Facile Generation of Potent Bispecific Fab via Sortase A and Click Chemistry for Cancer Immunotherapy

Abstract

Bispecific antibodies (BsAbs) for T cell engagement have shown great promise in cancer immunotherapy, and their clinical applications have been proven in treating hematological malignance. Bispecific antibody binding fragment (BiFab) represents a promising platform for generating non-Fc bispecific antibodies. However, the generation of BiFab is still challenging, especially by means of chemical conjugation. More conjugation strategies, e.g., enzymatic conjugation and modular BiFab preparation, are needed to improve the robustness and flexibility of BiFab preparation. We successfully used chemo-enzymatic conjugation approach to generate bispecific antibody (i.e., BiFab) with Fabs from full-length antibodies. Paired click handles (e.g., N₃ and DBCO) was introduced to the C-terminal LPETG tag of Fabs via sortase A mediated transpeptidation, followed by site-specific conjugation between two click handle-modified Fabs for BiFab generation. Both BiFab^CD20/CD3 (EC₅₀ = 0.26 ng/mL) and BiFab^Her2/CD3 exhibited superior efficacy in mediating T cells, from either PBMC or ATC, to kill target tumor cell lines while spared antigen-negative tumor cells in vitro. The BiFab^CD20/CD3 also efficiently inhibited CD20-positive tumor growth in mouse xenograft model. We have established a facile sortase A-mediated click handle installation to generate homogeneous and functional BiFabs. The exemplary BiFabs against different targets showed superior efficacy in redirecting and activating T cells to specifically kill target tumor cells, demonstrating the robustness of sortase A-mediated "bio-click" chemistry in generating various potent BiFabs. This approach also holds promise for further efficient construction of a Fab derivative library for personalized tumor immunotherapy in the future.

Keywords: BiFab; Fab; anti-CD20 antibody; bispecific antibody; chemo-enzymatic approach; sortase A.

Figure 1

Generation and characterization of BiFabs. (a) Schematic diagram of sortase A-mediated click chemistry installation for BiFab preparation. (b) Characterization of the purified Fabs by SDS-PAGE. Lane 1, high molecular weight protein marker; Lane 2, the reduced Fab^CD20; Lane 3, the intact Fab^CD20; Lane 3, the reduced Fab^CD3; Lane 4, the intact Fab^CD3. (c) Reverse-phase HPLC analysis of Fab-click handle conjugation through sortase A-mediated transpeptidation, under different reaction conditions. (d) Characterization of BiFabs by SDS-PAGE. Lane 1, high molecular weight protein marker; Lane 2, the reduced BiFab^Her2/CD3; Lane 3, the intact BiFab^Her2/CD3; Lane 4, the intact Fab^Her2; Lane 5, the intact Fab^CD3. (e) Reverse phase high-performance liquid chromatography (RP-HPLC) analysis of the purity of BiFab^CD20/CD3.

Figure 2

In vitro efficacy of BiFabs. (a) The binding abilities of Fabs and BiFab with CD20-positive Ramos and Jurkat cells. (b) The in vitro efficacy of the BiFab^CD20/CD3 on T cell activation. After CD20-positive B cell depletion, fresh PBMCs were treated with serial concentrations of BiFab^CD20/CD3 in the presence of target tumor cells at an E:T ratio of 5:1 for 48 h. The expression levels of CD69 and CD25 on T cells, two biomarkers for T cell activation, were evaluated after immuno-staining via flow cytometry. (c) Evaluation of the binding abilities of BiFab^Her2/CD3 with CD3-positive Jurkat cells and HER2-positive SK-OV-3 cells by flow cytometry. (d) The quantification of interferon-γ release from T cells activated by BiFab^CD20/CD3. Fresh PBMCs were incubated with Daudi or Raji cells at an E:T ratio for 5:1 for 48 h. The secreted interferon-γ from T cells was quantified by ELISA Kit. (e) BiFab^CD20/CD3 mediated T cell proliferation in the presence of CD20-negative K562 cells or CD20-positive Ramos cells at an E:T ratio of 5:1 for 48 h. (f) After treatment with various concentrations of BiFab^CD20/CD3 with an E:T ratio of 5:1 for 48 h, T cell proliferation was analyzed by flow cytometry.

Figure 3

The in vitro and in vivo antitumor activities of BiFabs. (a) The in vitro efficacy of BiFab^CD20/CD3. Target cells (Ramos and Daudi) and active T cells (E:T = 2:1) were incubated with serial diluted BiFab^CD20/CD3 for 24 h (data shown as mean ± SD, n = 3). LDH release was determined by ELSIA kit and used to calculate cell viability. (b) The in vitro cytotoxicity of BiFab^CD20/CD3 was analyzed by Annexin V/PI apoptosis detection kit, by using the same condition as described in (a). (c) Study on potential Fc-related cytotoxicity of BiFab^CD20/CD3. The K562 cells and PBMCs were co-cultured with serial concentrations of non-binding IgG-based bispecific antibody or BiFab. The apoptosis rate was determined by Annexin V-Cy5 Apoptosis Detection Kit. (d) The in vitro cytotoxicity of BiFab^Her2/CD3. Target tumor cells (SK-OV-3 or MDA-MB-468) and PBMC (E:T = 4:1) were incubated with serial concentrations of BiFab^Her2/CD3 for 72 h, and the LDH release in the supernatant was determined by LDH detection kit. All data were shown as mean ± SD, n = 3. (e) The in vivo antitumor activities of BiFab^CD20/CD3 in mouse xenograft model. Mice were inoculated subcutaneously with 2.5 × 10⁶ Ramos cells in the presence of 1 × 10⁷ fresh human PBMC from healthy donors at an E:T ratio of 4:1. All samples were administered intravenously via the tail vein at following dosages, 1 mg/kg of Fab^CD3 and 1 mg/kg or 3 mg/kg of BiFab^CD20/CD3 at every two days for four times.

Figure 4

Schematic diagram of modular BiFab generation. Fabs could be adapted from full-length IgGs targeting tumor antigens or T cell/NK cell activating receptors. Fabs are genetically modified to have a C-terminal sortase A recognition motif (e.g., LPETG). Then, the paired click chemistry could be installed to the Fabs via sortase A mediated transpeptidation, followed by click reaction between two Fabs to generate BiFab.