Bispecific Antibodies: A Success Story in Advancing Cancer Immunotherapy

Introduction

In the four decades since the first monoclonal antibody therapy was approved for market distribution, antibody-based therapeutics have become the go-to strategy in the treatment of diverse diseases, including many types of cancers. In order to address therapeutic resistance and to enhance drug efficacy, antibody engineers have increasingly been designing and developing bispecific and multispecific antibodies, which can target two or more antigenic epitopes on a single cell or bring together two different cells via their respective epitopes. The potential applications in immune-oncology are numerous, and the explosion of drugs being currently investigated in research pipelines or being monitored in clinical trials speaks to the importance of bispecific antibodies as an avenue for innovation.

To provide proof of concept and contribute to humanity’s ongoing battle against cancer, Yurogen’s scientists set out to combine immune cell activation with targeted cancer therapy to enhance anti-tumor efficacy. Yurogen’s expertise in antibody development and engineering, combined with the powerful SMAB? platform, has allowed the development of a potent bispecific antibody that offers fresh insights into antibody design and could provide a blueprint for future anticancer therapeutic strategies.

Involving the Host Immune System

The initial binding of the T cell receptor with a corresponding peptide-MHC complex is crucial to the specificity of an immune response, but that response relies heavily on co-stimulatory molecules to generate robust and long-lived humoral and cell-mediated immunity. CD40 is one such co-stimulatory molecule expressed on the surface of antigen-presenting cells (APCs). A member of the tumor necrosis factor (TNF) superfamily, CD40 is a characteristic marker of many APCs, including B cells, macrophages, and dendritic cells (DCs). The ligand of CD40, termed CD40 ligand (CD40L) or CD154, is expressed by activated T cells to interact with CD40 and drive subsequent immune functions against specific antigens, pathogens, and other foreign objects. The CD40-CD40L interaction is critical to proper immunity, and emulating this interaction via anti-CD40 antibodies can induce DC maturation and increase tumor immunity.

?Despite encouraging preclinical results, limited anti-tumor efficacy was observed in cancer patients during clinical trials of anti-CD40 monoclonal antibody treatments, which stalled the exploration of this seemingly logical therapeutic avenue. Yurogen sought to engineer an anti-human CD40 agonist rabbit monoclonal antibody, and to combine immune cell activation via CD40 with a targeted cancer therapy against HER2-positive tumors to enhance therapeutic efficacy. The principle is to bring APCs into proximity of tumors, priming them to subsequently activate the host immune system to specifically seek out and destroy the tumors.

Building an Effective Anti-CD40 Antibody

The first step was to establish a robust anti-human CD40 (hCD40) rabbit monoclonal antibody using SMAB? technology. Rabbits were immunized with a specially designed hCD40 antigen formulation to maximize immunogenicity and titer. After a suitable immunization period and having assessed bleed samples (Figure [KL1]?1A), the rabbits were sacrificed and single-sorted B cells were cultured in Yurogen media for 10 to 14 days. The ex vivo B cell cultures were screened by high throughput ELISA, and positive B cell supernatants were cherry picked and tested against Daudi B cells by flow cytometry (Figure 1B).

Next, a HEK-Blue CD40L reporter assay (Invivogen) was performed to assess the ability of each of the best candidate anti-hCD40 antibody clones in their agonistic activities (Figure 2[KL1]?). From this assay, the best clones (1A8, 8G3, and 9C1) were incubated with human PBMCs to assess agonist function (Figure 3[KL2]?[KL3]?). Gating on CD19-positive B cells, it was demonstrated [KL4]?[KL5]?that the selected clones could significantly induce expression of the coreceptor CD86 in MHC class II-positive cells, confirming the agonist effect of the anti-hCD40 antibodies. The best clone, 9C1, was selected and carried over to the bispecific antibody engineering project.

Constructing a Robust Bispecific Antibody

The best-performing anti-hCD40 rabbit mAb clone was first humanized and subsequently combined with trastuzumab, the prototypical anti-HER2 mAb, to craft a therapeutic bispecific antibody. Various formats were employed to generate candidate antibodies with both the anti-hCD40 antigen-binding domain and well as the anti-HER2 binding domain from trastuzumab (Figure [KL1]?4A), and the candidates that could be efficiently expressed and purified were tested for binding to CD40 (Figure 4B) and HER2 (Figure 4C).

The best bispecific antibody candidates from the binding screen were carried forward to determine their binding effectiveness on live cells by flow cytometry (Figure 5A[KL1]?). The candidates that could effectively bind both CD40 expressing cells (such as the Raji B cell line) and HER2 expressing cancer cells (SK-BR-3 and SK-OV-3) were CD40xHER2-8 and CD40xHER2-9. These two candidates were then tested for cytotoxicity against SK-BR-3 cell line in the presence of human PBMCs by lactate dehydrogenase release using commercially available LDH cytotoxicity detection kits (Figure 5B). Both candidates were able to induce cytotoxicity to a much greater extent than the anti-hCD40 clone or trastuzumab alone, demonstrating a successful development of a CD40-HER2 bispecific therapeutic.

The Future of Bispecific Therapeutics

The successful development of the agonistic rabbit monoclonal antibodies against human CD40 as well as the subsequent engineering of an effective CD40-HER2 bispecific therapeutic antibody demonstrates the potential of activating the host immune system against tumors through intelligent antibody design. Leveraging additional immune and tumor targets can offer new avenues for cancer immunotherapy. These innovative antibody molecules represent a more precise and effective treatment option to provide more personalized medicine and combination therapy strategies. While this is merely the first step in demonstrating the potential efficacy of such a therapeutic antibody, there is cause for much excitement as future studies work towards understanding the functional mechanisms and assessing safety in human patients.

With a decade of experience in the development of more than 3,000 monoclonal antibodies targeting diverse targets，Yurogen has broad experiences in the design, development, and engineering of optimal antibody reagents. Based on the CD40-HER2 case study discussed in this article, Yurogen can produce robust bispecific and multispecific candidate antibodies that can be used for the desired workflow and project as well as downstream applications. Employing our innovative design process and the powerful SMAB? platform, Yurogen boasts extraordinary success for obtaining the desired monoclonal antibodies that can be further engineered to improve or modify antigen-specific binding and immune effects for downstream applications.

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