Many methods have been developed to produce bispecific antibodies (BsAbs) for industrial application. However, huge challenges still remain in synthesizing whole length BsAbs, including their assembly, stability, immunogenicity, and pharmacodynamics. Here we present for first time a generic technology platform of generating bispecific IgG antibodies, “Bispecific Antibody by Protein Trans-splicing (BAPTS)”. Different from published methods, we assembled two parental antibody fragments in the hinge region by the protein trans-splicing reaction of a split intein to generate BsAbs without heavy/heavy and light/heavy chain mispairing. Utilizing this simple and efficient approach, there have been several BsAbs (CD3×HER2, CD3×EGFR, EGFR×HER2) synthesized to demonstrate its broad applicability. Correctly paired mAb arms were assembled to form BsAbs that were purified through protein A affinity chromatography to demonstrate industrial applicability at large scale. Further, the products were characterized through physical-biochemistry properties and biological activities to confirm expected quality of the products from “BAPTS”. More importantly, correct pairing was confirmed by mass spectrum. Proof-of-concept studies with CD3×HER2 BsAb (T-cell recruitment) demonstrated superior bioactivity compared with trastuzumab. The results of undetectable mispairing and high biological activity have indicated that this method has the potential to be utilized to manufacture BsAbs with high efficiency at industrial scale.

Background: Prolactin receptor (PRLR) is highly expressed in a subset of human breast cancer and prostate cancer, which makes it a potential target for cancer treatment. In clinical trials, the blockade of PRLR was shown to be safe but with poor efficacy. It is therefore urgent to develop new therapies against PRLR target. Bispecific antibodies (BsAbs) could guide immune cells toward tumor cells, and produced remarkable effects in some cancers. Methods: In this study, a bispecific antibody targeting both tumor antigen PRLR and T cell surface CD3 antigen (PRLR-DbsAb) was constructed by split intein mediated protein transsplicing (BAPTS) system for the first time. Its binding activity was determined by Biacore and Flow cytometry, and target-dependent T cell mediated cytotoxicity was detected using LDH release assay. ELISA was utilized to study the secretion of cytokines by immune cells. Subcutaneous tumor mouse models were used to analyze the in vivo anti-tumor effects of PRLR-DbsAb. Results: PRLR-DbsAb in vitro could recruit and activate T cells to promote the release of Th1 cytokines IFN-γ and TNF-α, which could kill PRLR expressed breast cancer cells. In xenograft models with breast cancer cell line T47D, NOD/SCID mice intraperitoneally injected with PRLR-DbsAb exhibited significant inhibition of tumor growth and a longer survival compared to mice treated with PRLR monoclonal antibody (PRLR mAb). Conclusions: Both in vitro and in vivo experiments showed PRLR-DbsAb had a potential therapy of cancer treatment potential therapy for cancer. Immunotherapy may be a promising treatment against the tumor target of PRLR.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) continue to wreak havoc across the globe. Higher transmissibility and immunologic resistance of VOCs bring unprecedented challenges to epidemic extinguishment. Here we describe a monoclonal antibody, 2G1, that neutralizes all current VOCs and has surprising tolerance to mutations adjacent to or within its interaction epitope. Cryo-electron microscopy structure showed that 2G1 bound to the tip of receptor binding domain (RBD) of spike protein with small contact interface but strong hydrophobic effect, which resulted in nanomolar to sub-nanomolar affinities to spike proteins. The epitope of 2G1 on RBD partially overlaps with angiotensin converting enzyme 2 (ACE2) interface, which enables 2G1 to block interaction between RBD and ACE2. The narrow binding epitope but high affinity bestow outstanding therapeutic efficacy upon 2G1 that neutralized VOCs with sub-nanomolar half maximal inhibitory concentration in vitro. In SARS-CoV-2, Beta or Delta variant-challenged transgenic mice and rhesus macaque models, 2G1 protected animals from clinical illness and eliminated viral burden, without serious impact to animal safety. Mutagenesis experiments suggest that 2G1 is potentially capable of dealing with emerging SARS-CoV-2 variants in the future. This report characterized the therapeutic antibodies specific to the tip of spike against SARS-CoV-2 variants and highlights the potential clinical applications as well as for developing vaccine and cocktail therapy.

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