Antibody Humanization Service

Antibody humanization or humanized antibodies are designed to reduce immunogenicity response while maintaining high specificity. Antibody humanization is a critical step in the development pathway of all therapeutic antibodies derived from non-human sources, such as hybridomas. The majority have been humanized as IgG1 molecules, and with some humanized IgG4.

Since the first report on antibody humanization by CDR grafting in the second half of the 1980s up to June 2007, the two main trends are rational and empirical methods to humanize antibodies.

One common method for producing humanized antibodies or antibody humanization includes framework-homology-based humanization, germline humanization, complementary determining regions (CDR)-homology-based humanization and specificity determining residues (SDR) grafting.

The procedure of Oak Biosciences antibody humanization services include in silico CDR grafting, computer-aided design, back mutation, focused humanized phage library design and construction and panning and optimization.

Oak Biosciences antibody humanization service will deliver a panel of 5-24 unique humanized variants from parent clone. We guarantee to produce a variant with comparable affinity to the parent antibody, and enhancing antibody manufacturability, such as increased expression, lowered aggregation, and good thermal stability and solubility.

Oak Biosciences scientists design an integrated strategy to accomplish this complex task effectively. Oak Biosciences have been delivered antibody humanization service since 2008, and over 300 successful projects have been done.

Oak Biosciences provides quality guaranteed antibody humanization service for mouse, rat, rabbit, chicken and camelid monoclonal antibodies. Using our proprietary technologies (extensive antibody database, bioinformatics software, unique antibody humanness score standards) and for rapid production of cell lines expressing full length recombinant antibodies, sequences of the antibody variable domains which determine its binding specificity are incorporated into human donor sequences, creating a panel of full length humanized antibodies for expression.

1. Murine/Rat Monoclonal Antibody Humanization

CDR grafting, from rodent antibodies into human antibody frameworks, is effective because the folding of the polypeptide backbone in the variable regions and the canonical structures are very similar between these species, despite sequence differences. Humanization by CDR grafting becomes a clinically proven technology for therapeutic mAbs. However, some CDR-grafted antibodies are still evoke immune responses. Thus, framework resurfacing, further mutation the murine framework surface residues in the most resembling human counterpart is necessary.

Oak Biosciences scientists design an integrated strategy to to accomplish this complex task effectively.

2. Rabbit Monoclonal Antibody Humanization

Rabbit is one of the best sources of high quality antibodies, due to its robust immune response and its propensity to produce very high-affinity and quality rabbit monoclonal antibodies (RabMabs) to a wide range of epitopes, to generate antibodies targeting uncommon epitopes, including interesting epitopes that are less immunogenic in mice and humans. However, there are big issues to humanize: 1) many rabbit kappa chains have a disulfide bond between the variable region and the constant region, causing protein folding and expression problems and dimerizing; 2) many rabbit heavy and light chains are short by one or two amino-acid residues, and cannot find a corresponding homologous human residue; 3) many rabbit heavy chain and/or light chain variable domains have extra paired cysteines; 4) many rabbit antibody CDRs do not belong to any previously known canonic structures, causing modeled inaccurately. Based on our structural and sequence analysis, Oak Biosciences scientists design a strategy to humanize RabMAbs by grafting the combined Kabat/IMGT/Paratome CDRs, which cover most antigen-contacting residues, into a human germline framework sequence.

3. Chicken Monoclonal Antibody Humanization

Birds (and in particular, chickens) are phylogenetically distant from humans, produce antibodies of high affinity and specificity, and can recognize unique epitopes not accessible in mice. Chickens express a single immunoglobulin structural framework consisting of the germline-encoded VH and VL regions, with somatic diversity accumulating mainly in the CDRs. Oak Biosciences develop a humanization of chicken mAbs by CDR-grafting, following by framework fine-tuning using a chicken phage-displayed mAbs, a phage-displayed combinatorial library with permutation of important framework residues.

