Production Of Bispecific Antibodies Research Articles

Construction of bispecific antibodies by specific pairing between the heavy chain and the light chain using removable SpyCatcher/SnoopCatcher units

During the production of bispecific antibodies (bsAbs), nonspecific pairing results in low yields of target bsAb molecules, an issue known as the “mispairing problem.” Several antibody engineering techniques have been developed to overcome mispairing issues. Here, we introduce “bsAb by external pairing and excision” (BAPE), a novel chain pairing method that induces specific chain pairing by fusing external SpyCatcher/Tag and SnoopCatcher/Tag units. These tags are then removed via protease cleavage. In this study, we applied this method to force the correct pairings of heavy and light chains while the heavy-chain pairing was achieved by the Knobs-into-Holes mutation. We then confirmed the formation of interchain bridges with covalent isopeptide bonds. Both anti-CD3/anti-Her2 and anti-CD3/anti-EGFR bsAbs displayed satisfactory target binding activities and in vitro cell-killing activity with activated T-cells.

Macrophage-engaging peptidic bispecific antibodies (pBsAbs) for immunotherapy via a facile bioconjugation strategy.

Bispecific antibodies are artificial molecules that fuse two different antigen-binding sites of monoclonal antibodies into one single entity. They have emerged as a promising next-generation anticancer treatment. Despite the fascinating applications of bispecific antibodies, the design and production of bispecific antibodies remain tedious and challenging, leading to a long R&D process and high production costs. We herein report an unprecedented strategy to cyclise and conjugate tumour-targeting peptides on the surface of a monoclonal antibody to form a novel type of bispecific antibody, namely the peptidic bispecific antibody (pBsAb). Such design combines the merits of highly specific monoclonal antibodies and serum-stable cyclic peptides that endows an additional tumour-targeting ability to the monoclonal antibody for binding with two different antigens. Our results show that the novel pBsAb, which comprises EGFR-binding cyclic peptides and an anti-SIRP-α monoclonal antibody, could serve as a macrophage-engaging bispecific antibody to initiate enhanced macrophage-cancer cell interaction and block the "don't eat me" signal between CD47-SIRP-α, as well as promoting antibody-dependent cellular phagocytosis and 3D cell spheroid infiltration. These findings give rise to a new type of bispecific antibody and a new platform for the rapid generation of new bispecific antibodies for research and potential therapeutic uses.

Generation of Bispecific Antibodies by Functionalized Poly-ADP-Ribose Polymers.

Bispecific antibodies have drawn considerate research interests for therapeutic development. Numerous genetic and chemical methods are established to produce bispecific antibodies with varied formats. This protocol describes a novel approach to the synthesis of bispecific antibodies by utilizing chemically functionalized poly-ADP-ribose polymers derived from post-translational poly-ADP-ribosylation. Basic Protocol 1 includes experimental procedures for expressing and purifying recombinant full-length human poly-ADP-ribose polymerase 1 (PARP1) as well as monoclonal antibodies targeting T-cell CD3 and breast cancer tumor-associated human epidermal growth factor receptor 2 (HER2) molecules. Basic Protocol 2 details methods for enzymatic preparation of functionalized poly-ADP-ribose polymers by PARP1 and chemical conjugation of antibody molecules for bispecific antibody production. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Expression and purification of PARP1 and antibodies Basic Protocol 2: PARP1 auto-modification and antibody conjugation.

Abstract 5658: MG-Ig (molecularly grafted immunoglobulin): An alternative platform for production of bispecific antibodies

