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HCAb-Based Bispecific Antibodies

Bispecific/Multi-Specific Antibody Engineering

HCAb-Based Bispecific Antibodies
Sophisticated Workflow

HCAb-Based Bispecific Antibodies

Monoclonal antibodies have significantly improved cancer treatment options, but their long-term efficacy is limited by resistance mechanisms such as pathway downregulation, upregulation of alternative pathways, and pathway crosstalk. Multispecific therapeutic reagents offer a potential solution by enhancing the efficacy of targeted therapy. Currently, numerous bispecific or multi-specific antibodies are under extensive investigation.

The structural design of bispecific antibodies is very critical. Natural bivalent IgG antibodies comprise two identical heavy chains and two identical light chains, each Fv pair with an identical antigen-binding site. Bispecific antibodies, on the other hand, require the incorporation of two different antigen-binding sites from two distinct IgG antibodies. This results in a molecular structure composed of two different pairs of Fv domains. Consequently, one of the main challenges in bispecific antibody development lies in addressing the issue of chain mismatch, which involves obtaining functional bispecific antibodies with the correct combination of heavy and light chains from more than 10 possible combinations.

To overcome this challenge, scientists have developed various strategies and technologies to improve the homogeneity and yield of desired bispecific antibodies. These approaches involve introducing different design features or functional properties. For instance, the “knobs-into-holes” technology and other mutations have been employed to address heavy chain mispairing, while the “common light chain” and “CrossMAb” technologies have been developed to tackle mispairing between heavy chain and light chain. The most commonly used strategy is introduction of scFv structures. However, scFv usually requires further antibody engineering to optimize its stability and solubility.

On the other hand, heavy chain only antibodies (HCAbs) offer unique advantages in the construction of bispecific or even multispecific antibodies. Since HCAb lacks light chains, it naturally avoids the issue of mispairing between heavy chain and light chain when used in the context of bispecific antibodies. This simplifies the antibody structure significantly. Additionally, the antigen-binding domain of HCAb is only one-quarter the size of the Fab region found in conventional antibodies. As a result, using HCAb and sdAb derived from it allows for the construction of bispecific or multispecific antibodies with smaller molecular weights, fewer polypeptide chains, and simpler structures.

Harbour Mice® HCAb transgenic mice enable the generation of a novel format of heavy chain antibodies with fully human variable region sequences. These fully human HCAb and their derived human VH single domains serve as fundamental building blocks for creating innovative bispecific modalities. Notably, human HCAb offers advantages in terms of immunogenicity and drugability compared to camelid HCAb.

Attributing to its smaller size and lack of light chain, human VH single-domain moieties from HCAb can be flexibly appended to another antibody no matter H2L2 or HCAb to generate bispecific antibodies with various geometries.

As shown in the example below, the VH domain from anti-B HCAb can be appended to existing anti-A H2L2 at different positions, such as the N-terminus or C-terminus of the heavy chain or light chain of anti-A H2L2, resulting in four distinct bispecific antibodies. When the anti-B VH domain is appended to the N-terminus of the anti-A H2L2, the generated bispecific antibodies (bsAb-1 and bsAb-2) can bind to both targets A and B with the similar activities to that of their parental antibodies (anti-A H2L2 or anti-B HCAb); when the anti-B VH domain is appended to the C-terminus of light chain, the generated bispecific antibody (bsAb-3) can retain the binding activity to target A but significantly reduces the binding activity to target B. This example illustrates the flexibility of designing bispecific antibodies from HCAb and demonstrates how different structural geometries, relative positions, binding valences, and other parameters can be adjusted to tailor the functional activities of bispecific antibodies for different targets.

Sophisticated Workflow for Bispecific Antibody Design and Characterization

Nona Biosciences has a top-tier scientific team in the field of bispecific antibody development. This team has established the sophisticated workflow for bispecific antibody design and characterization. Our expertise has led to the successful advancement of a first-in-class B7H4×4-1BB bispecific antibody into clinical development, along with the creation of multiple innovative bispecific antibodies currently undergoing pre-clinical studies.

HBICE® – HCAb-Based Immune Cell Engager

Harbour Mice® HCAb platform enables the generation of diverse and stable fully human VH single-domain moieties, facilitating the construction of novel bispecific or multi-specific antibodies. Building upon this platform, Nona Biosciences has established the proprietary HBICE® (HCAb Based Immune Cell Engager) platform, which allows for the rapid development of multi-specific antibodies designed to redirect immune cells to the tumor microenvironment (TME) and effectively eliminate tumors.

HBICE® molecules are designed to recognize and bind specific tumor-associated antigens (TAA) on tumor cells, as well as CD3 or co-stimulatory molecules on immune cells like T cells or NK cells. This dual binding mechanism leads to the selective activation of immune cells within the TME, while minimizing non-specific activation of peripheral immune cells. The HBICE® technology provides flexibility in generating molecules with different architectures and avidities, enabling the achievement of diverse mechanisms of action that are not attainable through combination therapies alone.

One exemplary HBICE® molecule is HBM7004, a novel bispecific antibody targeting B7H4 on tumor cells and CD3 on T cells. It features a unique “2+1” asymmetric format, comprising one optimized CD3 binding moiety and two B7H4 binding moieties arranged in tandem. With its relatively weak binding activity to CD3 and avidity-dependent binding activity to B7H4, HBM7004 selectively binds to tumor cells exhibiting high expression of B7H4. This preferential binding reduces systemic exposure in peripheral tissues while enhancing distribution to tumor sites. As a result, HBM7004 elicits robust and specific T-cell dependent cellular cytotoxicity (TDCC) against B7H4-positive tumors, while exhibiting minimal cytotoxicity in the absence of B7H4.


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