Bispecific Antibody Service

Based on different antibody preparation technologies and platforms, Alpha Lifetech provides customized services for monoclonal and polyclonal antibodies of different species. In addition to antibody customization, we can also provide a series of bispecific antibody production, bispecific antibody purification, antibody sequencing and other services. We can provide our customers with quality assured antibody and recombinant protein products and services. We can prepare antibodies with high efficacy, strong specificity, and good stability.

Introduction to Bispecific Antibody

Bispecific Antibody (BsAb) has two specific antigen binding sites that can simultaneously bind and recognize two different antigens, or two different epitopes of an antigen. Bispecific antibodies can associate target cells with immune cells to mediate the occurrence of reactions. Bispecific antibodies are prepared by antibody cloning using genetic engineering technology. The concept of bispecific antibodies was first proposed in 1960, but due to its high technical difficulty, its initial development was also relatively slow. But with the continuous improvement of genetic engineering technology, the development of bispecific antibodies has also made phased progress. At present, the preparation methods of bispecific antibodies mainly include chemical conjugation, hybridoma fusion, and genetic engineering. Among them, genetic engineering has become the most commonly used preparation method due to its high efficiency and flexibility.

Bispecific Antibody Production-Alpha Lifetech

Fig. 1 Multiple types of bispecific antibodies

Classification and mechanism of action of bispecific antibodies

According to their different structures, bispecific antibodies are divided into two categories: non IgG like bispecific antibodies and IgG like bispecific antibodies. Among them, non IgG like bispecific antibodies, also known as Fc fragment free bispecific antibodies, can bind multiple antigen binding fragments to molecules without Fc regions, thereby avoiding chain cross-linking problems. Non IgG like bispecific antibodies also have advantages such as low immunogenicity, fast clearance rate, and short half-life. IgG like bispecific antibodies, also known as Fc fragment containing bispecific antibodies, are composed of two monoclonal antibodies targeting different targets. They possess Fc fragments and can exert Fc mediated effector functions.

Bispecific antibodies are mainly used for the treatment of tumors, recruiting T cells or natural killer cells to directly target tumor cells with effector cells to enhance their cytotoxicity. Bispecific antibodies can simultaneously block two different signaling pathways during the pathogenesis process to affect the growth and proliferation of tumor cells. The use of bispecific antibodies can connect different types of immune cells, such as T cells and tumor cells, promote interactions between immune cells, and enhance immune responses.

Different methods for production of bispecific antibodies

Chemical Coupling

The advantage of using chemical coupling method of bispecific antibody production is that it is fast and simple, but the antigen binding site is easily destroyed, which affects the antibody activity. At the same time, the safety and carcinogenicity of the crosslinking agent itself are also uncertain. Coupling agents such as phthalimide, N-succinyl-3- (2-pyridine disulfide) propionate, and dithioacylbenzoic acid are used to couple two intact IgG antibody fragments or two F (ab ') 2 antibody fragments into a bispecific antibody.

Hybridoma Technology

The advantage of using hybridoma fusion method to prepare bispecific antibodies is that the bispecific antibodies prepared by this method have good biological activity, stable antibody structure, but low antibody yield and difficult screening process. Firstly, two single specific hybridoma cells are prepared, and then they are fused into double hybridoma cells using cell fusion technology. Two heavy chain and two light chain molecules are generated, which can be paired randomly to produce BsAb.

Gene Engineering

The advantage of using genetic engineering to prepare bispecific antibodies lies in its high efficiency, flexibility, and controllability. Through genetic engineering technology, the structure and function of antibodies can be accurately designed, then antibody cloning be performed. Common technical methods include gene recombination, gene knockout, gene insertion, etc. After modification, antibodies can form bispecific antibodies with the required functions.

Other Methods

In addition, there are other methods for preparing bispecific antibodies. Two Fab fragments of a single specific antibody can be combined through cross-linking reaction to form a hybrid immunoglobulin. Through chain recombination technology, two VHH fragments can be combined to form a double stranded variable region antibody. A molecule formed by connecting the heavy chain variable region and light chain variable region of two different specific antibodies can bind to two antigens simultaneously in the form of a dimer.

Bispecific Antibodies Service Workflow

Steps	Service Content	Timeline
Gene Synthesis	Generation of up to 3 different design schemes of sequences, de novo generation of recombinant antibody DNA plasmids	2-3 weeks
Small Scale Test	Production of bispecific antibody proteins → Small scale expression in mammalian cell lines → Validation by SDS-PAGE → Binding analysis with recombinant protein antigens by ELISA → Comparison of clones with parental antibodies	5-6 weeks
Identify	Expression of the first two full-length bispecific antibodies → Binding analysis by ELISA → ELISA evaluation of antibodies recognized by anti mouse antibodies	1 week
Antibody Production	Large scale antibody production	3-4 weeks

Our Advantages

We cover the upstream and downstream services of antibody production, and can provide technical services from antibody preparation, bispecific antibody purification and antibody separation and purification, antibody sequencing, antibody validation, etc, , which can be used for bispecific antibody therapy.

High Expression

High protein expression in CHO cells.
Stable Storage

>2 weeks in serum at 37 °C.
No Solubility Issue

>30 mg/ml
Stable Cell Lnes

Many stable cell lines with tumor antigens have been developed to validate many bispecific antibodies