“Superhumanized” Antibodies: Reduction of Immunogenic Potential by Complementarity-Determining Region Grafting with Human Germline Sequences: Application to an Anti-CD28

Abstract

Humanized Abs are created by combining, at the genetic level, the complementarity-determining regions of a murine mAb with the framework sequences of a human Ab variable domain. This leads to a functional Ab with reduced immunogenic side effects in human therapy. In this study, we report a new approach to humanizing murine mAbs that may reduce immunogenicity even further. This method is applied to humanize the murine anti-human CD28 Ab, 9.3. The canonical structures of the hypervariable loops of murine 9.3 were matched to human genomic V gene sequences whose hypervariable loops had identical or similar canonical structures. Framework sequences for those human V genes were then used, unmodified, with the 9.3 complementarity-determining regions to construct a humanized version of 9.3. The humanized 9.3 and a chimeric 9.3 control were expressed in Escherichia coli as Fab. The humanized Fab showed a moderate loss in avidity in a direct binding ELISA with immobilized CD28-Ig fusion protein (CD28-Ig). Humanized 9.3 blocked ligation of CD28-Ig to cells expressing the CD28 receptor CD80. Lastly, the humanized 9.3 showed biological activity as an immunosuppressant by inhibiting a MLR.

Camelid Ig V genes reveal significant human homology not seen in therapeutic target genes, providing for a powerful therapeutic antibody platform

“…These "canonical structures" (canonical folds) were proven to be critical to retain affinity when humanizing antibodies, 9 ultimately leading to the approach of "super humanization," which provides the opportunity to match murine to human canonical structures. 8 One important driver of the breakthrough of antibody-based therapeutics was the sequencing/characterization of human, rodent and other species germline immunoglobulin (Ig) heavy and light chain (V, (D), J and C) genes. [10][11][12] This revealed the extensive diversity of the Ig repertoire and explained its ability to recognize and bind virtually any antigen with a different level of affinity.…”

“…5 Disappointingly, the question of how humanized and even fully human antibodies are still more or less immunogenic in patients remains to be explained. [6][7][8] The observation that the CDRs of most humanized antibodies exhibit different structural fold combinations compared with naturally occurring human antibodies, offers a plausible explanation to the induction of undesirable immune responses against therapeutic antibodies. These "canonical structures" (canonical folds) were proven to be critical to retain affinity when humanizing antibodies, 9 ultimately leading to the approach of "super humanization," which provides the opportunity to match murine to human canonical structures.…”

Beyond CDR-grafting: Structure-guided humanization of framework and CDR regions of an anti-myostatin antibody

“…9,17,18 "Superhumanization" uses a CDR homology based approach to antibody humanization and effectively selects a germline with CDRs that most closely match the murine CDRs. 19,20 An additional method includes screening for germline segments while only grafting the CDR3 of the heavy and light chain. 21 This method can also be combined with affinity optimization using somatic hypermutation.…”

Application of Monoclonal Antibody Therapies in Autoimmune Diseases

“…specificity determining residue (SDR) engrafting or superhumanization had been proposed. While the first is based on the computational analysis of the three-dimensional structure of the antigen-antibody complex, suggesting that only 20-33% of CDR residues are in contact with the antigen, the latter relies on the in silico selection of the best matching canonical structures of both the nonhuman and human sequences (59,60). For the engraftment, most frequently human germline sequences are used to minimize potential immunogenicity (61).…”