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Bispecific Antibody Development Services

TekBiotech focuses on developing high-quality targeted drug antibodies for customers. Bispecific antibodies (bsAbs) refer to antibodies with two antigen-binding capabilities, that is, one antibody contains a V region that binds to two different antigens. Compared with therapeutic monoclonal antibodies, bsAbs have been shown to have better efficacy prospects and higher safety in clinical studies, especially in the fields of tumor verification and viral infection, becoming a hot research direction. According to the mechanism of action, bsAbs are mainly divided into 4 categories: cell bridging, dual-target blocking, immune cell activation, and bridging of the same cell surface protein. They can also be divided into full-length bsAbs and fragment-type bsAbs according to the structure. The structure of bsAbs is crucial to the therapeutic effect of bsAbs, and the structural design of bsAbs mainly depends on their targets and mechanisms of action.

█ Common Structural Types of Bispecific Antibodies

Common bispecific antibody structures include KIH (knob into hole), Crossmab, DVD-I and IgG fusion scFV structures, as shown in Figure 1:

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Figure1 Common bispecific antibody structural types

Tekbiotech has been engaged in nanoantibody research for many years, and is good at nanodevelopment, antibody humanization and other therapeutic antibody drug development. At the same time, it has designed the IgG-VHH bispecific mode, which has simple, safe and effective structural characteristics.

█ Bispecific Antibody Recombinant Expression Service

Tekbiotech has a mature recombinant antibody mammalian expression platform, which can provide customers with high-quality antibody expression customization services. A full set of Gibco serum-free mammalian cell culture, combined with 293F and CHO suspension cell lines with clear sources, the entire system meets the in vitro recombinant expression needs of different customers for natural conformational proteins. The full set of cost-effective research services provided by TekBiotech to customers meet the GMP-like system, including the cell seed bank source document record system, cell seed batch verification, quality control of animal-derived ingredients, standardized cell culture and transformation operation procedures, etc.

Table2 Contents of bispecific antibody recombinant expression service

Steps	Service Content	Cycle
Step1: Design and construction of bispecific antibodies	-- Confirmation and verification of affinity of parent sequence; -- Bispecific antibody structure design (confirmed with customers); -- Linker system screening and yield verification; -- Delivery: sequencing raw data, verification data and experimental report;	4-5 Weeks
Step2: In vitro functional verification	-- Recombinant plasmid transfected cells (293F, CHO) transient expression + purification + SDS-PAGE and WB detection; -- In vitro binding and blocking verification (EC50, FACS, etc.); -- In vitro functional verification; -- Delivery: experimental data, related materials, experimental report;	6-10 Weeks
Step3: Stable expression (optional)	-- Construction and screening of recombinant protein CHO stable expression cell line; -- Delivery: stable cell line, construction report; marginal product, 3 clones, 50ml fermentation product and purified product for each clone;	12–14 Weeks

Tekbiotech can also provide customers with one-stop technical services such as antibody humanization, CAR-T/CAR-NK lead sequence design and cell killing verification, to help customers' scientific research projects and drug antibody development.

█ Advantages of Bispecific Antibody Development Service Platform

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Mature platform, high success rate

Diversified bispecific antibody forms

Rich expression experience, strict QC

control system

Customized bispecific antibody structure

Bispecific Antibody Development Services Frequently Asked Questions

What are bispecific antibodies?

Since most diseases involve multiple parallel signaling pathways, multiple inhibition of receptors and ligands can lead to better interference with various ligand-receptor signaling pathways. While this effort to use combination therapies has been used in small molecule drug discovery, its scope is expanding to include therapeutic antibodies. Bispecific antibodies can react with two different epitopes on the same target antigen or against binding sites on two different antigens, respectively. The extensive efforts to construct immune cell redirection platforms to deliver cytotoxicity to target cells can only be realized through the use of bispecifics. The challenge lies in identifying a specific set of BsAbs and determining the effective dosage window to produce the desired effect when targeting different specificities. For extensive protein engineering of BsAbs, there is a good evaluation of: antibody fragments without the Fc structural domain; antibody fragments with the Fc structural domain.
What are the applications of bispecific antibodies?

