Autocrine-based selection of ligands for personalized CAR-T therapy of lymphoma

Abstract

We report the development of a novel platform to enhance the efficacy and safety of follicular lymphoma (FL) treatment. Since lymphoma is a clonal malignancy of a diversity system, every tumor has a different antibody on its cell surface. Combinatorial autocrine-based selection is used to rapidly identify specific ligands for these B cell receptors on the surface of FL tumor cells. The selected ligands are used in a chimeric antigen receptor T cell (CAR-T) format for redirection of human cytotoxic T lymphocytes. Essentially, the format is the inverse of the usual CAR-T protocol. Instead of being a guide molecule, the antibody itself is the target. Thus, these studies raise the possibility of personalized treatment of lymphomas using a private antibody binding ligand that can be obtained in a few weeks.

Fig. 1. Workflow for selection of ligands for the personalized FL–CAR-T therapy.

A lymph node biopsy sample from a patient with FL is isolated, and the collected tumor cells are used for identification of the malignant BCR genes, after which they are reconstituted as a membrane-bound BCR using platelet-derived growth factor receptor (PDGFR) as a membrane anchor. The reconstituted malignant BCR, coexpressed with the cyclopeptide-CAR library on the surface of the Jurkat cell line, is used as a reporter-cell system for selection of the tumor cell–targeting ligand. Following several rounds of panning, the selected peptide ligands, fused to the CAR, are sequenced and may be immediately used for generation of the therapeutic T lymphocytes modified by tumor-specific CAR. V_H, variable region of Ig heavy chain; V_L, variable region of Ig light chain.

Fig. 2. Autocrine-based selection of malignant FL-BCR ligands.

(A) The reporter system format. PMA, phorbol 12-myristate 13-acetate. TM, transmembrane. (B) Verification of the reporter cell system by Myc-CAR/anti-Myc antibody pair interaction. (C) Patient BCR-specific peptides on CAR activate reporter Jurkat cells transduced by membrane-tethered FL BCRs.

Fig. 3. The selected peptide ligands specifically interact with the FL-BCRs and redirect CTLs to kill tumor cells.

(A) Surface staining of Raji cells transduced with lymphoma BCR scFv by synthetic biotinylated peptides and antibody against IgG Fc. For IgG Fc staining, the same Raji cell population flow cytometry result was used as control in the three histograms. (B) FL–CAR-Ts were cocultured with Raji cells transduced with different lymphoma BCRs. Mock-transduced T cells and CD19–CAR-Ts were used as a comparison. Cytotoxicity was determined by measuring LDH release after 6 hours. (C) Cells from the patient’s biopsy or control B cells were stained with the synthetic biotinylated FL1 peptide. The B cell population was identified by a B220-specific antibody, and the FL1 peptide was labeled with biotin and detected with fluorescein isothiocyanate (FITC)–labeled streptavidin. (D) Lysis of B cells derived from the lymphoma biopsy sample by FL1–CAR-Ts compared to Myc–CAR-Ts and Mock-transduced T cells.

Fig. 4. CTLs redirected by FL1-CAR suppress lymphomagenesis in vivo.

(A) Experimental design indicating the engraftment of NOD SCID mice with 5 × 10⁶ Raji-FL1 cells. At day 15, animals (12 per group) were randomized according to the tumor volume and intravenously received 3 × 10⁶ FL1–CAR-Ts, CD19–CAR-T, or Myc–CAR-Ts per mouse at day 17. (B) Transduction efficacy of activated, CD3/CD28 bead-expanded human CD8⁺ T cells with lentiviral-based vectors expressing FL1–CAR-T, Myc–CAR-T, and CD19–CAR-T constructs. Cells were stained with an IgG1-specific antibody or protein L. (C) Survival of Raji-FL xenografted mice treated on day 17 after tumor initiation with 3 × 10⁶ CTLs (n = 12 mice per group). Overall survival curves were plotted using the Kaplan-Meier method and compared using the log-rank (Mantel-Cox) test (*P < 0.01). (D) Tumor growth curve in groups of mice (n = 12) treated with 3 × 10⁶ FL1–CAR-Ts, CD19–CAR-Ts, or Myc–CAR-Ts administered intravenously on day 17 after injection of Raji-FL1 (**P < 0.05, unpaired t test). Absolute counts of adoptively transferred modified T cells were monitored in blood obtained from retro-orbital puncture using flow cytometry analysis with a CD3⁺-specific antibody (inset). (E) Flow cytometry analysis of the phenotype of FL1–CAR-Ts before injection and on day 21 following the injection. (F) Relative percentages of naïve, central memory (CM), and effector memory (EM) CAR-Ts on day 21 following the injection.