GMP development and preclinical validation of CAR-T cells targeting a lytic EBV antigen for therapy of EBV-associated malignancies

Xi Zhang; Tiaoxia Wang; Xiaona Zhu; Yong Lu; Mingpeng Li; Zhihong Huang; Deping Han; Longzhen Zhang; Yang Wu; Liantao Li; Frank Klawonn; Renata Stripecke

doi:10.3389/fimmu.2023.1103695

GMP development and preclinical validation of CAR-T cells targeting a lytic EBV antigen for therapy of EBV-associated malignancies

Front Immunol. 2023 Feb 2:14:1103695. doi: 10.3389/fimmu.2023.1103695. eCollection 2023.

Authors

Xi Zhang¹, Tiaoxia Wang¹, Xiaona Zhu¹, Yong Lu¹, Mingpeng Li¹, Zhihong Huang¹, Deping Han¹, Longzhen Zhang², Yang Wu², Liantao Li², Frank Klawonn^{3

4

5}, Renata Stripecke^{5

6

7

8}

Affiliations

¹ Biosyngen/Zelltechs Pte. Ltd., Singapore, Singapore.
² Department of Radiotherapy, the Affiliated Hospital of Xuzhou Medical University, Xuzhou, China.
³ Biostatistics Group, Helmholtz Centre for Infection Research, Braunschweig, Germany.
⁴ Institute for Information Engineering, Ostfalia University, Wolfenbuettel, Germany.
⁵ German Centre for Infection Research (DZIF), Partner Site Hannover-Braunschweig and Partner Site Cologne-Bonn, Cologne, Hannover, Germany.
⁶ Laboratory of Regenerative Immune Therapies Applied, Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany.
⁷ Clinic I for Internal Medicine, University Hospital Cologne, University of Cologne, Cologne, Germany.
⁸ Institute for Translational Immune-Oncology, Cancer Research Center Cologne-Essen (CCCE), University of Cologne, Cologne, Germany.

Abstract

Introduction: Epstein-Barr virus (EBV) is a widely spread pathogen associated with lymphoproliferative diseases, B/ T/ NK cell lymphomas, nasopharyngeal carcinoma (NPC) and gastric carcinoma (GC). EBV lytic reactivations contribute to the genomic instability, inflammation and tumorigenesis of NPC, promoting cancer progression. Patients with NPC refractory to standard therapies show dismal survival. EBV gp350 is an envelope protein detectable in NPC specimens intracellularly and on the cell membrane of malignant cells, and is a potential viral antigen for T cell-directed immunotherapies. The potency of T cells engineered with a chimeric antigen receptor (CAR) targeting gp350 against EBV⁺ lymphoproliferative disease was previously shown.

Methods: Here, we advanced towards preclinical and non-clinical developments of this virus-specific CAR-T cell immunotherapy against NPC. Different gp350CAR designs were inserted into a lentiviral vector (LV) backbone.

Results: A construct expressing the scFv 7A1-anti-gp350 incorporating the CD8 transmembrane and CD28.CD3ζ signaling domain (ZT002) was selected. High titer ZT002 (~1x10⁸ TU/ml) was manufactured in HEK 293T/17 suspension cells in serum free media as large-scale production under good manufacturing practices (GMP). A LV multiplicity of infection (MOI) of 1 resulted in high frequencies of functional gp350CAR⁺ T cells (>70%) at a low (<2) vector copy numbers in the genome. ZT002 was therefore used to establish gp350CAR-T batch run production methods. GMP upscaling and validation of T cell transduction and expansion in several runs resulted in average 3x10⁹ gp350CAR-T cells per batch. >80% CD3⁺ gp350CAR-T cells bound to purified gp350 protein. In vitro cytotoxicity and cytokine secretion assays (IFN-γ and TNF-α) confirmed the specificity of gp350CAR-T cells against gp350⁺ NPC, GC and lymphoma cell targets. Immunocompromised B-NDG mice (NOD.CB17-PrkdcscidIl2rgtm1/Bcgen) were challenged s.c. with a EBV⁺ NPC C666.1 cell line expressing gp350 and then treated with escalating doses of gp350CAR-T cells or with non-transduced T cells. gp350CAR-T cells promoted antitumor responses, bio-distributed in several tissues, infiltrated in tumors and rejected gp350⁺ tumor cells.

Discussion: These results support the use of gp350CAR-T cells generated with ZT002 as an Innovative New Drug to treat patients with solid and liquid EBV-associated malignancies.

Keywords: CAR-T cell; EBV; GMP; gastric carcinoma; gp350; lymphoma; nasopharyngeal carcinoma.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Epstein-Barr Virus Infections*
Herpesvirus 4, Human
Mice
Mice, Inbred NOD
Nasopharyngeal Carcinoma
Nasopharyngeal Neoplasms*
Receptors, Chimeric Antigen / immunology
T-Lymphocytes

Substances

Receptors, Chimeric Antigen

Grants and funding

Upscaling and GMP adaptation for clinical development of gp350CAR-T cells and preclinical testing was financed by Biosyngen/ Zelltechs Pte. Ltd. Academic development and preclinical testing of additional CAR-T cells in mouse models in R.S.’s laboratories is ongoing and financed by grants of the German Center for Infections Research (DZIF-TTU07.912 to RS), by the German Cancer Aid (Deutsche Krebshilfe Nr. 70114234 to RS), by the Jackson Laboratory (JAX, USA, grant HLA-LV) and by the Cancer Research Center Cologne-Essen (CCCE), University of Cologne, Cologne, Germany.