A novel bispecific antibody drug conjugate targeting HER2 and HER3 with potent therapeutic efficacy against breast cancer.

Antibody drug conjugate (ADC) therapy has become one of the most promising approaches in cancer immunotherapy. Bispecific targeting could enhance the efficacy and safety of ADC by improving its specificity, affinity and internalization. In this study we constructed a HER2/HER3-targeting bispecific ADC (BsADC) and characterized its physiochemical properties, target specificity and internalization in vitro, and assessed its anti-tumor activities in breast cancer cell lines and in animal models. The HER2/HER3-targeting BsADC had a drug to antibody ratio (DAR) of 2.89, displayed a high selectivity against the target JIMT-1 breast cancer cells in vitro, as well as a slightly higher level of internalization than HER2- or HER3-monospecific ADCs. More importantly, the bispecific ADC potently inhibited the viability of MCF7, JIMT-1, BT474, BxPC-3 and SKOV-3 cancer cells in vitro. In JIMT-1 breast cancer xenograft mice, a single injection of bispecific ADC (3 mg/kg, i.v.) significantly inhibited the tumor growth with an efficacy comparable to that caused by combined injection of HER2 and HER3-monospecific ADCs (3 mg/kg for each). Our study demonstrates that the bispecific ADC concept can be applied to development of more potent new cancer therapeutics than the monospecific ADCs.

Re-Engineering Therapeutic Anti-Aß Monoclonal Antibody to Target Amyloid Light Chain.

Amyloidosis involves the deposition of misfolded proteins. Even though it is caused by different pathogenic mechanisms, in aggregate, it shares similar features. Here, we tested and confirmed a hypothesis that an amyloid antibody can be engineered by a few mutations to target a different species. Amyloid light chain (AL) and ß-amyloid peptide (Aß) are two therapeutic targets that are implicated in amyloid light chain amyloidosis and Alzheimer's disease, respectively. Though crenezumab, an anti-Aß antibody, is currently unsuccessful, we chose it as a model to computationally design and prepare crenezumab variants, aiming to discover a novel antibody with high affinity to AL fibrils and to establish a technology platform for repurposing amyloid monoclonal antibodies. We successfully re-engineered crenezumab to bind both Aß42 oligomers and AL fibrils with high binding affinities. It is capable of reversing Aß42-oligomers-induced cytotoxicity, decreasing the formation of AL fibrils, and alleviating AL-fibrils-induced cytotoxicity in vitro. Our research demonstrated that an amyloid antibody could be engineered by a few mutations to bind new amyloid sequences, providing an efficient way to reposition a therapeutic antibody to target different amyloid diseases.

Characterization of a novel bispecific antibody targeting tissue factor-positive tumors with T cell engagement.

T cell engaging bispecific antibody (TCB) is an effective immunotherapy for cancer treatment. Through co-targeting CD3 and tumor-associated antigen (TAA), TCB can redirect CD3+ T cells to eliminate tumor cells regardless of the specificity of T cell receptor. Tissue factor (TF) is a TAA that involved in tumor progression. Here, we designed and characterized a novel TCB targeting TF (TF-TCB) for the treatment of TF-positive tumors. In vitro, robust T cell activation, tumor cell lysis and T cell proliferation were induced by TF-TCB. The tumor cell lysis activity was dependent upon both CD3 and TF binding moieties of the TF-TCB, and was related to TF expression level of tumor cells. In vivo, in both tumor cell/human peripheral blood mononuclear cells (PBMC) co-grafting model and established tumor models with poor T cell infiltration, tumor growth was strongly inhibited by TF-TCB. T cell infiltration into tumors was induced during the treatment. Furthermore, efficacy of TF-TCB was further improved by combination with immune checkpoint inhibitors. For the first time, our results validated the feasibility of using TF as a target for TCB and highlighted the potential for TF-TCB to demonstrate efficacy in solid tumor treatment.

IgG-like Bispecific Antibody CD3×EpCAM Generated by Split Intein Against Colorectal Cancer.