Initially, therapeutic applications focused on effector cell repositioning for cancer therapy, including T cells, which cannot be recruited into tumor cells by normal antibodies. As technology has evolved, a variety of bispecific antibody therapies are now being developed. In addition to effector molecule, cellular and genetic vector retargeting, dual targeting and pre-targeting strategies, half-life extension, and delivery through biological barriers such as the blood-brain barrier have been explored. It can also be used for T-cell retargeting, where one Fv arm targets the CD3 structural domain in the T-cell receptor complex and the other Fv arm binds to an epitope on the target cell, thereby bringing cytotoxic T cells in close proximity to the target cell to promote target cell lysis and elimination.
What forms of bispecific antibodies exist? What is the difference?

Most bivalent or multivalent molecules in natural antibodies have the same antigen-binding site, however, IgG4 molecules are different. The instability of its hinge region confers a special ability to randomly swap its Fab arms to generate bivalent molecules with doubly different specificities. To date, bispecific antibody fragments exist in three main forms: BiTE, DART, and TandAbs. Of these, BiTE molecules have been widely used in immunotherapy of tumors, aiming to relocate T cells to the tumor microenvironment in order to attack tumor cells or related tumor-associated cells. They utilize scFv fragments from two different monoclonal antibodies that are linked by peptide junctions capable of retaining binding activity . Dual Affinity Retargeting Protein (DART), which consists of two Fv fragments that, when heterodimerized, form two unique antigen-binding sites. The small size of scFvs contributes to higher renal clearance compared to natural antibodies. Size issues can be helped by generating tandem doublets (TandAbs).
How should the junctions in bispecific antibodies be designed?

The junction region between the light and heavy chain structural domains plays an important role in stabilizing antibodies and is therefore an important target for optimizing scFvs. Studies on the orientation of heavy and light chain structural domains have shown that both orientations are favorable in different situations. The two central considerations in the process of junction design are the amino acid composition and the length of the sequence. First and foremost, amino acid composition is critical for creating functional and flexible junction peptides; for example, hydrophilic sequences are essential to prevent peptides from embedding within or between V domains during protein folding. Glycine and serine are preferred because of the conformational flexibility and very low immunogenicity afforded by short side chains. In addition to the amino acid composition, the length of the heavy- and light-chain junctions in the Fv structural domain is also critical for the assembly of single-chain antibody fragments (scFv) with the correct conformation. The length of the junctions profoundly affects the multimer formation of the antibody, so by carefully planning the length of the junctions, we can efficiently facilitate the formation of scFv molecules designed for bis-antibodies, especially TandAbs molecules, or enable their multivalent form to exhibit superior performance compared to their monovalent form.
For bispecific antibody development services, how do you ensure the stability of the antibody?

The stability of the scFv molecule is a key factor as there is thought to be a direct correlation between stability and biological activity, and a stable scFv molecule is an important component in development services. One way to optimize protein stability is to directly modify the scFv framework. The common methods used in building structures are the ring grafting technique and mutagenesis. Ring grafting techniques enable the simultaneous enhancement of stability and humanization through a one-step operation in which an antigen-specific complementary determining region (CDR) is grafted onto a framework that possesses desirable biophysical properties, such as stability. Alternatively, for mutagenesis approaches, stability enhancement is achieved through rational site mutation or directed evolution. In addition to shared methods, other approaches alter residues to achieve stability goals, such as creating inter-domain disulfide bonds, creating intramolecular hydrogen bonds, and optimizing hydrophobicity.

What are bispecific antibodies?

