PD-1 downregulation enhances CAR-T cell antitumor efficiency by preserving a cell memory phenotype and reducing exhaustion

Abstract

Background: Despite the encouraging outcome of chimeric antigen receptor T cell (CAR-T) targeting B cell maturation antigen (BCMA) in managing relapsed or refractory multiple myeloma (RRMM) patients, the therapeutic side effects and dysfunctions of CAR-T cells have limited the efficacy and clinical application of this promising approach.

Methods: In this study, we incorporated a short hairpin RNA cassette targeting PD-1 into a BCMA-CAR with an OX-40 costimulatory domain. The transduced PD-1^KD BCMA CAR-T cells were evaluated for surface CAR expression, T-cell proliferation, cytotoxicity, cytokine production, and subsets when they were exposed to a single or repetitive antigen stimulation. Safety and efficacy were initially observed in a phase I clinical trial for RRMM patients.

Results: Compared with parental BCMA CAR-T cells, PD-1^KD BCMA CAR-T cell therapy showed reduced T-cell exhaustion and increased percentage of memory T cells in vitro. Better antitumor activity in vivo was also observed in PD-1^KD BCMA CAR-T group. In the phase I clinical trial of the CAR-T cell therapy for seven RRMM patients, safety and efficacy were initially observed in all seven patients, including four patients (4/7, 57.1%) with at least one extramedullary site and four patients (4/7, 57.1%) with high-risk cytogenetics. The overall response rate was 85.7% (6/7). Four patients had a stringent complete response (sCR), one patient had a CR, one patient had a partial response, and one patient had stable disease. Safety profile was also observed in these patients, with an incidence of manageable mild to moderate cytokine release syndrome and without the occurrence of neurological toxicity.

Conclusions: Our study demonstrates a design concept of CAR-T cells independent of antigen specificity and provides an alternative approach for improving the efficacy of CAR-T cell therapy.

Keywords: Immunotherapy; Multiple Myeloma; Receptors, Chimeric Antigen.

Figure 1

Characterization of BCMA/PD-L1/PD-L2 expressions in the bone marrow and extramedullary disease sites of MM patients. (A) IHC staining of BCMA/PD-L1/PD-L2 expressions in patient tumor tissues. Representative images of extramedullary bone-related disease arising from the hand and extramedullary extraosseous disease arising from intracranial plasmacytoma. (B) Representative immunofluorescence images showing the expression of BCMA and PD-L1 and their colocalization in BM samples from RRMM patients. Scale bar, 20 µm. Flow cytometry assays showing expressions of PD-L1(C) and PD-L2 (D) on various cell types in BMMCs from seven RRMM patients. (E) Representative histograms of PD-L1(left) and PD-L2 (right) expression in BMMC samples from seven RRMM patients. (F) Representative results showing induced PD-L1 expression in BMMCs after 24 hours coculture or 48 hours of coculture with BCMA CAR-T cells (n=3 independent healthy donors). BCMA, B cell maturation antigen; EM-B, extramedullary bone-related; EM-E, extramedullary extraosseous; IHC, immunohistochemical; MM, multiple myeloma; RRMM, relapsed/refractory MM.

Figure 2

Characteristics of the PD-1^KD BCMA CAR-T cells. (A) Diagrams of the anti-BCMA CAR with and without shPD-1. (B) Expression of PD-1 in the CAR+cell population (9 days after virus infection) after 24 hours of coculture with 8226 cells. During coculture, shPD-1 significantly decreased the expression of PD-1 (p=0.0394 < 0.05). (C) Representative expression of PD-1 in the CAR+cell population (9 days after virus infection) determined by flow cytometry after 24 hours of coculture with 8226 cells. (D) Median fluorescence intensity (MFI) of PD-1 expression in CAR+cells detected after 24 hours of coculture with 8226 cells. CD3+ (untransfected T cells, control) or CD3+CAR+ cells were analyzed. Compared with that in BCMA CAR-T cells, shPD-1 significantly decreased the expression of PD-1 (p=0.01< 0.05). (E) Expansion of CAR-T cells after transduction (n=3 independent healthy donors). (F) Representative CAR expression was determined by flow cytometry in PD-1^KD BCMA CAR-T cells (left) and BCMA CAR-T cells (right). (G) The percentages of CAR+ cells were compared between PD-1^KD BCMA CAR-T cells and BCMA CAR-T cells. (H) Representative CD4+ and CD8+ CAR+T cell proportions were determined by flow cytometry in PD-1^KD BCMA CAR-T cells (left) and BCMA CAR-T cells (right). (I) The ratios of CD4/CD8 in CAR+T cells were compared between PD-1^KD BCMA CAR-T cells and BCMA CAR-T cells. (J) The differentiation status of PD-1^KD BCMA CAR-T cells (left) and BCMA CAR-T cells (right) were determined via flow cytometry using the surface markers CD45RA and CD62L. (K) Ratios of naive (Tnaive-like; CD45RA+CD62L+), central memory (Tcm; CD45RA−CD62L+), effector memory (Tem; CD45RA−CD62L−), and most differentiated T (Temra; CD45RA+CD62 L−) cells in PD-1^KD BCMA CAR-T cells and BCMA CAR-T cells were compared. BCMA, B cell maturation antigen; NS, not significant; UT, untransfected T cells.

