Overview of DutaMab Technology

Backgrounds of DutaMab Technology

Antibodies are a class of immunoglobulins that can specifically recognize antigens, with a wide range of biological functions and clinical applications. Conventional monoclonal antibodies (MoAbs) can only recognize one antigen or one epitope, thus limiting their therapeutic effects. To improve the efficacy of antibodies, people have developed bispecific antibodies (bsAbs), which are antibodies that can simultaneously recognize two different antigens or two different epitopes. BsAbs have various advantages, such as recruiting immune cells to kill tumor cells, blocking two signaling pathways at the same time, and enhancing the specificity and affinity of antibodies. BsAbs have broad application prospects in tumor immunotherapy, infectious diseases, autoimmune diseases and other fields.

BsAbs have various formats, ranging from small molecules of dual Fab fragments to large molecules of IgG-like structures, with different design and preparation methods. Among them, the most attractive one is the two-in-one antibody, which is a conventional IgG that can bind to two different antigens. This format retains the Fc-mediated functions of IgG and has a similar structure to wild-type IgG, thus having low immunogenicity and standardized development methods. However, generating two-in-one antibodies is difficult, time-consuming and expensive. To solve this problem, people invented the DutaMab technology platform, which can combine two different heavy chain variable regions (VH) on one Fab arm, thus generating a bispecific IgG with four independent antigen binding sites.

Principle of DutaMab Technology

DutaMab technology platform is a novel approach to create bispecific antibodies by combining two different variable heavy (VH) domains in each Fab arm of an IgG, resulting in a tetra-VH IgG format. The VH domains are used as building blocks, where one VH is placed at its usual position, and the second VH replaces the variable light (VL) domain in a conventional IgG. This way, each Fab arm of the IgG has two independent antigen binding sites, one on the H-side paratope and one on the L-side paratope. The H-side paratope consists of HCDR1, HCDR3 and LCDR2, while the L-side paratope consists of LCDR1, LCDR3 and HCDR2. The tetra-VH IgGs are functionally tetravalent, meaning that they can bind two antigens on each arm of the IgG molecule simultaneously.

The DutaMab technology platform differs from other bispecific antibody formats in several aspects. First, it maintains the wild-type IgG structure and stability, without introducing any artificial linkers or mutations. Second, it does not require any light chain engineering or matching, which simplifies the generation process and reduces the immunogenicity risk. Third, it allows for modular and flexible combination of different VH domains, which can be selected from a large repertoire of natural or synthetic VH libraries. Fourth, it enables simultaneous binding of two antigens with high affinity and specificity, without interfering with each other.

Production Processes of DutaMab

The production process of the DutaMab is similar to the conventional IgG production process, but with some specific steps and considerations:

• VH discovery: Using large-scale natural or synthetic VH libraries, VHs that can specifically bind to target antigens are discovered by immunological or non-immunological methods. Each VH has a unique sequence and structure, which can be used as a building block. To ensure the stability and expression of VHs, some optimization is needed, such as adding a C-terminal cysteine to form an endogenous disulfide bond.
• VH selection: According to the desired combination of bispecific antibodies, two different VHs are selected from the discovered VHs, one as the H-side paratope and one as the L-side paratope. The selection criteria include the affinity, specificity, stability, and expression of VHs. To avoid interaction or interference between VHs, some tests are needed, such as bivalent affinity test, competition test, thermal stability test, etc.
• VH combination: Two different VHs are connected to one Fab arm by gene synthesis and recombinant DNA technology, forming a bispecific Fab fragment (DutaFab). One VH remains in its original position, and the other VH replaces the original VL. To ensure the correct folding and assembly of DutaFab fragments, a linker peptide is introduced between the heavy and light chains, and an N-terminal cysteine is added to the light chain to form an exogenous disulfide bond with the C-terminal cysteine on the heavy chain.
• VH expression: The DutaFab fragment is connected to the constant regions (CH1 and CL) to form a complete IgG molecule. It is expressed and purified by mammalian cells or other suitable host cells, resulting in a bispecific IgG (DutaMab) with four independent antigen binding sites. Since DutaMab has a similar structure and stability to wild-type IgG, standardized production processes and quality control methods can be used.

