CrossMab technology is an innovative method for generating bispecific antibodies that ensures the correct pairing of antibody light and heavy chains, thereby improving the production efficiency, stability, developability and functionality of bispecific antibodies. CrossMab technology was invented by researchers from Roche in 2011. It is based on the Knobs-into-holes (KiH) technology, and achieves the cross-linking of light and heavy chains by designing KiH heterodimeric connections in the Fc region and exchanging the CH1 and CL domains in the Fab region. This design principle applies not only to heterodimeric antibodies (1+1 format), but also to monovalent antibodies (MonoMab), bivalent antibodies (DuoMab) and multispecific antibodies with additional or tandem Fab formats.
Fig.1 Schematic diagram of CrossMab technology (Schaefer,2011)
Since the invention of CrossMab technology, it has developed into one of the most mature, versatile and widely used technologies in the field of bispecific antibodies in the past decade. As of mid-2021, at least 19 bispecific antibodies and fusion proteins based on CrossMab technology developed by Roche and other companies have entered clinical trials, of which 16 are still being evaluated in ongoing clinical trials. Among them, the most advanced ones are the Ang-2/VEGF bispecific antibody faricimab, which is currently undergoing regulatory review, and the CD20/CD3 T cell-directed bispecific antibody glofitamab, which is currently in a pivotal phase 3 trial. CrossMab technology has shown a wide range of application potential and excellent clinical performance in tumor treatment, HIV-1 prevention and treatment, autoimmune diseases, ocular angiogenic diseases and other fields.
The basic principle of CrossMab technology is to use Knobs-into-holes (KiH) technology and Fab domain exchange technology to achieve the correct pairing of antibody light and heavy chains and the formation of heterodimeric antibodies. KiH technology is to design a protrusion (knob) and a groove (hole) on the heavy chains of the antibody Fc region, so that two different heavy chains can bind together through knob-hole interaction, forming a heterodimeric antibody. Fab domain exchange technology is to swap the CH1 and CL domains of two different antibody molecules, so that each Fab fragment is composed of CH1 and CL from the same antibody molecule, thereby avoiding the problem of light chain mismatch. By combining these two technologies, CrossMab technology can produce bispecific antibodies with two different antigen binding sites.
CrossMab technology has high flexibility and versatility, and can be used to construct various forms and functions of bispecific or multispecific antibodies. For example, by fusing an additional Fab fragment on each heavy chain, trivalent (2+1) or tetravalent (2+2) bispecific antibodies can be produced; by linking two Fab fragments together, multispecific antibodies with three or four antigen binding sites can be produced; by connecting an Fc fragment or a fusion protein with a Fab fragment, monovalent (MonoMab) or bivalent (DuoMab) bispecific antibodies can be produced. These different forms and functions of bispecific or multispecific antibodies can be used to achieve various therapeutic strategies, such as dual checkpoint inhibition, T cell redirection, cytokine neutralization, receptor activation or blockade, and others.
The generation methods of CrossMab technology are to use mammalian cell culture and in vitro assembly to produce bispecific or multispecific antibodies. Specifically, the production process of CrossMab technology includes the following steps:
The production process of CrossMab technology also requires strict bioanalysis and quality control methods to ensure the quality and safety of bispecific or multispecific antibodies. These methods include but are not limited to the following:
The application of CrossMab technology in cancer treatment is to use bispecific or multispecific antibodies to target tumor-related targets, thereby achieving anti-tumor and immune modulation effects. There are many bispecific antibodies developed using CrossMab technology targeting tumor-related targets,such as VEGF, Ang-2, FAP, CD20, CD3.
VEGF and Ang-2 are two important angiogenic factors, which are related to tumor growth, invasion and metastasis in various solid tumors by their expression and secretion. Bispecific antibodies targeting VEGF/Ang-2 can simultaneously block these two factor signaling pathways, thereby inhibiting tumor angiogenesis and improving tumor microenvironment. The bispecific antibodies targeting VEGF/Ang-2 developed using CrossMab technology are vanucizumab (RG7221) and faricimab (RG7716). Vanucizumab is a bivalent (1+1) bispecific antibody that showed effective anti-tumor and anti-angiogenic effects in preclinical models, but failed to show significant advantages in clinical trials and has been discontinued. Faricimab is a trivalent (2+1) bispecific antibody that showed superior efficacy and safety than bevacizumab or abicipar monotherapy in clinical trials for the treatment of wet age-related macular degeneration (wAMD) and diabetic macular edema (DME) and other eye diseases. Faricimab is the most advanced bispecific antibody developed using CrossMab technology and has submitted regulatory review applications.
FAP is a fibroblast activation protein that is highly expressed in most solid tumors and almost not expressed in normal tissues. TNFRSF10B is a receptor that induces cell apoptosis and is widely expressed on tumor cells. Bispecific antibodies targeting FAP/TNFRSF10B can use FAP as a conditional target to deliver TNFRSF10B activators to tumor cells, thereby inducing tumor cell apoptosis. The bispecific antibody targeting FAP/TNFRSF10B developed using CrossMab technology is RG7386, which is a tetravalent (2+2) bispecific antibody that effectively triggered FAP-dependent, affinity-driven TNFRSF10B oligomerization and subsequent tumor cell apoptosis in preclinical models, but after completing phase 1 study, RG7383's clinical development was discontinued due to portfolio reprioritization.