Figure 3

PD-1 inhibition enhances antitumor efficiency and proliferation capacity of BCMA CAR-T cells. (A) In vitro cytotoxicity of BCMA CAR-T or PD-1^KD BCMA CAR-T cells against 8226 cells, MM.1S cells (B), BCMA overexpressing K562 cells (C) and PD-L1/BCMA overexpressing K562 cells (D) was determined by a luciferase-based cytotoxicity assay. E/T ratio, effector/target ratio (n=3 independent healthy donors). (E) Cytokines in the supernatant after coculture with PD-L1/BCMA overexpressing K562 cells for 24 hours. GSEA of the genes associated with (F) the positive regulation of cytokines is listed in online supplemental table 1, and (G) the genes related to the proliferation of T cells are listed in online supplemental table 2. The genes from the left to the right of the rank-ordered list are enriched in PD-1^KD BCMA CAR-T cells and BCMA CAR-T cells. (H) Cell proliferation was compared among non-CAR T cells, PD-1^KD BCMA CAR-T cells and BCMA CAR-T cells on stimulation with one dose of antigen. (I) Analysis of apoptotic PD-1^KD BCMA CAR-T cells and BCMA CAR-T cells on day 5 after one dose of antigen stimulation. BCMA, B cell maturation antigen; FDR, false discovery rate; GSEA, gene set enrichment analysis; NES, normalized enrichment score.

Figure 4

PD-1 inhibition promotes the memory phenotype and reduces the exhaustion of BCMA CAR-T cells. (A) Schematic diagram of the CAR-T cell cytotoxicity assay after long-term repetitive antigen stimulation. (B) CAR expression was determined by flow cytometry in PD-1^KD BCMA CAR-T cells (upper) and BCMA CAR-T cells (lower) after fourth repetitive stimulation (d13). Representative (B) and summarized (E) data from three representative healthy donors are shown. (C) Annexin V and PI-staining of PD-1^KD BCMA CAR+T and BCMA CAR+T cells after four repetitive stimulations (d13). Representative (C) and summarized (F) data from three representative healthy donors are shown. (D) Representative differentiation statuses of PD-1^KD BCMA CAR-T cells (upper) and BCMA CAR-T cells (lower) cells were determined by flow cytometry using the surface markers CD45RA and CD62L after four repetitive stimulations (d13). (G, H) Ratios of central memory (Tcm; CD45RA−CD62L+) and effector memory (Tem; CD45RA−CD62L−) cells were compared for PD-1^KD BCMA CAR-T and BCMA CAR-T cells. (I) GSEA of central memory genes. The central memory genes are shown in online supplemental table 3. The genes from the left to the right of the rank-ordered list are enriched in PD-1^KD BCMA CAR-T cells and BCMA CAR-T cells. (J) GSEA of effector versus exhausted up genes. Effector versus exhausted upgenes are shown in online supplemental table 4. The genes from the left to the right of the rank-ordered list are enriched in PD-1^KD BCMA CAR-T cells and BCMA CAR-T cells. (K, L) Volcano plots of genes differentially expressed between PD-1^KD BCMA CAR+T cells and BCMA CAR+T cells sorted after one antigen stimulation (d1) or after four repetitive stimulations (d13). Significantly differentially expressed genes associated with the memory phenotype are highlighted with an orange circle. (M) Heatmap of normalized RNA-seq reads for exhaustion-related genes differentially expressed between PD-1^KD BCMA CAR+ and BCMA CAR+T cells after four repetitive stimulations (d13). The results are from two representative healthy donors with two duplicates. BCMA, B cell maturation antigen; GSEA, gene set enrichment analysis; HD, heathy donor; NES, normalized enrichment score; FDR, false discovery rate.

Figure 5

BCMA CAR-T cells with PD-1 inhibition had enhanced antitumor efficacy in a PD-L1+CDX tumor model. (A) Experimental timeline of the CDX tumor model. (B) Bioluminescence imaging of tumor cell growth following different treatments on the indicated days after CAR-T cell infusion (n=5). (C) Kaplan-Meier curves for overall survival are shown. Statistical significance was determined by the MantelCox test and is presented by **p≤0.01. (D) H&E-stained tissues from mice treated with either tumor only (upper) or PD-1^KD BCMA CAR-T cells (lower) 7 days after CAR-T cell infusion. Scale bar: 20 µm. BCMA, B cell maturation antigen.

Figure 6

Clinical efficacy of PD-1^KD BCMA CAR-T cells in patients with RRMM. (A) Schematic diagram of the clinical PD-1^KD BCMA CAR-T cell treatment regimen and clinical/laboratory monitoring. *Patients may receive therapy during manufacturing to maintain disease control. **After the first 28 days, follow-up was every 4 weeks up to 6 months and then every 3 months up to 2 years. ***Pre-tx, pretreatment, 3–7 days before CAR-T cell infusion. Flu indicates fludarabine. Cy indicates cyclophosphamide. (B) Measurements of CAR-T cells assessed by means of qPCR assay in peripheral blood of patients treated with cyclophosphamide/fludarabine combination conditioning and three-infusion CAR T delivery. (C–F) Cytokine concentrations in the serum of all patients who received infusions of PD-1^KD BCMA CAR-T cells, as determined by ELISA. (G) Swimmer plot depicting each subjects’ response category over time and the results of MRD detection via flow cytometry on bone marrow aspirates. (H) Response of extramedullary lesion. Extramedullary infiltration lesions disappeared or were reduced after PD-1^KD BCMA CAR-T cell infusion. Representative PET-CT images of patient 3 and patient 5 and MRIs of patient 4 before and after PD-1^KD BCMA CAR-T cell treatment. Extramedullary diseases are indicated by red arrows. Light blue arrows indicate tumor reduction. BCMA, B cell maturation antigen; MRD, minimal residual disease; PR, partial response; RRMM, relapsed/refractory multiple myeloma; sCR, stringent complete response; SD, stable disease.