Abstract The generation of bispecific molecules, in which two target-specific moieties are engineered into a single entity, offers an attractive approach to optimize biological activity which may improve clinical outcomes. Among various formats of bispecific molecules, the heterodimeric Fc currently is a favorable scaffold for the design of bispecific antibodies because of its structural similarity to the natural antibody. However, development of molecules based on this concept is challenged by the presence of potential homodimer contamination, reduced yield, and stability loss relative to the natural Fc. Here, we report the development of the MG-Ig platform by grafting a single chain fragment variable (scFv) onto the hinge region of a full-size immunoglobulin with a different specificity to produce a trivalent IgG-like bispecific antibody that retains natural homodimer Fc. Whereas the MG-Ig platform is evolved from the Dock-and-Lock (DNL) technology which exploits the interaction between the anchoring domain (AD) of A kinase anchor proteins and the dimerization and docking domain (DDD) of regulatory subunit of cAMP-dependent protein kinase, the main distinctions include 1) the AD-fused scFv monomer is judiciously designed to graft onto the DDD dimer inserted into the hinge region of IgG, forming a bispecific antibody with three (1+2) binding arms and 2) all modular subunits are expressed in a single host cell and self-assemble to generate the bioactive conjugate in situ. To validate the method, bispecific T-cell engagers, designated 3scFv × hL0125C (CD3 × Trop2) and 3scFv × hACD20C (CD3 × CD20), which redirect T cells to Trop2 and CD20, respectively, have been produced with high yield and purity. Flow cytometry analysis demonstrated the bispecific binding of 3scFv × hL0125C to both CD3 on Jurkat and Trop2 on ME-180 with unaltered affinities. These T-cell redirecting bispecific antibodies mediated dose-dependent killing of target cancer cells when co-incubated with human PBMCs. Based on the 60-h viability assays using MTS, the IC50 of 3scFv × hL0125C was about 6 pM for MDA-MB-468, 13 pM for MDA-MB-231, 20 pM for BT-474, and 18 pM for HCT116. In 3D culture of HCT116 cells, the formation of multicellular tumor spheroids was impeded by 3scFv × hL0125C at 19.5 pM or higher, which is consistent with the cytotoxicity assays for tumor cells grown in monolayer. In addition, 3scFv × hL0125C was stable in human plasma for 7 days at 37°C, retaining bispecific integrity and binding activity. These data are promising and merit further development of MG-Ig bispecific antibodies for preclinical and clinical studies. Citation Format: Donglin Liu, Li Zeng, Berenice E. Liu, Chien-Hsing Chang. MG-Ig (molecularly grafted immunoglobulin): An alternative platform for production of bispecific antibodies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5658.

Enabling the next steps in cancer immunotherapy: from antibody-based bispecifics to multispecifics, with an evolving role for bioconjugation chemistry.

In the past two decades, immunotherapy has established itself as one of the leading strategies for cancer treatment, as illustrated by the exponentially growing number of related clinical trials. This trend was, in part, prompted by the clinical success of both immune checkpoint modulation and immune cell engagement, to restore and/or stimulate the patient's immune system's ability to fight the disease. These strategies were sustained by progress in bispecific antibody production. However, despite the decisive progress made in the treatment of cancer, toxicity and resistance are still observed in some cases. In this review, we initially provide an overview of the monoclonal and bispecific antibodies developed with the objective of restoring immune system functions to treat cancer (cancer immunotherapy), through immune checkpoint modulation, immune cell engagement or a combination of both. Their production, design strategy and impact on the clinical trial landscape are also addressed. In the second part, the concept of multispecific antibody formats, notably MuTICEMs (Multispecific Targeted Immune Cell Engagers & Modulators), as a possible answer to current immunotherapy limitations is investigated. We believe it could be the next step to take for cancer immunotherapy research and expose why bioconjugation chemistry might play a key role in these future developments.

Rapid Production of Bispecific Antibodies from Off-the-Shelf IgGs with High Yield and Purity.

Bispecific antibodies (BsAb) refer to a class of biomacromolecules that are capable of binding two antigens or epitopes simultaneously. This can elicit unique biological effects that cannot be achieved with either individual antibody or two unlinked antibodies. Bispecific antibodies have been used for targeting effector cells to tumor cells, preferential targeting of cells expressing two target biomarkers over cells expressing either target biomarker individually, or to couple two molecular targets on the same cell surface to trigger unique intracellular signaling pathways. Here, we present two related methods that enable direct, rapid assembly of bispecific antibodies from any two "off-the-shelf" Immunoglobulin G (IgG) antibodies, in as little as 1 day. Both workflows can be summarized into two steps: (1) attach a small photoreactive antibody binding domain (pAbBD) fused to SpyCatcher or SpyTag (peptide-protein partners derived from the S. pyogenes fibronectin-binding protein FbaB) to each component IgG, respectively; (2) assemble the BsAb through the spontaneous isopeptide bond formation that occurs between SpyTag and SpyCatcher. These approaches enable production of BsAbs from any two IgG molecules without the need to elucidate their amino acid sequences or genetically alter their structure. Binding assays and T cell-mediated cytolysis assays were performed to validate the binding and functional properties of Trastuzumab × Cetuximab BsAb and Cetuximab × OKT3 BsAb, respectively. This approach enables rapid, low-cost production of highly homogeneous tetravalent BsAbs in a modular fashion, presenting an opportunity to quickly evaluate antibody pairs in a BsAb format for unique or synergistic functionalities.

