Preclinical characterization and comparison between CD3/CD19 bispecific and novel CD3/CD19/CD20 trispecific antibodies against B-cell acute lymphoblastic leukemia: targeted immunotherapy for acute lymphoblastic leukemia

doi:10.1007/s11684-021-0835-8

Front. Med.

2022, Vol. 16

Issue (1) : 139-149 https://doi.org/10.1007/s11684-021-0835-8

RESEARCH ARTICLE

Preclinical characterization and comparison between CD3/CD19 bispecific and novel CD3/CD19/CD20 trispecific antibodies against B-cell acute lymphoblastic leukemia: targeted immunotherapy for acute lymphoblastic leukemia

Sisi Wang^1,², Lijun Peng^1,², Wenqian Xu^1,², Yuebo Zhou^1,², Ziyan Zhu³, Yushan Kong⁴, Stewart Leung⁴, Jin Wang^1,²(

), Xiaoqiang Yan⁴(

), Jian-Qing Mi^1,²(

)

¹. Shanghai Institute of Hematology, State Key Laboratory of Medical Genomics, National Research Center for Translational Medicine at Shanghai, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
². Pôle Franco-Chinois de Recherche en Sciences du Vivant et Genomique, Shanghai 200025, China
³. Shanghai Blood Center, Shanghai 200051, China
⁴. Generon Biomed, Shanghai 201210, China

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Abstract

The CD19-targeting bispecific T-cell engager blinatumomab has shown remarkable efficacy in patients with relapsed/refractory B-cell precursor acute lymphoblastic leukemia. However, several studies showed that blinatumomab has a short plasma half-life due to its low molecular weight, and thus its clinical use is limited. Furthermore, multiple trials have shown that approximately 30% of blinatumomab-relapsed cases are characterized by CD19 negative leukemic cells. Here, we design and characterize two novel antibodies, A-319 and A-2019. Blinatumomab and A-319 are CD3/CD19 bispecific antibodies with different molecular sizes and structures, and A-2019 is a novel CD3/CD19/CD20 trispecific antibody with an additional anti-CD20 function. Our in vitro, ex vivo, and in vivo experiments demonstrated that A-319 and A-2019 are potent antitumor agents and capable of recruiting CD3 positive T cells, enhancing T-cell function, mediating B-cell depletion, and eventually inhibiting tumor growth in Raji xenograft models. The two molecules are complementary in terms of efficacy and specificity profile. The activity of A-319 demonstrated superior to that of A-2019, whereas A-2019 has an additional capability to target CD20 in cells missing CD19, suggesting its potential function against CD19 weak or negative CD20 positive leukemic cells.

Keywords B-cell acute lymphoblastic leukemia bispecific antibody trispecific antibody CD19 CD20

Corresponding Author(s): Jin Wang,Xiaoqiang Yan,Jian-Qing Mi

Just Accepted Date: 12 July 2021 Online First Date: 31 August 2021 Issue Date: 28 March 2022

Cite this article:

Sisi Wang,Lijun Peng,Wenqian Xu, et al. Preclinical characterization and comparison between CD3/CD19 bispecific and novel CD3/CD19/CD20 trispecific antibodies against B-cell acute lymphoblastic leukemia: targeted immunotherapy for acute lymphoblastic leukemia[J]. Front. Med., 2022, 16(1): 139-149.

URL:

https://academic.hep.com.cn/fmd/EN/10.1007/s11684-021-0835-8
https://academic.hep.com.cn/fmd/EN/Y2022/V16/I1/139

Fig.1 Schematic and purification of A-319 and A-2019. A-319 and A-2019 were produced by using expression vector-transfected 293 HEK cells and through purification from culture supernatants with antibody beads against constant heavy 1 (CH1) domain of Fab and SDS-PAGE analysis. (A) Structures of A-319 and A-2019. A-319 is composed of anti-CD3 scFv and anti-CD19 Fab fragment, and A-2019 is composed of anti-CD3 Fab fragment, anti-CD19 scFv, and anti-CD20 scFv. (B) SDS-PAGE and Coomassie blue staining of purified A-319 (left) and A-2019 (right). Nonreducing (nr) and reducing (r) SDS-PAGE of A-319 and A-2019 are shown.

