CRISPR-engineered T cells in patients with refractory cancer

Abstract

CRISPR-Cas9 gene editing provides a powerful tool to enhance the natural ability of human T cells to fight cancer. We report a first-in-human phase 1 clinical trial to test the safety and feasibility of multiplex CRISPR-Cas9 editing to engineer T cells in three patients with refractory cancer. Two genes encoding the endogenous T cell receptor (TCR) chains, TCRα (TRAC) and TCRβ (TRBC), were deleted in T cells to reduce TCR mispairing and to enhance the expression of a synthetic, cancer-specific TCR transgene (NY-ESO-1). Removal of a third gene encoding programmed cell death protein 1 (PD-1; PDCD1), was performed to improve antitumor immunity. Adoptive transfer of engineered T cells into patients resulted in durable engraftment with edits at all three genomic loci. Although chromosomal translocations were detected, the frequency decreased over time. Modified T cells persisted for up to 9 months, suggesting that immunogenicity is minimal under these conditions and demonstrating the feasibility of CRISPR gene editing for cancer immunotherapy.

Fig. 1.. Feasibility of CRISPR-Cas9 NYCE T cell engineering.

(A) Schematic representation of CRISPR-Cas9 NYCE T cells. (B) Large-scale expansion of NYCE T cells. Autologous T cells were transfected with Cas9 protein complexed with single guide RNAs (ribonucleoprotein; RNP complex) against TRAC, TRBC (i.e., endogenous TCR deletion) and PDCD1 (i.e., PD-1 deletion) and subsequently transduced with a lentiviral vector to express a transgenic NY-ESO-1 cancer-specific TCR. Cells were expanded in dynamic culture for 8 to 12 days. On the final day of culture, NYCE T cells were harvested and cryopreserved in infusible medium. The total number of enriched T cells during culture is plotted for all four subjects (UPN07, UPN27, UPN35 and UPN39). (C) NY-ESO-1 TCR transduction efficiency was determined in harvested infusion products by flow cytometry. Data are gated on live CD3-expressing and Vβ8.1 or dextramer positive lymphocytes and further gated on CD4 and CD8 positive cells. (D) The frequencies of TRAC, TRBC and PDCD1 gene-disrupted total cells in NYCE infusion products were measured using chip-based digital PCR. All data are representative of at least two independent experiments. Error bars represent mean +/- SEM.

Fig. 2.. Potency and immunogenicity of CRISPR-Cas9 engineered T cells.

(A) Cytotoxicity of NYCE T cells co-cultured with HLA-A*0201 positive Nalm-6 tumor cells engineered to express NY-ESO-1 and luciferase. Patient T cells transduced with the NY-ESO-1 TCR without CRISPR-Cas9 editing (NY-ESO-1 TCR) and untransduced T cells with CRISPR-Cas9 editing of TRAC, TRBC and PDCD1 (denoted as here as CRISPR) were included as controls (n = 4 patient T cell infusion products). Asterisks indicate statistical significance determined by paired t-tests between groups (*P < 0.05). Error bars represent SEM. (B) Levels of soluble interferon-γ produced by patient NYCE T cell infusion products (denoted as NYCE) following a 24-hour co-culture with anti-CD3/CD28 antibody-coated beads or NY-ESO-1-expressing Nalm-6 target cells. Patient NY-ESO-1 TCR transduced T cells (NY-ESO-1 TCR) and untransduced, CRISPR-Cas9 edited T cells (denoted as CRISPR) served as controls. Error bars indicate SEM. (C) Quantification of residual Cas9 protein in NYCE T cell infusion products in clinical-scale manufacturing is shown over time. Asterisks depict statistical significance determined by paired t-tests between time points (*P < 0.05). (D) Results from the fluorescence-based indirect ELISA screen performed to detect antibodies against Cas9 protein in the sera of three patients treated with NYCE T cells. Each dot represents the amount of anti-Cas9 signals detected in patient serum prior to T cell infusion (denoted by a vertical black arrow) and at various time points following NYCE T cell transfer. RFU = relative fluorescent units. (E) Immunoreactive Cas9-specific T cells in baseline patient leukapheresis samples were detected. Representative flow cytometry plots (left panel) from two patients whose T cells were positive for interferon-γ in response to Cas9 peptide stimulation. Unstimulated T cells treated with vehicle alone (dimethyl sulfoxide, DMSO) served as a negative control, while matched T cells stimulated with phorbol myristate acetate (denoted as PMA)/Ionomycin served as a positive control. Bar graphs (right panel) show the frequency of ex vivo CD4⁺ and CD8⁺ T cells from patients or healthy donor controls (n = 6) that secrete interferon-γ in response to stimulation with three different Cas9 peptide pools. The background frequency of interferon-γ-expressing T cells (unstimulated control group, DMSO alone) is subtracted from the values shown in the bar graph. Error bars depict SD.

Fig. 3.. Sustained in vivo expansion and persistence of CRISPR-Cas9 engineered T cells in patients.

(A) The total number of vector copies per microgram of genomic DNA of the NY-ESO-1 TCR transgene in the peripheral blood (UPN07, UPN35 and UPN39), bone morrow (UPN07 and UPN35; multiple myeloma), and tumor (UPN39; sarcoma) is shown pre- and post-NYCE T cell infusion. (B) Calculated absolute numbers of NY-ESO-1 TCR-expressing T cells per microliter of whole blood from the time of infusion to various post-infusion time points in the study are shown. The limit of detection is approximately 2.5 cells/μL of whole blood. (C) Frequencies of CRISPR-Cas9-edited T cells (TRAC, TRBC and PDCD1 knockout) before and following adoptive cell transfer are depicted. Error bars indicate SD.

