Overview of Diabody-CH3

What is Diabody-CH3

Diabody-CH3 is a bispecific antibody fragment composed of two different polypeptide chains, each containing a heavy chain variable region (V_H) and a light chain variable region (V_L). The two chains are linked by a short peptide linker (usually 5 amino acids) to form a single-chain Fv (scFv) fragment. Two scFv fragments form a dimer through VH-VH interactions, called a Diabody. Each Diabody molecule has two antigen-binding sites, formed by the pairing of VH and VL from different chains, giving Diabody the ability to recognize two different antigens simultaneously, resulting in bispecificity.

Fig.1 A schematic diagram of Diabody-CH3

Diabody-CH3 is derived from the Diabody structure by fusing one of the chains with a covalently linked CH3 domain or an Fc domain. This fusion increases the molecular weight and stability of Diabody, extends its half-life in vivo, and provides the ability to bind to Fc receptors or complement. Diabody-CH3 is a tetravalent bispecific molecule composed of two chains: one is a VH-VL-CH3 or VH-VL-Fc chain, and the other is a VH-VL chain.

Diabody-CH3 has some advantages and limitations compared to conventional monoclonal antibodies or bispecific antibodies. These advantages include its simple structure, ease of expression and purification, the ability to target two antigens and to modulate Fc functions, etc. However, Diabody-CH3 also has some limitations. For instance, its antigen-binding sites may not be stable enough; it may have immunogenicity, and linker length and sequence optimization may be required.

Generation Methods of Diabody-CH3

Diabody-CH3 can be generated by different technical platforms and strategies, such as phage display, yeast display, DNA recombination, etc. Each of these methods has its own advantages, disadvantages, and applicability.

Phage display is a widely used method for generating diabodies by screening a large library of scFv fragments with short linkers. The selected scFv fragments are then cloned into a phagemid vector and expressed as diabodies on the surface of phages. The diabodies can be further fused with a CH3 domain or an Fc domain to form diabody-CH3 molecules. Phage display allows for high-throughput screening and affinity maturation of diabodies, but it may also introduce biases and limitations due to the bacterial expression system and the phage packaging constraints.

Yeast display is another method for generating diabodies. It entails displaying scFv fragments with short linkers on the surface of yeast cells. The yeast cells can be sorted by fluorescence-activated cell sorting (FACS) based on their binding to antigens. Similar to phage display, the diabodies generated through yeast display can be fused with a CH3 domain or an Fc domain to form diabody-CH3 molecules. Yeast display offers advantages such as eukaryotic expression system, post-translational modifications, and direct affinity measurement. However, it may also suffer from low expression levels, low diversity, and high background noise.

DNA recombination is a method for generating diabodies by recombining two different scFv genes with short linkers using PCR or restriction enzymes. The resulting diabody genes can be cloned into an expression vector and transfected into mammalian cells or other hosts for production. The diabodies can be also fused with a CH3 domain or an Fc domain to form diabody-CH3 molecules. DNA recombination enables precise control over the diabody sequence and orientation, but it may also require extensive optimization and validation of the recombination efficiency and specificity.

Clinical Applications of Diabody-CH3

Diabody-CH3 has been applied for clinical purposes, such as cancer immunotherapy, dual targeting of disease mediators, and receptor activation or blockade. Several diabody-CH3 products have been approved or are undergoing clinical trials for various indications.

One example of an approved diabody-CH3 product is catumaxomab (Removab®), a tetravalent bispecific antibody that targets EpCAM on tumor cells and CD3 on T cells. It was approved in Europe in 2009 for the treatment of malignant ascites in patients with EpCAM-positive tumors. Catumaxomab can induce tumor cell lysis by redirecting T cells to the tumor site, as well as by activating other immune effector cells and complement.

Another example of a diabody-CH3 product in clinical development is emicizumab (ACE910), a tetravalent bispecific antibody that mimics the function of factor VIII by bridging factor IXa and factor X. It was designed for the treatment of hemophilia A patients with or without inhibitors to factor VIII. Emicizumab has shown promising results in phase I and phase III clinical trials, demonstrating a significant reduction in bleeding events and a favorable safety profile.

References

1. Byrne H, et al. A tale of two specificities: bispecific antibodies for therapeutic and diagnostic applications. Trends Biotechnol. 2013 Nov;31(11):621-32.
2. Chan AC, et al. Therapeutic antibodies for autoimmunity and inflammation. Nat Rev Immunol. 2010 May;10(5):301-16.
3. Kontermann RE. Dual targeting strategies with bispecific antibodies. MAbs. 2012 Jan-Feb;4(1):182-97.
4. Lu D, et al. A novel tetravalent bispecific antibody (diabody) for cancer therapy with improved pharmacokinetics and enhanced biological activity by design. Mol Immunol. 2003 Aug;40(1):7-17.
5. Seifert O, et al, Richter F, Kontermann RE. Diabody-Ig: a novel platform for the generation of multivalent and multispecific antibody molecules. MAbs. 2019 Jul;11(5):919-29.
6. Spiess C, et al. Alternative molecular formats and therapeutic applications for bispecific antibodies. Mol Immunol. 2015 Oct;67(2 Pt A):95-106.
7. Weiner LM, et al. Monoclonal antibodies: versatile platforms for cancer immunotherapy. Nat Rev Immunol. 2010 May;10(5):317-27.