A new immunotherapy strategy targeted CD30 in peripheral T-cell lymphomas: CAR-modified T-cell therapy based on CD30 mAb

Abstract

Chimeric antigen receptor T-cell immunotherapy (CAR-T) has shown remarkable efficacy in treating tumors of lymphopoietic origin. Herein, we demonstrate an effective CAR-T cell treatment for recurrent and malignant CD30-positive peripheral T-cell lymphomas (PTCL) has been demonstrated. The extracellular fragment gene sequences of CD30 were obtained from tumor tissues of PTCL patients and cloned into a plasmid vector to express the CD30 antigen. The CD30 targeting single-chain antibody fragment (scFv) was obtained from CD30-positive monoclonal hybridoma cells, which were obtained from CD30 antigen immunized mice. After a second-generation of CAR lentiviral construction, CD30 CAR T cells were produced and used to determine the cytotoxicity of this construct toward Karpas 299 cells. The results of CD30 CAR T-mediated cell lysis show that 9C11-2 CAR T cells could significantly promote the lysis of CD30-positive Karpas 299 cells in both LDH and real-time cell electronic sensing (RTCA) assays. In vivo data show that 9C11-2 CAR T cells effectively suppress the tumor growth in a Karpas 299 cell xenograft NCG mouse model. The CD30 CAR T cells exhibited an efficient cytotoxic effect after being co-cultured with the target cells and they also exhibited a significant tumor-inhibiting ability after being intravenously injected into PTCL xenograft tumors; these observations suggest that the new CD30 CAR-T cell may be a promising therapeutic candidate for cancer therapy.

Fig. 1. Construction and expression of the recombinant CD30 antigen protein.

A Immunohistochemical assay of a CD30-positive biopsy sample (left CD30⁻, right CD30⁺); B gel electrophoresis assay of the recombinant pET28a-CD30 construct (1, 2) plasmid, C the coomassie blue staining of the CD30 antigen; (1. Negative control without IPTG, 2. Negative control with IPTG, 3. pET28a-CD30 without IPTG, 4. pET28a-CD30 with IPTG), D Western blot assay of CD30 antigen (1. Negative control with IPTG, 2. pET28a-CD30 without IPTG, 3. pET28a-CD30 with IPTG).

Fig. 2. Characterization of CD30-specific monoclonal antibody.

A Flow cytometry assay the specificity of CD30 antibody from 6B1 and 9C11 monoclonal hybridoma cells, B immunofluoresence assay the specificity of CD30 antibody at 20× amplification, C Western blot assay the specificity of CD30 antibody (9C11, middle line), Abcam CD 30 antibody (up line) and GAPDH (bottom line) used as a control, D, E immunohistochemical assay the specificity of CD30 antibody.

Fig. 3. Synthesized of CD30 CAR and prepared CD30 CAR T cell.

A Gel electrophoresis assay of VH and VL genes from monoclonal hybridoma cells, B restriction enzyme analysis of the CD30-specific recombinant plasmid, C the diagram of CD30-specific recombinant plasmid. D Generation and proliferation of CD30 CAR T cells (9C11-2 CAR-T) and flow cytometer assay of the CD30 expression rate of 9C11-2 CAR T cells over 14 days, E fluorescence microscopy monitoring of CD30 CAR T cells, F, G CD3, CD4, CD8, and CD30 expression rates of CD30 CAR T cells over 14 days.

Fig. 4. In vitro cytotoxicity of CD30 CAR-T cells.

The LDH assay of cytotoxicity of CD30 CAR T cells to Karpas 299 (A CD30⁺, **p < 0.01 vs. NC CART), SU-DHL-1 (B CD30⁺, **p < 0^.01 vs. NC CART) and Jurkat (C CD30⁻); D the RTCA assay of the impedance of Karpas 299 cells, the reduction in impedance of Karpas 299 cells means more cells were killed by CD30 CAR T cells at E:T of 20:1, E the cytokine release of CD30 CAR T cells measured by Luminex 3D, F flow cytometry was used to evaluate the apoptosis rate of Karpas 299 cells, **p < 0.01 vs. NC CART.

Fig. 5. In vivo xenograft tumor suppression by CD30 CAR T cells.

A The average tumor dimensions were monitor after intravenously injected with CD30 CAR T cells, NC CAR T cells, or saline at the day of 1 (arrow show, **p < 0.01 vs. saline), B the imaging of tumor tissue treated with CD30 CAR T (bottom line), NC CAR T (middle line), and saline (up to line) at 30 days, C the tumor weight at 30 days, **p < 0.01 vs. saline, D the survival rate of xenograft tumor-bearing mice, **p < 0.01 vs. saline, immunohistochemical analysis of cytokine levels in tumor sections at day 30, GM-CSF (E), TNF-α (F), and INF-γ (G) expression in tumors is shown as percentages of positive area.

Fig. 6. Immunohistochemical and tunnel assay of tumor tissue.

Tumors were collected from Kappas-299 xenograft mice treated with saline (top), NC CAR T (middle) or CD30 CAR T (bottom). Formalin-fixed, paraffin-embedded tumor sections were consecutively cut and stained. (bar of G-CSF, TNF-α, INF-γ is 50 μm, a bar of the tunnel is 20 μm).