Genetic Engineering of Antibodies to Harness Their Clinical Potential

Kashmiri SVS, De Pascalis R, Gonzales NR, and Schlom J. SDR grafting—a new approach to antibody humanization. Methods 36: 25–34, 2005.

Dr. Syed V.S. Kashmiri of the NCI, Center for Cancer Research, Laboratory of Tumor Immunology and Biology, passed away on July 19, 2005. He will be remembered for his intellect, seminal and highly innovative contributions to the field of genetic engineering of antibody molecules, and for his extreme kindness. He will be very much missed.

ince the advent of hybridoma technology, a vast repertoire of murine monoclonal antibodies (mAbs) has been generated. The utility of these antibodies for the diagnosis and treatment of human cancers and infectious diseases is, however, limited—mainly because they elicit human anti-murine antibody (HAMA) responses in patients. To improve their clinical potential, murine antibodies have been genetically manipulated to replace their murine content with the amino acid residues present in their human counterparts, rendering them potentially less immunogenic in patients.

Initially, human-mouse chimeric antibodies were generated by replacing the murine-constant regions with those of the human antibodies. To further reduce the murine content, mAbs have been humanized by grafting their complementarity-determining regions (CDRs)—the segments of their variable regions that confer antigen binding specificity and affinity on antibodies—onto the variable light and heavy frameworks of human immunoglobulin molecules. However, the xenogeneic CDRs of the humanized antibodies may still evoke an anti-idiotypic response in patients. To circumvent this, we have developed a new approach to humanize antibodies based on grafting only those residues of the xenogeneic CDRs that are critical in the antigen-antibody interaction onto the human antibody frameworks. An analysis of the three-dimensional structures of antibody-combining sites suggests that only 20% to 33% of CDR residues are critical in the antigen-antibody interaction. These residues, which are located in the regions of high variability and which are most likely to be unique to each antibody, are designated as specificity-determining residues (SDRs) (Padlan E et al. FASEB J 9: 133–9, 1995). SDR-grafted humanized antibody has a substantially reduced number of non-human residues as compared with those present in its CDR-grafted counterpart (Tamura M et al. J Immunol 164: 1432–41, 2000).

The first step in the humanization by SDR grafting is to choose the most appropriate human frameworks to be used as templates and to identify the framework residues, which are deemed essential for the antigen-binding properties of an antibody to be retained. The next step is to identify the SDRs. When the three-dimensional structure of the antigen-antibody complex (based on X-ray crystallographic studies) is known, the residues of the combining site directly involved in ligand contact can be easily identified. In the absence of a three-dimensional structure, potential SDRs could be identified by examining the known crystal structures of antibody:ligand complexes that are currently available in the Protein Data Bank database. The database does not always lead to a definite conclusion about the dispensability of some SDRs, and may require experimental validation to assess the dispensability of the murine residues.

We have also used a more conservative approach to humanize an anti-CEA antibody. (CEA, or carcinoembryonic antigen, is overexpressed in a wide range of human carcinomas and is an excellent target for immunotherapy approaches.) This approach is based on grafting of the “abbreviated” CDRs—the stretches of CDR residues that include all the SDRs (De Pascalis R et al. J Immunol 169: 3076–84, 2002) (Figure 1). Since this approach does not require extensive genetic manipulation, it minimizes the risk of a loss in the antigen-binding of the antibody.

Figure 1. Schematic representation of the humanization protocols of the VL (variable light) region of an antibody, showing the VL region of a murine, complementarity-determining region (CDR)–grafted, “abbreviated” CDR-grafted, and specificity-determining residue (SDR)–grafted humanized antibody.

For a humanization protocol to achieve the desirable goal, it is important that the structural features of the target antibody are preserved. Humanization often results in a significant modification of the antigen-combining site structure and a consequent loss in the antigen-binding affinity of the antibody. To offset this loss, we have also used in vitro affinity maturation (De Pascalis R et al. Clin Cancer Res 9: 5521–31, 2003) to generate humanized antibodies with enhanced antigen-binding affinity and reduced immunogenic potential in human patients. It is anticipated that these novel recombinant technologies will result in the more effective use of monoclonal antibodies for the diagnosis and/or therapy of a range of human cancers.

The NCI has filed seven patent applications to establish its intellectual property rights on the humanized antibodies generated in our laboratory and one to claim proprietary rights to the technique of humanizing antibodies by SDR grafting. Several pharmaceutical and biotechnology companies have already executed agreements and/or are in negotiation toward the development of the humanized antibodies we have generated.

Syed VS Kashmiri, PhD
Principal Investigator
Laboratory of Tumor Immunology and Biology

back to top