Development of a recombinant anti-VEGFR2-EPCAM bispecific antibody to improve antiangiogenic efficiency

Tumor progression and metastasis, especially in invasive cancers (such as triple-negative breast cancer [TNBC]), depend on angiogenesis, in which vascular epithelial growth factor (VEGF)/vascular epithelial growth factor receptor [1] has a decisive role, followed by the metastatic spread of cancer cells. Although some studies have shown that anti-VEGFR2/VEGF monoclonal antibodies demonstrated favorable results in the clinic, this approach is not efficient, and further investigations are needed to improve the quality of cancer treatment. Besides, the increased expression of epithelial cell adhesion molecule (EpCAM) in various cancers, for instance, invasive breast cancer, contributes to angiogenesis, facilitating the migration of tumor cells to other parts of the body. Thus, the main goal of our study was to target either VEGFR2 or EpCAM as pivotal players in the progression of angiogenesis in breast cancer. Regarding cancer therapy, the production of bispecific antibodies is easier and more cost-effective compared to monoclonal antibodies, targeting more than one antigen or receptor; for this reason, we produced a recombinant antibody to target cells expressing EpCAM and VEGFR2 via a bispecific antibody to decrease the proliferation and metastasis of tumor cells. Following the cloning and expression of our desired anti-VEGFR2/EPCAM sequence in E. coli, the accuracy of the expression was confirmed by Western blot analysis, and its binding activities to VEGFR2 and EPCAM on MDA-MB-231 and MCF-7 cell lines were respectively indicated by flow cytometry. Then, its anti-proliferative potential was indicated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and apoptosis assay to evaluate inhibitory effects of the antibody on tumor cells. Subsequently, the data indicated that migration, invasion, and angiogenesis were inhibited in breast cancer cell lines via the bispecific antibody. Furthermore, cytokine analysis indicated that the bispecific antibody could moderate interleukin 8 (IL-8) and IL-6 as key mediators in angiogenesis progression in breast cancer. Thus, our bispecific antibody could be considered as a promising candidate tool to decrease angiogenesis in TNBC.

Characterization of Bispecific Antibody Production in Cell Cultures by Unique Mixed Mode Size Exclusion Chromatography.

Bispecific antibodies have received wide attention as promising immunotherapeutic agents because of their high specificity and the ability to target immune cells to tumors. However, analysis of bispecific antibodies is challenging because multiple forms of antibodies are potentially generated during production in cell culture. Most analyses of bispecific antibodies rely on liquid chromatography with mass spectrometry (LC-MS), which could miss detection or becomes less quantitative if those forms are not physically separated. Here, we report a novel and sensitive mixed mode size exclusion chromatography (MM SEC) coupled with multiangle light scattering (MALS) to analyze different forms of bispecific IgG molecules under native conditions. The method displayed great ability to separate various antibody forms with peak resolutions unmatched by other methods we tested, isolating desired bispecific molecules, parental homodimers, half molecules, and antibodies with mispaired light and heavy chains. Each peak was analyzed by online MALS and then identified and confirmed by intact and reduced LC-MS of isolated forms. MM SEC in this study performs by a novel mechanism through the interactions of resin with protein surface hydrophobic clusters distributed across CDRs of light chains. This novel MM SEC allows quantitative detection of even low abundance forms and provides a new tool for screening expression profiles of cell culture clones, monitoring purification, and evaluating drug substance purity.

