Augmented Binary Substitution: Single-pass CDR germ-lining and stabilization of therapeutic antibodies

Abstract

Although humanized antibodies have been highly successful in the clinic, all current humanization techniques have potential limitations, such as: reliance on rodent hosts, immunogenicity due to high non-germ-line amino acid content, v-domain destabilization, expression and formulation issues. This study presents a technology that generates stable, soluble, ultrahumanized antibodies via single-step complementarity-determining region (CDR) germ-lining. For three antibodies from three separate key immune host species, binary substitution CDR cassettes were inserted into preferred human frameworks to form libraries in which only the parental or human germ-line destination residue was encoded at each position. The CDR-H3 in each case was also augmented with 1 ± 1 random substitution per clone. Each library was then screened for clones with restored antigen binding capacity. Lead ultrahumanized clones demonstrated high stability, with affinity and specificity equivalent to, or better than, the parental IgG. Critically, this was mainly achieved on germ-line frameworks by simultaneously subtracting up to 19 redundant non-germ-line residues in the CDRs. This process significantly lowered non-germ-line sequence content, minimized immunogenicity risk in the final molecules and provided a heat map for the essential non-germ-line CDR residue content of each antibody. The ABS technology therefore fully optimizes the clinical potential of antibodies from rodents and alternative immune hosts, rendering them indistinguishable from fully human in a simple, single-pass process.antibody | paratope | plasticity | humanization | immunogenicity

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

“…24 Finally, a method of generating a binary substitution library between mouse and germline CDR allows for germlining any non-essential CDR residue. 25 Beyond the potential risk of immunogenicity associated with non-human sequence content, the additional constraint of manufacturability must be considered when selecting germlines. Properties such as stability and expression vary among particular families of V-genes.…”

Sym021, a promising anti-PD1 clinical candidate antibody derived from a new chicken antibody discovery platform

“…Humanization based on the frameworks of the most homologous human germlines IGHV3-23 and IGLV3-19 was used in combination with sequence variation at selected Vernier residues. [26][27][28] With this approach, variation in the chicken FRs was not carried forward, but binding specificities encoded by CDRs were successfully grafted onto human FRs by means of de novo gene synthesis (Supplementary Figure 3). The simple one-step "mass CDR grafting" procedure obviates the need for time-consuming iterative optimization cycles and structure-based homology modeling, which are usually used in, for example, mouse humanization campaigns.…”

“…To avoid immunogenicity of chimeric antibodies, a humanization step was performed by grafting the cognate unique chicken CDRs onto human frameworks as previously described for mouse antibodies 25 ( Supplementary Figure 3). Vernier positions are known to play an important role in antibody structure, stability and function, hence the gene designs also incorporated variation between human and chicken frameworks at selected Vernier residue positions both in VH and VL framework, [26][27][28] resulting in the generation of up to four humanization variants for each antibody. Apart from Vernier residue variants, the in-silico CDR grafting step did not include variants with mutations in the chicken immunoglobulin frameworks.…”

Affinity improvement of the fully human anti‑TSLP recombinant antibody

“…With the development of hybridoma technology, mass spectrometry engineering, phage display and transgenic animal technology, the preparation of antibodies has successively transitioned from polyclonal to monoclonal antibodies (20,21). a priority has been the humanization of murine antibodies by genetic engineering, in order to obtain a fully humanized monoclonal antibody with high affinity, stability and biological activity (22,23). using phage display technology, human ethical issues have been avoided and large numbers of human monoclonal antibodies can be prepared (24).…”