Humanization of rabbit monoclonal antibodies via grafting combined Kabat/IMGT/Paratome complementarity-determining regions: Rationale and examples

Abstract

Rabbit monoclonal antibodies (RabMAbs) can recognize diverse epitopes, including those poorly immunogenic in mice and humans. However, there have been only a few reports on RabMAb humanization, an important antibody engineering step usually done before clinical applications are investigated. To pursue a general method for humanization of RabMAbs, we analyzed the complex structures of 5 RabMAbs with their antigens currently available in the Protein Data Bank, and identified antigen-contacting residues on the rabbit Fv within the 6 Angstrom distance to its antigen. We also analyzed the supporting residues for antigen-contacting residues on the same heavy or light chain. We identified "HV4" and "LV4" in rabbit Fvs, non-complementarity-determining region (CDR) loops that are structurally close to the antigen and located in framework 3 of the heavy chain and light chain, respectively. Based on our structural and sequence analysis, we designed a humanization strategy by grafting the combined Kabat/IMGT/Paratome CDRs, which cover most antigen-contacting residues, into a human germline framework sequence. Using this strategy, we humanized 4 RabMAbs that recognize poorly immunogenic epitopes in the cancer target mesothelin. Three of the 4 humanized rabbit Fvs have similar or improved functional binding affinity for mesothelin-expressing cells. Interestingly, 4 immunotoxins composed of the humanized scFvs fused to a clinically used fragment of Pseudomonas exotoxin (PE38) showed stronger cytotoxicity against tumor cells than the immunotoxins derived from their original rabbit scFvs. Our data suggest that grafting the combined Kabat/IMGT/Paratome CDRs to a stable human germline framework can be a general approach to humanize RabMAbs.

Engineered Anti‐GPC3 Immunotoxin, HN3‐ABD‐T20, Produces Regression in Mouse Liver Cancer Xenografts Through Prolonged Serum Retention

“…Although deimmunization of the bacterial domain may be possible, this would most certainly have a negative impact on the affinity for albumin. An easier, more cost‐effective method may be to humanize the ALB1 antibody to reduce immunogenicity …”

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

“…2, VL1, VH1, VH2). Third, residues determined to preserve affinity (29) were back mutated from the human germline residues to the original rabbit residues. From these 3 steps comprised of CDR grafting and rational back mutations, 4 heavy chain (VH1-VH4) and 2 light chain variants (VL1, VL2) were formed (Fig.…”

“…Humanization of X3.12 was done by finding the closest human germline(s) using IgBlast (www.ncbi.nlm.nih.gov/igblast/) with the least amount of polymorphisms, which was determined using IMGT's IGHV and IGKV mammalia human (Homo sapiens) links (www.imgt.org/IMGTrepertoire/Proteins/). Rational mutations (29) were performed in varying severity to determine mutations that were necessary to retain ROR2 affinity.…”

Exploration and Modulation of Antibody Fragment Biophysical Properties by Replacing the Framework Region Sequences

“…However, the combination of clusters II and III mutations seemed additional, S1D4 exhibited an additional higher Tm increased by +0.5 • C. This profile could be explained by the spatial proximity and compatibility between VH-RPGHG (positions 45 to 49) and the nearby VH-V101. Thus, creating better complementarity between the AAs of the same domain or at the interface between the variable domains may improve stability of scFv (16). Moreover, the scFv S1D4 exhibited a very high thermal stability [19,41,42] even without additional disulfide bridge [43], which could greatly increase long-term storage stability compared to other scFvs [30].…”

“…When re-engineering variable domains, affinity maturation and antibody humanization are carefully considered and worked on [11][12][13][14][15][16]. In some cases, mutations in the antibody complementarity determining regions (CDRs) have shown to improve molecule stability [17,18] thus being able to repair intrinsic flaws in the packing between two V-domains [19].…”