High-throughput antibody screening from complex matrices using intact protein electrospray mass spectrometry

Toward the goal of increasing the throughput of high-resolution mass characterization of intact antibodies, we developed a RapidFire-mass spectrometry (MS) assay using electrospray ionization. We achieved unprecedented screening throughput as fast as 15 s/sample, which is an order of magnitude improvement over conventional liquid chromatography (LC)-MS approaches. The screening enabled intact mass determination as accurate as 7 ppm with baseline resolution at the glycoform level for intact antibodies. We utilized this assay to characterize and perform relative quantitation of antibody species from 248 samples of 62 different cell line clones at four time points in 2 h using RapidFire-time-of-flight MS screening. The screening enabled selection of clones with the highest purity of bispecific antibody production and the results significantly correlated with conventional LC-MS results. In addition, analyzing antibodies from a complex plasma sample using affinity-RapidFire-MS was also demonstrated and qualified. In summary, the platform affords high-throughput analyses of antibodies, including bispecific antibodies and potential mispaired side products, in cell culture media, or other complex matrices.

Tailoring translational strength using Kozak sequence variants improves bispecific antibody assembly and reduces product‐related impurities in CHO cells

Optimal production of bispecific antibodies (bsAb) requires efficient and tailored co-expression and assembly of two distinct heavy and two distinct light chains. Here, we describe a novel technology to modulate the translational strength of antibody chains via Kozak sequence variants to produce bsAb in a single cell line. In this study, we designed and screened a large Kozak sequence library to identify 10 independent variants that can modulate protein expression levels from approximately 0.2 to 1.3-fold compared with the wild-type sequence in transient transfection. We used a combination of several of these variants, covering a wide range of translational strength, to develop stable single cell Chinese hamster ovary bispecific cell lines and compared the results with those obtained from the wild-type sequence. A significant increase in bispecific antibody assembly with a concomitant reduction in the level of product-related impurities was observed. Our findings suggest that for production of bsAbit can be advantageous to modify translational strength for selected protein chains to improve overall yield and product quality. By extension, tuning of translational strength can also be applied to improving the production of a wide variety of heterologous proteins.

Design and Production of Bispecific Antibodies.

With the current biotherapeutic market dominated by antibody molecules, bispecific antibodies represent a key component of the next-generation of antibody therapy. Bispecific antibodies can target two different antigens at the same time, such as simultaneously binding tumor cell receptors and recruiting cytotoxic immune cells. Structural diversity has been fast-growing in the bispecific antibody field, creating a plethora of novel bispecific antibody scaffolds, which provide great functional variety. Two common formats of bispecific antibodies on the market are the single-chain variable fragment (scFv)-based (no Fc fragment) antibody and the full-length IgG-like asymmetric antibody. Unlike the conventional monoclonal antibodies, great production challenges with respect to the quantity, quality, and stability of bispecific antibodies have hampered their wider clinical application and acceptance. In this review, we focus on these two major bispecific types and describe recent advances in the design, production, and quality of these molecules, which will enable this important class of biologics to reach their therapeutic potential.

Monitoring removal of hole-hole homodimer by analytical hydrophobic interaction chromatography in purifying a bispecific antibody

Despite the use of knobs-into-holes (KiH) strategy to promote heterodimerization in recombinant production of bispecific antibody (bsAb), homodimer (especially the hole-hole homodimer) can still be generated in small amount. This by-product needs to be removed by downstream process. However, as homodimer and the target bsAb are usually very close in size, these two species may not be readily differentiated using size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC). Thus, method other than SEC-HPLC needs to be developed to monitor removal of this by-product. Here, through a case study we demonstrate that analytical hydrophobic interaction chromatography (HIC) is a powerful tool for quantitatively monitoring removal of hole-hole homodimer in bsAb purification.

Effect of allotypic variation of human IgG1 on the thermal stability of disulfide-linked knobs-into-holes mutants of the Fc for stable bispecific antibody design.

BackgroundDisulfide-linked knobs-into-holes (dKiH) mutation is a well-validated antibody engineering technique to force heterodimer formation of different Fcs for efficient production of bispecific antibodies. An artificial disulfide bond is created between mutated cysteine residues in CH3 domain of human IgG1 Fc whose positions are 354 of the “knob” and 349 of the “hole” heavy chains. The disulfide bond is located adjacent to the exposed loop with allotypic variations at positions 356 and 358. Effects of the variation on the biophysical property of the Fc protein with dKiH mutations have not been reported.MethodsWe produced dKiH Fc proteins of high purity by affinity-tag fusion to the hole chain and IdeS treatment, which enabled removal of mispaired side products. Thermal stability was analyzed in a differential scanning calorimetry instrument.ResultsWe firstly analyzed the effect of the difference in allotypes of the Fcs on the thermal stability of the heterodimeric Fc. We observed different melting profiles of the two allotypes (G1m1 and nG1m1) showing slightly higher melting temperature of G1m1 than nG1m1. Additionally, we showed different characteristics among heterodimers with different combinations of the allotypes in knob and hole chains.ConclusionAllotypic variations affected melting profiles of dKiH Fc proteins possibly with larger contribution of variations adjacent to the disulfide linkage.

