Affinity Tune-Up & Humanization

ProMab Biotechnologies has pioneered an artificial intelligence platform designed to simulate and guide the natural process of antibody affinity maturation. This innovative evolutionary approach aims to enhance binding affinity, modify specificity, and improve the stability of antibodies. Our method harnesses ESM models, which are trained on extensive natural protein databases, and involves intricate calculations to validate potential antibody mutations. 

Mouse monoclonal antibodies were one of the first antibodies to be produced in vivo; however, they were quickly discovered to induce an immune response in humans for therapeutic resources. This became known as the human anti-mouse antibody (HAMA) response, and it led to the rapid development of antibody humanization technology. 

An antibody, or immunoglobulin, is a protein that consists of four peptide chains - two identical heavy chains (H) and two identical light chains (L), with each chain containing a variable (V) and constant (C) domain. The complementarity-determining regions (CDRs), located on each variable domain (VH-VL), are the parts of the antibody that directly interact with the antigen. 

Antibody humanization works by replacing non-human antibody structures with human ones. More specifically, humanization produces an antibody where only the CDRs of the variable regions are of xenogenic origin. The benefits of antibody humanization are that it can reduce the immunogenicity of monoclonal antibodies derived from non-human sources, while also improving its efficacy, safety due to toxicity, and activation within the immune system. 

Depending on your application, ProMab offers an affinity tune-up and maturation service to increase antibody function and specificity to your needs. Antibodies derived from other sources than humans may also have low or varying affinity, so when it’s combined with our antibody humanization services, we can help you tune the antibody to the required goal. 

Affinity tune-up and maturation go hand in hand with antibody humanization services. Our scientists with 20 years of antibody engineering experience will utilize many different methods for humanization including state-of-the-art 3D modeling software to guarantee the best outcome for your desired application. 



ProMab Biotechnologies Custom Humanization Service: A complete service, from antigen to clinically relevant solution.

At ProMab Biotechnologies, we perform high-throughput screening of our human antibody library using ELISA or SPR (surface plasmon resonance). Clones from the human antibody library are individually tuned, and varying binding activities are generated for preclinical and future clinical use. The goal of this high-throughput screening assay is to generate antibodies with no (or low) affinity for antigens in normal cells but with high affinity for cancer cells. Additional assays can be performed such as flow cytometry, western blot, IHC, and more.

For SPR, our service can measure the on/off-rate (Kon / Koff), dissociation constant (Kd), and affinity (KD), for antibody-antigen interaction utilizing the Biacore 3000.

To decrease the immunogenicity of monoclonal antibodies derived from rodents, we use a rational approach based on antibody sequence and 3D structure by computer modeling and docking programs. The standard service is outlined below: 



Application of Antibody Humanization in CAR-T 

The model shown below is what ProMab uses for many of its proprietary CAR-T technologies as humanizing the antibody scFv sequence can possibly lead to improved efficacy in the CAR-T setting.



Figure 1. The scheme for antibody humanization for generating CAR-T cells.

In addition to ProMab's antibody humanization platform, our scientists can test your humanized scFvs in a CAR-T/NK setting using various in vitro or in vivo studies. 



Figure 2. An illustration of the stepwise process of humanizing a rodent mAb for generating a CAR-T cell with a humanized scFv.

The scFv generated from our antibody humanization service will be cloned into a lentivirus vector with selected CAR domains and transduced in activated T-cells. The newly generated CAR-T cells will then be subject to biological assays to confirm binding affinity and cytotoxicity against target cells. ProMab Biotechnologies has developed many CAR-T products based on nearly 20 years' experience in hybridomas, monoclonal antibodies (rabbit, mouse, and human), recombinant proteins, and cancer stem cells. Click here to learn more about our CAR-T Platform



Other Custom Services:

Mouse monoclonal antibodies

Human monoclonal antibodies


Hybridoma Sequencing