CD20 and BCMA are two molecules that are highly expressed on malignant B cells, while CD3ε is an essential signaling molecule on T cells. T cell-directed bispecific antibodies targeting CD20/CD3ε or BCMA/CD3ε etc. can physically link T cells with malignant B cells and activate T cells to kill malignant B cells. The T cell-directed bispecific antibodies developed using CrossMab technology include cibisatamab (RG7802), glofitamab (RG6026), CC-93269, TYRP1-TCB (RG6232), etc., which are all trivalent (2+1) bispecific antibodies and show good safety and efficacy in clinical trials. Among them, glofitamab is the most advanced T cell-directed bispecific antibody developed using CrossMab technology and has entered pivotal phase 3 trials.
Target | Drug name | Developer/Institution | Clinical stage | Indication | Country/Region |
---|---|---|---|---|---|
Ang-2/VEGF | faricimab | Roche/Genentech | Phase 3 (completed) | Wet age-related macular degeneration, diabetic macular edema, diabetic retinopathy | Global |
CD20/CD3 | glofitamab | Roche/Genentech | Phase 3 (ongoing) | Relapsed/refractory B-cell non-Hodgkin lymphoma | Global |
CD20/CD3 | mosecabtagene autoleucel (mosunetuzumab) | Roche/Genentech | Phase 2 (ongoing) | Relapsed/refractory B-cell non-Hodgkin lymphoma | Global |
MUC16/CD3 | cevostamab (BAY 1834942) | Bayer/ImmunoGen | Phase 1 (ongoing) | Relapsed/refractory ovarian cancer, endometrial cancer, non-small cell lung cancer | USA, Europe |
MUC16/CD3 | murlentamab (ABTL0812) | Ability Pharma/Merck KGaA/Pierre Fabre/Servier/Celgene/BMS/Syndax Pharmaceuticals/Oncolytics Biotech Inc. | Phase 1 (ongoing) | Relapsed/refractory ovarian cancer, endometrial cancer, non-small cell lung cancer | USA, Europe |
Moreover, at present, two bispecific antibodies based on CrossMab technology have been approved by the regulatory agency for marketing. On October 15, 2021, the U.S. Food and Drug Administration (FDA) approved faricimab for the treatment of wet age-related macular degeneration (wet AMD) and diabetic retinopathy (DR). Bispecific antibodies for disease. On November 5, 2021, the European Medicines Agency (EMA) approved glofitamab for the treatment of relapsed or refractory B-cell non-Hodgkin's lymphoma (B-NHL), which is the first dual drug for this type of cancer. specific antibody.
References
1. Klein C, et al. Progress in overcoming the chain association issue in bispecific heterodimeric IgG antibodies. MAbs. 2012;4(6):653-663.
2. Surowka M, et al. Ten years in the making: application of CrossMab technology for the development of therapeutic bispecific antibodies and antibody fusion proteins. MAbs. 2021 Jan-Dec;13(1):1967714.
3. Klein C, et al. The use of CrossMAb technology for the generation of bi- and multispecific antibodies. MAbs. 2016 Aug-Sep;8(6):1010-20.
4. Regula JT, et al. Targeting key angiogenic pathways with a bispecific CrossMAb optimized for neovascular eye diseases. EMBO Mol Med. 2016 Nov 2;8(11):1265-1288.
5. Dumontet C, et al. Engineering therapeutic bispecific antibodies using CrossMab technology. Methods. 2019 Feb 1;154:21-31.
6. Qian W, et al. Combating non-Hodgkin lymphoma by targeting both CD20 and HLA-DR through CD20-243 CrossMab. MAbs. 2014 May-Jun;6(3):740-8.
7. Briguet A, et al. Detailed Analytical Characterization of a Bispecific IgG1 CrossMab Antibody of the Knob-into-Hole Format Applying Various Stress Conditions Revealed Pronounced Stability. ACS Omega. 2022 Jan 19;7(4):3671-3679.
8. Kienast Y, et al. Ang-2-VEGF-A CrossMab, a novel bispecific human IgG1 antibody blocking VEGF-A and Ang-2 functions simultaneously, mediates potent antitumor, antiangiogenic, and antimetastatic efficacy. Clin Cancer Res. 2013 Dec 15;19(24):6730-40.
9. Mueller T, et al. Efficacy of a Bispecific Antibody Co-Targeting VEGFA and Ang-2 in Combination with Chemotherapy in a Chemoresistant Colorectal Carcinoma Xenograft Model. Molecules. 2019 Aug 7;24(16):2865
10. Hartmann G. Targeting key angiogenic pathways with a bispecific CrossMAb optimized for neovascular eye diseases. EMBO Mol Med. 2019 May;11(5):e10666.
11. Schaefer, Wolfgang, et al. "Immunoglobulin domain crossover as a generic approach for the production of bispecific IgG antibodies." Proceedings of the National academy of Sciences 108.27 (2011): 11187-11192.
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