Advantages and Disadvantages of DutaMab Technology

The advantages of the DutaMab technology platform in the field of bispecific antibodies include high affinity, simultaneous binding of two antigens, good stability, low immunogenicity, and Fc-mediated functions. DutaMab can bind to two different antigens simultaneously, and each antigen binding site has high affinity, which can reach nM or pM level. High affinity helps to improve the therapeutic effect and selectivity of DutaMab. DutaMab can bind to two different antigens on one Fab arm, rather than one antigen on each Fab arm, which can avoid some potential problems, such as the difference in antigen expression level, the restriction of antigen distance, the influence of antibody angle, etc. Simultaneous binding of two antigens can achieve various complex mechanisms, such as recruiting immune cells, blocking signaling pathways, and enhancing targeting effect. DutaMab maintains the structure and stability of wild-type IgG, without introducing any artificial linkers or mutations. DutaMab has good physicochemical and biological stability, and can withstand high temperature, low pH, freeze-thaw and other conditions. Good stability helps to improve the quality and safety of DutaMab. DutaMab is composed entirely of humanized VHs, without introducing any heterologous or unnatural components. DutaMab has no light chain engineering or matching problems, nor light chain exchange risk. Low immunogenicity helps to reduce the adverse reactions and neutralizing antibodies caused by DutaMab. DutaMab retains the Fc region of IgG, so it can exert Fc-mediated functions, such as antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP). Fc-mediated functions can enhance the therapeutic effect and diversity of DutaMab.

However, the DutaMab technology platform also has several disadvantages or potential challenges in the field of bispecific antibodies. DutaMab can only use VH as building blocks, and cannot use VL or other types of single-domain antibodies. This means that DutaMab can only select antigens or epitopes that can be recognized by VHs, and cannot select antigens or epitopes that can only be recognized by VLs or other types of single-domain antibodies. This may limit the range and combination possibilities of targets that DutaMab can cover. Although DutaMab has a similar structure and stability to wild-type IgG, its generation process still requires some specific steps and considerations, such as VH optimization, testing, and linking. This may increase the production cost and time of DutaMab, compared to conventional monospecific IgG or other types of bispecific antibodies. Moreover, although DutaMab has low immunogenicity and high specificity, its clinical safety still needs to be further verified and evaluated. Since DutaMab can bind to two different antigens simultaneously and has Fc-mediated functions, there may be some potential safety risks, such as target-related toxicity, off-target toxicity, allergic reactions, and cytokine storm. These risks need to be strictly monitored and managed in clinical trials.

Clinical Application of DutaMab Technology

Currently, the bispecific antibodies developed using the DutaMab technology platform are mainly aimed at the fields of ophthalmology and oncology, and some of them have entered the clinical trial or marketing stage. Currently, only one bispecific antibody developed using the DutaMab technology platform has been approved by the US FDA.

At present, several bispecific antibodies developed using the DutaMab technology platform are in different stages of clinical trials, mainly targeting ophthalmology and oncology fields. For example, RO7297089 is a novel bispecific BCMA/CD16A-directed innate cell engager (ICE®) designed to induce BCMA+ MM cell lysis through high affinity binding of CD16A and retargeting of NK cell cytotoxicity and macrophage phagocytosis. RO7297089 selectively binds to CD16A with no binding of other Fcγ receptors, including CD16B on neutrophils, and irrespective of 158V/F polymorphism. RO7297089 is less affected by competing IgG, suggesting activity in the presence of M-protein.

References

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2. Kakiuchi-Kiyota S, et al. A BCMA/CD16A bispecific innate cell engager for the treatment of multiple myeloma. Leukemia. 2022;36(3):1006-1014.
3. Plesner T, et al. Phase I Study of Safety and Pharmacokinetics of RO7297089, an Anti-BCMA/CD16a Bispecific Antibody, in Patients with Relapsed, Refractory Multiple Myeloma. Clin Hematol Int. 2023;5(1):43-51.
4. Schaefer W, et al. Immunoglobulin domain crossover as a generic approach for the production of bispecific IgG antibodies. Proc Natl Acad Sci U S A. 2011;108(27):11187-11192.
5. Spiess C, et al. Alternative molecular formats and therapeutic applications for bispecific antibodies. Mol Immunol. 2015;67(2 Pt A):95-106.
6. Labrijn AF, et al. Efficient generation of stable bispecific IgG1 by controlled Fab-arm exchange. Proc Natl Acad Sci U S A. 2013;110(13):5145-5150.
7. Beckmann R, et al. DutaFabs are engineered therapeutic Fab fragments that can bind two targets simultaneously. Nat Commun. 2021;12(1):708.