:
- A variant, V59, demonstrated significantly reduced reactivity to patient anti-variable region antibodies compared to the parental HuCC49V10.
- Variant V59 exhibited slightly increased binding affinity for the TAG-72 antigen.
- The findings suggest V59 is potentially less immunogenic.
Conclusions:
- Experimental validation of framework residues is crucial for antibody humanization, rather than relying solely on structural data (e.g., Protein Data Bank).
- Variant V59 shows promise as a clinically useful reagent against human carcinomas due to reduced immunogenicity and maintained antigen binding.
- This approach optimizes humanized antibodies for improved therapeutic efficacy and patient tolerance.