"BIClonals": Production of Bispecific Antibodies in IgG Format in Transiently Transfected Mammalian Cells.

Bispecific antibodies (bsAbs) are antibodies with two binding sites directed at different antigens, enabling therapeutic strategies not possible with conventional monoclonal antibodies (mAbs). Since bispecific antibodies are regarded as promising therapeutic agents, many different bispecific design modalities have been evaluated. Many of these are based on antibody fragments or on inclusion of non-antibody components. For some therapeutic applications, full-size, native IgG-like bsAbs may be the optimal format.To prepare bsAbs in IgG format, two challenges should be met. One is that each heavy chain will only pair with the heavy chain of the second specificity and that heavy chain homodimerization will be prevented. The second is that each heavy chain will only pair with the light chain of its own specificity and that pairing with the light chain of the second specificity will be prevented. The first solution to the first criterion (known as knobs into holes, KIH) was presented in 1996 by Genentech and additional solutions were presented more recently. However, until recently, out of >120 published formats, only a handful of solutions for the second criterion that make it possible to produce a bispecific IgG by a single expressing cell were suggested.Here, we present a protocol for preparing bsAbs in IgG format in transfected mammalian cells. For heavy chain dimerization we use KIH while as a solution for the second challenge-correct pairing of heavy and light chains of bispecific IgGs we present our "BIClonals" technology; an engineered (artificial) disulfide bond between the antibodies' variable domains that asymmetrically replaces the natural disulfide bond between CH1 and CL.During our studies of bsAbs we found that H-L chain pairing seems to be driven by VH-VL interfacial interactions that differ between different antibodies; hence, there is no single optimal solution for effective and precise assembly of bispecific IgGs that suits every antibody sequence, making it necessary to carefully evaluate the optimal solution for each new antibody.

Variable heavy-variable light domain and Fab-arm CrossMabs with charged residue exchanges to enforce correct light chain assembly.

Technologies for the production of bispecific antibodies need to overcome two major challenges. The first one is correct heavy chain assembly, which was solved by knobs-into-holes technology or charge interactions in the CH3 domains. The second challenge is correct light chain assembly. This can be solved by engineering the Fab-arm interfaces or applying the immunoglobulin domain crossover approach. There are three different crossovers possible, namely Fab-arm, constant domain and variable domain crossovers. The CrossMabCH1–CL exchange does not lead to the formation of unexpected side products, whereas the CrossMabFab and the CrossMabVH–VL formats result in the formation of typical side products. Thus, CrossMabCH1–CL was initially favored for therapeutic antibody development. Here, we report a novel improved CrossMab design principle making use of site-specific positional exchanges of charged amino acid pairs in the constant domain of these CrossMabs to enable the correct light chain assembly in the CrossMabVH–VL and improvements for the CrossMabFab design.

A HER2 bispecific antibody can be efficiently expressed in Escherichia coli with potent cytotoxicity.

Bispecific antibodies have been actively studied for cancer therapy due to their potent cytotoxicity against tumor cells. A number of bispecific antibody formats have exhibited strong tumor cytotoxicity in vitro and in vivo. However, effective production of bispecific antibodies remains challenging for the majority of bispecific antibody formats. In the present study, a bispecific antibody was designed that links a conventional antigen-binding fragment (Fab) against cluster of differentiation 3 antigen (CD3) to a camel single domain antibody (VHH) against human epidermal growth factor receptor 2 (HER2). This bispecific antibody may be secreted and purified efficiently from Escherichia coli culture medium. The purified bispecific antibody is able to trigger T cell-mediated HER2-specific cytotoxicity in vitro and in vivo. The data gathered in the present study suggest that this bispecific format may be applied to other tumor antigens to produce bispecific antibodies more efficiently.

Public antibodies to malaria antigens generated by two LAIR1 insertion modalities.