Since most diseases involve multiple parallel signaling pathways, multiple inhibition of receptors and ligands can lead to better interference with various ligand-receptor signaling pathways. While this effort to use combination therapies has been used in small molecule drug discovery, its scope is expanding to include therapeutic antibodies. Bispecific antibodies can react with two different epitopes on the same target antigen or against binding sites on two different antigens, respectively. The extensive efforts to construct immune cell redirection platforms to deliver cytotoxicity to target cells can only be realized through the use of bispecifics. The challenge lies in identifying a specific set of BsAbs and determining the effective dosage window to produce the desired effect when targeting different specificities. For extensive protein engineering of BsAbs, there is a good evaluation of: antibody fragments without the Fc structural domain; antibody fragments with the Fc structural domain.
What are the applications of bispecific antibodies?

Initially, therapeutic applications focused on effector cell repositioning for cancer therapy, including T cells, which cannot be recruited into tumor cells by normal antibodies. As technology has evolved, a variety of bispecific antibody therapies are now being developed. In addition to effector molecule, cellular and genetic vector retargeting, dual targeting and pre-targeting strategies, half-life extension, and delivery through biological barriers such as the blood-brain barrier have been explored. It can also be used for T-cell retargeting, where one Fv arm targets the CD3 structural domain in the T-cell receptor complex and the other Fv arm binds to an epitope on the target cell, thereby bringing cytotoxic T cells in close proximity to the target cell to promote target cell lysis and elimination.
What forms of bispecific antibodies exist? What is the difference?

Most bivalent or multivalent molecules in natural antibodies have the same antigen-binding site, however, IgG4 molecules are different. The instability of its hinge region confers a special ability to randomly swap its Fab arms to generate bivalent molecules with doubly different specificities. To date, bispecific antibody fragments exist in three main forms: BiTE, DART, and TandAbs. Of these, BiTE molecules have been widely used in immunotherapy of tumors, aiming to relocate T cells to the tumor microenvironment in order to attack tumor cells or related tumor-associated cells. They utilize scFv fragments from two different monoclonal antibodies that are linked by peptide junctions capable of retaining binding activity . Dual Affinity Retargeting Protein (DART), which consists of two Fv fragments that, when heterodimerized, form two unique antigen-binding sites. The small size of scFvs contributes to higher renal clearance compared to natural antibodies. Size issues can be helped by generating tandem doublets (TandAbs).
How should the junctions in bispecific antibodies be designed?

The junction region between the light and heavy chain structural domains plays an important role in stabilizing antibodies and is therefore an important target for optimizing scFvs. Studies on the orientation of heavy and light chain structural domains have shown that both orientations are favorable in different situations. The two central considerations in the process of junction design are the amino acid composition and the length of the sequence. First and foremost, amino acid composition is critical for creating functional and flexible junction peptides; for example, hydrophilic sequences are essential to prevent peptides from embedding within or between V domains during protein folding. Glycine and serine are preferred because of the conformational flexibility and very low immunogenicity afforded by short side chains. In addition to the amino acid composition, the length of the heavy- and light-chain junctions in the Fv structural domain is also critical for the assembly of single-chain antibody fragments (scFv) with the correct conformation. The length of the junctions profoundly affects the multimer formation of the antibody, so by carefully planning the length of the junctions, we can efficiently facilitate the formation of scFv molecules designed for bis-antibodies, especially TandAbs molecules, or enable their multivalent form to exhibit superior performance compared to their monovalent form.
For bispecific antibody development services, how do you ensure the stability of the antibody?

The stability of the scFv molecule is a key factor as there is thought to be a direct correlation between stability and biological activity, and a stable scFv molecule is an important component in development services. One way to optimize protein stability is to directly modify the scFv framework. The common methods used in building structures are the ring grafting technique and mutagenesis. Ring grafting techniques enable the simultaneous enhancement of stability and humanization through a one-step operation in which an antigen-specific complementary determining region (CDR) is grafted onto a framework that possesses desirable biophysical properties, such as stability. Alternatively, for mutagenesis approaches, stability enhancement is achieved through rational site mutation or directed evolution. In addition to shared methods, other approaches alter residues to achieve stability goals, such as creating inter-domain disulfide bonds, creating intramolecular hydrogen bonds, and optimizing hydrophobicity.

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