Background: Colorectal cancer is a commonly diagnosed cancer with high mortality worldwide. Postoperative recidivation and metastasis still are the main challenges in clinical treatments. Thus, it is urgent to develop new therapies against colorectal cancer. Epithelial Cell Adhesion Molecule (EpCAM) is overexpressed in colorectal cancer cells and strongly associated with cancer development. Bispecific antibody (BsAb) is a kind of promising immunotherapy, which could recognize T cells and cancer cells simultaneously to achieve the anti-tumor effects. Methods: A bispecific antibody targeting EpCAM and CD3 with IgG format was genereated by split intein based on the Bispecific Antibody by Protein Splicing" platform. In vitro, the affinity of CD3×EpCAM BsAb was determined by Biolayer interferometry, its cytotoxicity was detected by LDH release assay, T cell recruitment and activation was detected by Flow Cytometry. In vivo, its pharmacokinetic parameters were detected, and anti-tumor effects were evaluated on the tumor cell xenograft mouse model. Results: The results showed that the CD3×EpCAM BsAb could activate and recruit T cells via binding colorectal cells and T cells, which could lead to more potent cytotoxicity to various colorectal cell lines than its parent EpCAM monoclonal antibody (mAb) in vitro. The CD3×EpCAM BsAb had similar pharmacokinetic parameters with EpCAM mAb and inhibits tumor growth on the SW480 tumor cell xenograft mouse model. Conclusion: The CD3×EpCAM BsAb could be a promising candidate for colorectal cancer treatment.

Kinetics study of the natural split Npu DnaE intein in the generation of bispecific IgG antibodies.

Rapid and efficient bispecific antibody (BsAb) production for industrial applications is still facing many challenges. We reported a technology platform for generating bispecific IgG antibodies, "Bispecific Antibody by Protein Trans-splicing (BAPTS)." While the "BAPTS" method has shown potential in high-throughput screening of BsAbs, further understanding and optimizing the methodology is desirable. A large number of BsAbs were selected to illustrate the conversion efficiency and kinetics parameters. The temperature of reaction makes no significant influence in conversion efficiency, which can reach more than 70% within 2 h, and CD3 × HER2 BsAb can reach 90%. By fitting trans-splicing reaction to single-component exponential decay curves, the apparent first-order rate constants at a series of temperatures were determined. The rate constant ranges from 0.02 to 0.11 min-1 at 37 °C, which is a high rate reported for the protein trans-splicing reaction (PTS). The reaction process is activated rapidly with activation energy of 8.9 kcal·mol-1 (CD3 × HER2) and 5.2 kcal·mol-1 (CD3 × EGFR). The BsAbs generated by "BAPTS" technology not only had the similar post-translation modifications to the parental antibodies, but also demonstrated excellent in vitro and in vivo bioactivity. The kinetics parameters and activation energy of the reaction illustrate feasible for high-throughput screening and industrial applications using the "BAPTS" approach. KEY POINTS: • The trans-splicing reaction of Npu DnaE intein in "BAPTS" platform is a rapid process with low reaction activation and high rate. • The BsAb generated by "BAPTS" remained effective in tumor cell killing. • The kinetics parameters and activation energy of the reaction illustrate feasible for high-throughput screening and industrial applications using the "BAPTS" approach.

A Novel Bispecific Antibody Targeting CD3 and Lewis Y with Potent Therapeutic Efficacy against Gastric Cancer.

Lewis Y antigen, a glycan highly expressed on most epithelial cancers, was targeted for cancer treatment but lacked satisfactory results in some intractable and refractory cancers. Thus, it is highly desirable to develop an effective therapy against these cancers, hopefully based on this target. In this work, we constructed a novel T cell-engaging bispecific antibody targeting Lewis Y and CD3 (m3s193 BsAb) with the IgG-[L]-scfv format. In vitro activity of m3s193 BsAb was evaluated by affinity assay to target cells, cytotoxicity assay, cytokines releasing assay, and T cells proliferation and recruiting assays. Anti-tumor activity against gastric cancer was evaluated in vivo by subcutaneous huPBMCs/tumor cells co-grafting model and huPBMCs intravenous injecting model. In vitro, m3s193 BsAb appeared to have a high binding affinity to Lewis Y positive cells and Jurkat cells. The BsAb showed stronger activity than its parent mAb in T cell recruiting, activation, proliferation, cytokine release, and cytotoxicity. In vivo, m3s193 BsAb not only demonstrated higher therapeutic efficacy in the huPBMCs/tumor co-grafting gastric carcinoma model than the parent mAb but also eliminated tumors in the model of intravenous injection with huPBMCs. Strong anti-tumor activity of m3s193 BsAb revealed that Lewis Y could be targeted in T cell-engaging BsAb for gastric cancer therapy.