Fig. 4.. Fidelity of CRISPR-Cas9 gene editing.

(A) Genomic distribution of oligonucleotide (dsODN) incorporation sites, which mark locations of double strand breaks. The ring indicates the human chromosomes aligned end-to-end, plus the mitochondrial chromosome (labeled M). The targeted cleavage sites are on chromosomes 2, 7, and 14. The frequency of cleavage and subsequent dsODN incorporation is shown on a log scale on each ring (pooled over 10 Mb windows). The purple inner most ring plots all alignments identified. The green ring shows “pile ups” of three or more overlapping sequences; the blue ring shows alignments extending along either strand from a common dsODN incorporation site (“flanking pairs”); the red ring shows reads with matches to the gRNA (allowing <6 mismatches) within 100 bp (“target matched”). (B) Distribution of inferred positions of cleavage and dsODN incorporation at an on-target locus. Incorporations in different strand orientations are shown on the positive (red) and negative (blue) y-axis. The percentage in the bottom right corner is an estimate of the number of incorporations associated with the on-target site (based on pileups) captured within the allowed window of 100 bps. (C) Sequences of sites of cleavage and dsODN incorporation are shown, annotated by whether they are on target or off target (“Target”); the total number of unique alignments associated with the site (“Abund”); and an identifier indicating the nearest gene (“Gene ID”). *Symbols after the gene name indicate that the site is within the transcription unit of the specific gene, whereas the ~ symbol indicates the gene appears on the allOnco cancer-associated gene list.

Fig. 5.. Detection of chromosomal translocations in engineered T cells following CRISPR-Cas9 gene editing.

(A) Evaluation of chromosomal translocations in NYCE T cell infusion products during the course of large-scale culture is shown. For the 12 monocentromeric translocation assays conducted, a positive reference sample that contains 1 × 10³ copies of the synthetic template plasmid was evaluated as a control and the percent difference between expected and observed marking was calculated. The absence of amplification from the 12 reactions that correspond to the different chromosomal translocations indicates assay specificity (see supplemental methods). (B) Longitudinal analysis of chromosomal translocations in vivo in three patients pre- and post-NYCE T cell product infusion is displayed. In panels A and B, error bars denote SD. For graphical purposes, the proportions of affected cells were plotted on a log scale; a value of 0.001% indicates that translocations were not detected.

Fig. 6.. Single-cell RNA sequencing of patient UPN39 CRISPR-Cas9 engineered NYCE T cells pre- and post-infusion.

(A) Venn diagram showing relative numbers of NY-ESO-1 TCR-positive cells with TRAC, TRBC and/or italic mutations in the NYCE T cell infusion product (Day 0). (B) Proportions of pre-infusion (Day 0) and post-infusion (Days 10 and 113) T cells without mutations (wild-type), with TRAC, TRBC, PDCD1 mutations or expressing the NY-ESO-1 TCR transgene. Numbers of cells belonging to each of these categories are listed below the graph. (C) Analysis of NY-ESO-1 TCR-positive (right) and TCR–negative (left) cells without mutations (wild-type) or with single, double or triple mutations at Day 0 (NYCE T cell infusion product) and Day 113 post-NYCE T cell infusion. (D) UMAP plots of gene expression data. Analysis was performed on all T cells integrated across time points, but only NY-ESO-1 TCR-expressing cells, split by time point, are shown (top panel). The increase in TCF7 expression is indicative of an acquired central memory phenotype (bottom panel, same cells). (E) Heat map showing scaled expression of differentially expressed genes in NY-ESO-1 TCR-positive T cells across time points. Color scheme is based on scaled gene expression from –2 (yellow) to 2 (purple).

Fig. 7.. Clinical responses and patient outcome following infusion of CRISPR-Cas9 engineered NYCE T cells

(A) Swimmer’s plot describing time on study for each patient, duration of follow-up off study (defined as survival beyond progression or initiation of other cancer therapy) and present status (differentially colored) is shown. Arrows indicate ongoing survival. (B) Changes in kappa light chain levels (mg/L × 10³) in patient UPN07 following NYCE T cell product infusion are depicted. Vertical black arrow indicates initiation of a D-ACE salvage chemotherapy regimen (defined as intravenous infusion of cisplatin, etoposide, cytarabine and dexamethasone). (C) Longitudinal M-Spike levels (g/dL) in patient UPN35 post-NYCE T cell product administration are shown. Vertical black arrows denote administration of combination therapy with elotuzumab, pomalidomide and dexamethasone. (D) Computed tomography scans demonstrating tumor regression in patient UPN39 following administration of an autologous NYCE T cell infusion product. Radiologic studies were obtained before therapy and after adoptive transfer of NYCE T cells. Tumor is indicated by red X. SD, stable disease; PD, progressive disease. (E) Cytolytic capacity of NY-ESO-1-specific CD8⁺ T cells recovered at the indicated month after infusion and expanded from patients is shown. PBMC samples collected after NYCE T cell product infusion were expanded in vitro in the presence of NY-ESO-1 peptide and IL-2. The ability of expanded effector cells to recognize antigen and elicit cytotoxicity was tested in a 4-hour ⁵¹CR release assay incorporating Nalm-6 NY-ESO-1⁺, parental Nalm-6 (NY-ESO-1⁻) and A375 melanoma cells (NY-ESO-1⁺). All target cell lines were HLA-A*02 positive. Assays were performed in triplicate and error bars represent SD.