(T1130-04-20) Development of CD40 and HER2 Bispecific Antibodies for Enhanced Efficacy against HER2-Positive Tumors

Purpose: CD40, a member of the tumor necrosis factor superfamily, is primarily expressed on antigen-presenting cells. This receptor plays a crucial role in facilitating the interaction between dendritic cells, activating antigen-specific T cells. Agonist antibodies targeting the CD40 receptor have the potential to mimic the natural CD40 ligand, fostering dendritic cell maturation and stimulating immunity against "cold" tumors. Although this approach has shown promise in preclinical studies, translating it to clinical success has been challenging. Only modest anti-tumor activity was observed in cancer patients, and none of the developed CD40 monoclonal antibodies (mAbs) progressed beyond early clinical trials, highlighting a need for improved anti-tumor efficacy.

Methods: In this study, we successfully developed an anti-human CD40 agonist rabbit monoclonal antibody using Yurogen's proprietary SMABTM platform. The most potent CD40 mAb clone, was combined with trastuzumab (an anti-HER2 mAb) to create a therapeutic bispecific antibody. The objective is to leverage immune cell activation through HER2-targeted cancer therapy to achieve enhanced anti-tumor activity.

Results: A variety of CD40xHER2 bispecific antibody designs were explored, with careful evaluation of expression yield, purity, and binding profiles for each target respectively. A few bispecific antibody designs demonstrated robust binding to both HER2 and CD40 antigen proteins, as well as receptor-expressing cell lines. Notably, two developed bispecific antibodies exhibited potent killing of HER2-positive cancer cells with the assistance of innate immune cells.

Conclusion: In sum, this study provides novel insights into designing bispecific antibodies against HER2 and CD40, offering a promising avenue for advancing cancer immunotherapy.

References: [1]David M. Richards, Julian P. et al. Concepts for agonistic targeting of CD40 in immuno-oncology, Human Vaccines & Immunotherapeutics, 16:2, 377-387 (2020).
[2] Huang, Y. et al. Engineered bispecific antibodies with exquisite HIV-1-neutralizing activity. Cell 165, 1621–1631 (2016).
Jason Allen, Michelle Gonzalez.et al. Development of Fully Human, Bispecific Antibodies that Effectively Block Omicron Variant Pseudovirus Infections.bioRxiv 2023.03.07.531527
[3]Anna Sall.(2022).NOVEL POLYPEPTIDES.United States Patent and Trademark Office.US2022064325
[4] Lingqiao Zhu.(2015).Recombinant anti-HER2/PS bispecific antibody as well as preparation method and application thereof. China National Intellectual Property Administration.CN201410129991.3.
[5]PU PU.(2022).ANTI-BCMA×CD3 bispecific antibody and use thereof. world intellectual property organization. WO2022135468.
Pengfei Zhou.(2015).Construction and application of bispecific antibody CD20*CD3. China National Intellectual Property Administration. CN104558191 (A).
[6]Bacac Marina.(2017). Type II anti-Cd20 antibody and anti-Cd20/Cd3 bispecific antibody for treatment of cancer. European Patent Application.EP3252078 (A1).
[7]Yang Xiao.(2021).Anti-CD47/anti-PD-L1 antibody and application thereof. China National Intellectual Property Administration.CN113321734 (A).
[8]Xiangdong Qu.(2021).TAA/CTLA-4/IL15 three-function fusion protein and application thereof .China National Intellectual Property


Figure 2. (A) hCD40 immunized rabbit bleed sample evaluation by ELISA and flow cytometry. (B) hCD40-specific rabbit B cells were cultured in Yurogen media for 10-14 days and screened via HT ELISA. Positive B cell supernatants were cherry-picked and tested against the Daudi B cell line by flow cytometry .


Figure 4. Agonist functional characterization of rabbit anti-CD40 mAbs (clone 1A8, 8G3, and 9C1). human PBMCs were incubated with rabbit anti-CD40 agonist mAbs, negative control anti-BTA rabbit mAb, and the ligand of CD40 receptor (CD40L) at various concentrations. The MHCII+ CD86+ cell subpopulations were quantified.


Figure 5. (A) A panel of different bi-specific mAbs were designed based on the proprietary CD40 mAb and HER2 specific mAb trastuzumab . Some bi-specific mAbs were excluded from the downstream analysis due to suboptimal expression yield or purification challenges. The remaining 12 bi-specific mAbs were subject to the in vitro CD40 binding assay (B) as well as HER2 binding assay (C).