4. Camelid Single-domain Antibody Humanization

Camelid single-domain antibody (sdAb or VHH or Nanobody) is a kind of single-domain antigen-binding fragments with camelid-specific heavy-chain only, offers special advantages in therapy over classic antibody fragments because of their smaller size, robustness, and preference to target unique epitopes. A Nanobody differs from a human heavy chain variable domain in about ten amino acids extending all over its surface, four hallmark nanobody-specific amino acids in the framework-2 (FW2) region (positions 42, 49, 50, and 52),and a longer third antigen-binding loop(CDRH3) folding over this area. For therapeutic applications the camelid-specific amino acid sequences in the frame work have to be humanized by mutation to their human heavy chain variable domain equivalent. Through constructing a focused mutated library integrated FW2 Region and CDR3 loop, Oak Biosciences scientist successfully humanize VHH and retain antigen affinity, solubility, expression yield, and stability of the parent counterpart.

What We Offer

Complete antibody humanization service or customized service (any stage integration) to decrease client cost;
6-week affinity guaranteed services for whole project from receiving your VH and VK sequences to get your humanized antibodies, an industry leader.
Quality (specificity and affinity) guaranteed humanization service. We guarantee the humanized candidates retain their epitope specificity and binding affinity (at least 100%-120% to its parent antibody tested by ELISA).

Process Overview

A humanized antibody is engineered in four main steps. At the end of each stage, customer will receive a full technical report of the work completed.

Stage I: Variable Region Sequencing, Numbering and Annotation

The antibody variable domains are amplified from your hybridoma cell line, cloned, and then sequenced. The sequences will be numbered according to the Kabat Numbering Scheme, the Chothia Numbering Scheme and the Martin (Enhanced Chothia) Numbering Scheme to identify variable HCDRs and LCDRs and framework regions.

Stage II: Antibody Humanization

Bioinformatics (sequence analysis and modeling) softwares are used to search the antibody variable regions from stage I to human donor sequences of our unique and non-redundant local database, which integrates sequence data from IMGT, Kabat collection , EMBLIG and V-BASE and the PDB with structural data from the Protein Databank, to create a panel of humanized heavy and light chains against. The candidates of humanized variants will be re-validated through BLAST to find their closest human germlines and human subgroup, thus to assess their humanness score (H-score and Z-score). The humanized chains will then be codon optimized and synthesized for expression in a mammalian system. Selections of human constant domain sequences or engineered constant sequence either enhancing/decreasing ADCC and CDC, increasing/decreasing binding to neonatal FcR (FcRn), FcyRIIIa and C1q , increasing /decreasing half-life are available, and the isotype, class, and subclass of the final antibody can also be specified at this stage.

Stage III: Small Scale Transient Transfection and chimeric/humanized antibody production

Each humanized antibody is cloned into our proprietary expression vector for small scale transient transfection. Each construct will undergo small scale expression and purification; the resulting antibodies will be tested for affinity to the target.

Case Study 5: Affinity Enhanced Antibody Humanization of Parent Rabbit Monoclonal Antibody

Some Citations Used Oak Biosciences Humanization Services

1.Lenka Kyrych Sadilkova, Iva Valentova, Simona Hoskova, Pavel Vopalensky, Lukas Bammert, Roger Beerli, Ulrich Moebius, Radek Spisek. SO-N102, a novel CLDN18.2-targeting antibody-drug conjugate with strong anti-tumor effect in various solid tumors expressing low target levels. Cancer Res (2021) 81 (13_Supplement): 1204. https://doi.org/10.1158/1538-7445.AM2021-1204 (The anti-CLDN18.2 antibody was from Oak Biosciences chicken scFv library and then humanization by Oak Biosciences)

2.Chen Y, Jia M, Wang S, Xu S and He N (2022) Antagonistic Antibody Targeting TNFR2 Inhibits Regulatory T Cell Function to Promote Anti-Tumor Activity. Front. Immunol. 13:835690. https://doi.org/10.3389/fimmu.2022.835690 (The anti-TNFR2 antibody was from Oak Biosciences Rabbit Immunized scFv library and then humanization by Oak Biosciences)

Deliverables

1) Humanized sequences (up to top 12 with highest binding);

2) Humanized antibodies (0.2-0.5 mg purified antibodies, up to 15 humanized and 1 chimeric) and their codon optimized DNA constructs (≥2 μg each for both heavy and light chain expression plasmids in commercial vectors for your own upscale humanized and/chimeric IgG1 production in your lab, if you need our upscale, we offer you discounted price);

3) Project report

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