What is Antibody Engineering?

With the help of antibody engineering, it has been possible to modify the molecular size, pharmacokinetics, immunogenicity, binding affinity, specificity and effector function of antibodies. After synthesizing antibodies, the specific binding of antibodies makes them highly valuable in clinical diagnosis and treatment. Through antibody engineering, they can meet the needs of drug and diagnostic early development.

The purpose of antibody engineering is to design and produce highly specific, stable functions that natural antibodies cannot achieve, laying the foundation for the production of therapeutic antibodies.

Alpha Lifetech, with its extensive project experience in antibody  engineering, can provide customized monoclonal and polyclonal antibody services for multiple species, as well as phage display antibody library construction and screening services. Alpha Lifetech can provide customers with quality biosimilar antibodies and recombinant protein products, as well as corresponding services, to produce efficient, highly specific, and stable antibodies. By utilizing comprehensive antibody, protein platforms and phage display systems, we provide services covering the upstream and downstream of antibody production, including technical services such as antibody humanization, antibody purification, antibody sequencing, and antibody validation.

The pioneering stage of antibody engineering is related to two technologies:

--Recombinant DNA technology

--Hybridoma technology

The rapid development of antibody engineering is related to three important technologies:

--Gene cloning technology and polymerase chain reaction

--Protein expression: Recombinant proteins are produced by expression systems such as yeast, rod-shaped viruses, and plants

--Computer aided structural design

The first generation of antibodies were humanized for the production of chimeric antibodies, where the variable region of mouse monoclonal antibodies was linked to the constant region of human IgG molecules. The antigen binding region (CDR) of the second-generation mouse monoclonal antibody was transplanted into human IgG. Except for the CDR region, all other antibodies are almost human antibodies, and efforts were made to avoid inducing human anti mouse antibody (HAMA) responses when using mouse clone antibodies for human treatment.

 

To construct a phage display library, the first step is to obtain the genes encoding antibodies, which can be isolated from B cells of immunized animals (immune library construction), extracted directly from non immunized animals (natural library construction), or even assembled in vitro with antibody gene fragments (synthetic library construction). Then, the genes are amplified by PCR, inserted into plasmids, and expressed in suitable host systems (yeast expression (usually Pichia pastoris), prokaryotic expression (usually E. coli), mammalian cell expression, plant cell expression, and insect cell expression infected with rod-shaped viruses). The most common is the E. coli expression system, which integrates a specific encoding antibody sequence onto the phage and encodes one of the phage shell proteins (pIII or pVIII). The gene fusion of, And displayed on the surface of bacteriophages. The core of this technology is to construct a phage display library, which has the advantage over natural libraries in that it can have specific binding. Subsequently, antibodies with antigen specificity are screened through a biological selection process, target antigens are fixed, unbound phages are repeatedly washed away, and bound phages are washed away for further enrichment. After three or more rounds of repetition, high specificity and high affinity antibodies are isolated.

Fig 3: Antibody Library Construction and Screening

Recombinant DNA technology can be used to generate antibody fragments. Fab antibodies can initially only be hydrolyzed by gastric protease to produce (Fab ') 2 fragments, which are then digested by papain to generate individual Fab fragments. The Fv fragment consists of VH and VL, which have poor stability due to the absence of disulfide bonds. Therefore, VH and VL are linked together through a short peptide of 15-20 amino acids to form a single chain variable fragment (scFv) antibody with a molecular weight of approximately 25KDa.

The study of antibody structure in Camelidae (Camel, LIama, and Alpaca) has elucidated that antibodies only have heavy chains and no light chains, hence they are called heavy chain antibodies (hcAb). The variable domain of heavy chain antibodies is called single domain antibodies or nanobodies or VHH, with a size of 12-15 kDa. As monomers, they have no disulfide bonds and are very stable, with a very high affinity for antigens.

Fig 5: Heavy Chain Antibody and VHH/ Nanobody

Cell free expression utilizes the expression of natural or synthetic DNA to achieve in vitro protein synthesis, typically using the E. coli expression system. It produces proteins quickly and avoids the metabolic and cytotoxic burden on cells when producing large amounts of recombinant proteins in vivo. It can also produce proteins that are difficult to synthesize, such as those that are difficult to modify after translation or synthesize membrane proteins.