Fig.2 Qualitative and quantitative analysis of ex vivo immune synapses of autologous B and T cells induced by A-319, A-2019, and no antibody. PBMCs from treatment-naïve B-ALL patients were cultured with either A-319 or A-2019 for 30 min at 37 °C. Cells were stained with labels against CD4 (red), CD8 (yellow), CD24 (purple), and F-actin (green) and B/T cell synapses were analyzed using imaging flow cytometry. (A) Representative images of B/T cell synapse (A-319, top panel and A-2019, middle panel) and non-synapse B/T cell doublets (No Ab, bottom panel) are shown. B/T cell synapses demonstrate fluorescence overlapping with CD24, F-actin, and CD4 (left) or CD8 (right) signals at 40× magnification. BF, bright-field. (B) Qualitative analysis of the immune synapse between autologous B and T cells induced by A-319 and A-2019. Percentage of CD4 or CD8 T cells involved in synapse formation with B cells are shown. Asterisks indicate statistical significance. * P<0.05.

Fig.3 A-319 and A-2019 mediated specific cytotoxicity in different cell lines in vitro. Redirected T cell cytotoxicity was assayed through flow cytometry using healthy donor PBMCs as effector cells and various cell lines as targets. Increasing concentrations of A-319 and A-2019 were incubated with CFSE-labeled target and effector cells in an E:T ratio of 10:1 for 24 h. (A–E) Percent of tumor target lysis in CD19⁻/CD20⁻ K562, CD19⁺/CD20⁺ Raji, CD19⁺/CD20⁻ NALM6, CD19⁺/CD20⁻ K562-CD19, and CD19⁻/CD20⁺ K562-CD20 cells were determined through flow cytometric analysis. (F) Culture supernatants were analyzed for perforin (left) and granzyme B (right) levels induced by A-319 and A-2019 after 24 h coculture of PBMC and Raji cells. Asterisks indicate statistical significance. * P<0.05; ** P<0.005; *** P<0.0005.

Tab.1 Quantification of CD19 and CD20 cell surface molecules of each cell line (K562, K562-CD19, K562-CD20, NALM6, and Raji) and the respective EC₅₀ value (pmol/L) of specific killing mediated by A-319 and A-2019

Fig.4 A-319 and A-2019 induced potent ex vivo T cell activation and proliferation, B cell depletion, and cytokine release in primary B-ALL patients. PBMCs from treatment-naïve B-ALL blood samples were isolated and cocultured with an increasing dose of A-319 or A-2019 at different time periods. Flow cytometry analysis was then performed for the assessment of the efficacy of the two antibodies. (A, B) Upregulation of CD69 and CD25 on CD4 and CD8 T cell induced by A-319 and A-2019 for early activation at 16 h (A) and intermediate activation at 24 h (B). (C) T cell proliferation induced by A-319 and A-2019 was demonstrated by the upregulation of Ki-67 of CD4 and CD8 T cells in a dose-dependent manner after 3 days of incubation. (D) B cell cytotoxicity mediated by A-319 and A-2019 in a dose-dependent manner after 3 days of incubation. (E) Supernatants of cell cultures were harvested after 3 days of incubation and levels of IL-6, IL-8, IL-10, IFN-α, and TNF-γ were measured with ELISA. Asterisks indicate statistical significance. * P<0.05; ** P<0.005. n.s., no significance.

Fig.5 In vivo antitumor activities of A-319 and A-2019 in the Raji xenograft models. The mice were injected with human PBMCs into the peritoneal cavity. Subcutaneous Raji tumor growth reached a volume of approximately 100 mm³ after 13 days. The mice were then allocated to the treatment groups and received daily treatment for 21 consecutive days with vehicle control PBST, A-319 (10 μg/kg), A-319 (50 μg/kg), A-2019 (50 μg/kg), or A-2019 (250 μg/kg) through intraperitoneal infusion. (A) RCBW of Raji xenografts in different treatment groups. (B) Tumor growth curve of each group. (C) TGI and P value of each group. (D) Percentages of human CD4 and CD8 T cells in peripheral blood lymphocytes of Raji xenografts showed the huPBMC reconstitution of each mouse.

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