We previously described two donors in whom the extracellular domain of LAIR1, a collagen-binding inhibitory receptor encoded on chromosome 191, was inserted between the V and the DJ segments of an antibody. This insertion generated, through somatic mutations, broadly reactive antibodies against RIFINs, a type of variant antigen expressed on the surface of Plasmodium falciparum-infected erythrocytes (IEs)2. To investigate how frequently such antibodies are produced in response to malaria infection, we screened plasma from two large cohorts of individuals living in malaria-endemic regions. We report that 5-10% of malaria-exposed individuals, but none of the European blood donors tested, have high levels of LAIR1-containing antibodies that dominate the response to IEs without conferring enhanced protection against febrile malaria. By analyzing the antibody-producing B cell clones at the protein, cDNA and gDNA level, we characterized additional LAIR1 insertions between the V and DJ segments and discovered a second insertion modality whereby the LAIR1 exon encoding the extracellular domain and flanking intronic sequences are inserted into the switch region. By exon shuffling, this mechanism leads to the production of bispecific antibodies in which the LAIR1 domain is precisely positioned at the elbow between the VH and CH1 domains. Additionally, in one donor the gDNA encoding the VH and CH1 domains was deleted, leading to the production of a camel-like LAIR1-containing antibody. Sequencing of the switch regions of memory B cells from European blood donors revealed frequent templated inserts originating from transcribed genes that, in rare cases, comprised exons with orientation and frame compatible with expression. Collectively, these results reveal different modalities of LAIR1 insertion that lead to public and dominant antibodies against IEs and suggest that insertion of templated DNA represents an additional mechanism of antibody diversification that can be selected in the immune response against pathogens and exploited for B cell engineering.

Body, Make Thy Own Bispecific Antibody.

Synthetic mRNA that's been optimized, modified, and formulated to spur the production of bispecific antibodies in the liver can eradicate large tumors in mice. The strategy circumvents some of the manufacturing challenges associated with conventional antibody production and creates a therapeutic that's easier and more convenient to administer.

Asymmetric Fc Engineering for Bispecific Antibodies with Reduced Effector Function.

Asymmetric bispecific antibodies are a rapidly expanding therapeutic antibody class, designed to recognize two different target epitopes concurrently to achieve novel functions not available with normal antibodies. Many therapeutic designs require antibodies with reduced or silenced effector function. Although many solutions have been described in the literature to knockout effector function, to date all of them have involved the use of a specific antibody subtype (e.g., IgG2 or IgG4), or symmetric mutations in the lower hinge or CH2 domain of traditional homodimeric monospecific antibodies. In the context of a heterodimeric Fc, we describe novel asymmetric Fc mutations with reduced or silenced effector function in this article. These heteromultimeric designs contain asymmetric charged mutations in the lower hinge and the CH2 domain of the Fc. Surface plasmon resonance showed that the designed mutations display much reduced binding to all of the Fc gamma receptors and C1q. Ex vivo ADCC and CDC assays showed a consistent reduction in activity. Differential scanning calorimetry showed increased thermal stability for some of the designs. Finally, the asymmetric nature of the introduced charged mutations allowed for separation of homodimeric impurities by ion exchange chromatography, providing, as an added benefit, a purification strategy for the production of bispecific antibodies with reduced or silenced effector function.

Structural basis of a novel heterodimeric Fc for bispecific antibody production.

Bispecific antibodies provide an efficient tool for combinational clinical therapy. Here we have engineered a heterodimeric Fc for bispecific antibodies production by combining the knob-into-hole and electrostatic steering strategies where a bulky hydrophobic residue Phe405 of the IgG CH3 interface is mutated to a charged residue Lys and Lys409 of the corresponding CH3 domain is mutated to Ala. The crystal structure of this Fc heterodimer solved here at 2.7Å resolution revealed how these two mutations resulted a complementary binding interface and explained why F405K mutation could effectively inhibit Fc homodimer formation during protein expression. An anti-HER2 bispecific antibody derived from trastuzumab and pertuzumab was generated by this heterodimeric Fc. It showed comparable or improved efficacy than the combination of trastuzumab and pertuzumab in inhibiting proliferation of cancer cells in vitro and in vivo. Overall this study shows that the heterodimeric Fc engineered here provides an efficient platform for generating active bispecific antibody for cancer treatment.