A Rational Designed Novel Bispecific Antibody for the Treatment of GBM.

Epidermal growth factor receptor variant III (EGFRvIII) is highly and specifically expressed in a subset of lethal glioblastoma (GBM), making the receptor a unique therapeutic target for GBM. Recently, bispecific antibodies (BsAbs) have shown exciting clinical benefits in cancer immunotherapy. Here, we report remarkable results for GBM treatment with a BsAb constructed by the "BAPTS" method. The BsAb was characterized through LC/MS, SEC-HPLC, and SPR. Furthermore, the BsAb was evaluated in vitro for bioactivities through FACS, antigen-dependent T-cell-mediated cytotoxicity, and a cytokine secretion assay, as well as in vivo for antitumor activity and pharmacokinetic (PK) parameters through immunodeficient NOD/SCID and BALB/c mouse models. The results indicated that the EGFRvIII-BsAb eliminated EGFRvIII-positive GBM cells by recruiting and stimulating effector T cells secreting cytotoxic cytokines that killed GBM cells in vitro. The results demonstrated the antitumor potential and long circulation time of EGFRvIII-BsAb in NOD/SCID mice bearing de2-7 subcutaneously heterotopic transplantation tumors and BALB/c mice. In conclusion, our experiments in both in vitro and in vivo have shown the remarkable antitumor activities of EGFRvIII-BsAb, highlighting its potential in clinical applications for the treatment of GBM. Additional merits, including a long circulation time and low immunogenicity, have also made the novel BsAb a promising therapeutic candidate.

A novel bispecific antibody targeting CD3 and prolactin receptor (PRLR) against PRLR-expression breast cancer.

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.

Bioprocess development of a stable FUT8-/--CHO cell line to produce defucosylated anti-HER2 antibody.

In recent years, an increasing number of defucosylated therapeutic antibodies have been applied in clinical practices due to their better efficacy compared to fucosylated counterparts. The establishment of stable and clonal manufacturing cell lines is the basis of therapeutic antibodies production. Bioprocess development of a new cell line is necessary for its future applications in the biopharmaceutical industry. We engineered a stable cell line expressing defucosylated anti-HER2 antibody based on an established α-1,6-fucosyltransferase (FUT8) gene knockout CHO-S cell line. The optimization of medium and feed was evaluated in a small-scale culture system. Then the optimal medium and feed were scaled up in a bioreactor system. After fed-batch culture over 13 days, we evaluated the cell growth, antibody yield, glycan compositions and bioactivities. The production of anti-HER2 antibody from the FUT8 gene knockout CHO-S cells in the bioreactor increased by 37% compared to the shake flask system. The N-glycan profile of the produced antibody was consistent between the bioreactor and shake flask system. The antibody-dependent cellular cytotoxicity activity of the defucosylated antibody increased 14-fold compared to the wild-type antibody, which was the same as our previous results. The results of our bioprocess development demonstrated that the engineered cell line could be developed to a biopharmaceutical industrial cell line.

Enhanced production of anti-PD1 antibody in CHO cells through transient co-transfection with anti-apoptotic genes Bcl-x L and Mcl-1.

Apoptosis has a negative impact on the cell survival state during cell cultivation. To optimize mammalian cell culture for production of biopharmaceuticals, one of the important approaches is to extend cell life through over-expression of anti-apoptotic genes. Here, we reported a cost-effective process to enhance cell survival and production of an antibody through transient co-transfection with anti-apoptotic genes Bcl-x L or Mcl-1 in Chinese hamster ovary (CHO) cells with polyethylenimine (PEI). Under the optimal conditions, it showed reduced levels of apoptosis and improved cell viability after co-transfected with Bcl-x L or Mcl-1. The overall production yield of the antibody anti-PD1 increased approximately 82% in CHO cells co-transfected with Bcl-x L , and 34% in CHO cells co-transfected with Mcl-1. This work provides an effective way to increase viability of host cells through delaying apoptosis onset, thus, raise production yield of biopharmaceuticals without the process of generating stable cell lines and subsequent screening.

Efficient generation of bispecific IgG antibodies by split intein mediated protein trans-splicing system.

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.