RU2787828C2 - Materials and methods for cancer treatment - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, in particular to a method for the production of T-cell with a chimeric antigen receptor, having a decreased level of polypeptides of a granulocyte-macrophage colony-stimulating factor (GM-CSF). A method for improvement of T-cell effector functions of T-cell with a chimeric antigen receptor is also disclosed.

EFFECT: invention is effective for the treatment of a mammal suffering from cancer.

17 cl, 21 dwg, 1 tbl, 6 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority over U.S. Patent Application Serial Number 62/679,348 filed June 1, 2018 and U.S. Patent Application Serial Number 62/753,485 filed October 31, 2018. Disclosure of these earlier applications should be considered as part (which is incorporated by reference) of the description of the present application.

BACKGROUND OF THE INVENTION

1. FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

This document relates to methods and materials used in the treatment of cancer. For example, this document relates to methods and materials for using chimeric antigen receptor T cells having reduced expression levels of one or more cytokines (e.g., GM-CSF) in adoptive cell therapy (e.g., chimeric antigen receptor T cell therapy). ) for treating a mammal (eg, a human) suffering from cancer.

2. STATE OF THE ART

Pivotal clinical trials evaluating the safety and efficacy of CD19-targeted chimeric antigen receptor T cells (CART19) have produced unprecedented results, leading to the recent FDA approval of CART19 (Tisagenlecleucel) for the treatment of relapsed refractory acute lymphoblastic leukemia (ALL) and CART19 (Axi-Cel) for the treatment of diffuse large B-cell lymphoma (DLBCL). The use of CART cell therapy is associated with toxicity leading to cytokine release syndrome (CRS) and neurotoxicity. In addition, the effectiveness of CART cell therapy is limited to only 40% stable remissions in lymphoma and 50-60% stable remissions in acute leukemia.

SUMMARY OF THE INVENTION

This document relates to methods and materials for obtaining T cells (eg, chimeric antigen receptor (CAR) T cells (CART)) having a reduced level of expression of one or more cytokine polypeptides (eg, GM-CSF). For example, a T cell (eg, CART) can be engineered to have a reduced level of expression of a GM-CSF polypeptide (eg, for use in adaptive cell therapy). In some instances, a T cell (eg, CART) can be engineered by knockout (KO) of a nucleic acid encoding one or more cytokine polypeptides (eg, a GM-CSF polypeptide) to reduce the level of expression of those cytokine polypeptides (eg, a GM-CSF) in this T cell. Also provided herein are methods and materials for the use of T cells (eg, CART) having reduced expression of one or more cytokines (eg, GM-CSF polypeptides). For example, T cells (eg, CART) having reduced levels of GM-CSF polypeptides can be administered (eg, in adoptive cell therapy) to a mammal suffering from cancer to treat the mammal.

As shown herein, GM-CSF KO CART produce reduced levels of GM-CSF and continue to function normally in both in vitro and in vivo models . The present description also shows that GM-CSF KO CART can enhance the function of CART cells and antitumor activity. For example, after GM-CSF, enhanced proliferation and antitumor activity of CART cells can be observed. Antigen-specific proliferation of CART19 in the presence of monocytes can be increased in vitro after GM-CSF depletion. In xenografts derived from ALL patients, CART19 cells in combination with lenzilumab may result in better disease control, and in NALM6 xenografts GM-CSF ^k/o CART cells may be more effective in combating leukemia. In some instances, GM-CSF KO CARTs may be included in an adoptive T cell therapy (eg, CART cell therapy) for the treatment of, for example, mammalian cancers without the occurrence of CRS and/or neurotoxicity. For example, GM-CSF KO CART can be included in adoptive T cell therapy (eg, CART cell therapy) to increase the therapeutic window after CART cell therapy. In some instances, a single construct can be used both to introduce a CAR into a cell (eg, a T cell) and to reduce or knock out the expression of one or more cytokine polypeptides in the same cell.

In general, one aspect of the present invention relates to a method for obtaining a CAR T cell having a reduced level of cytokine polypeptides. The methods may comprise, or essentially consist of, ex vivo introducing a nucleic acid construct into a T cell, the nucleic acid construct comprising: a) a nucleic acid encoding a guide RNA (gRNA) complementary to a cytokine messenger RNA (mRNA); b) a nucleic acid encoding a Cas nuclease; and c) a nucleic acid encoding a chimeric antigen receptor. Cytokine polypeptides may include granulocyte-macrophage colony-stimulating factor (GM-CSF) polypeptides, interleukin 6 (IL-6) polypeptides, IL-1 polypeptides, m-CSF polypeptides, and/or MIP-1B polypeptides. The cytokine polypeptides may be GM-CSF polypeptides and the gRNA may include the nucleic acid sequence shown in SEQ ID NO: 1. The Cas nuclease may be a Cas9 nuclease. The nucleic acid encoding a CAR may include the nucleic acid sequence shown in SEQ ID NO: 2. The nucleic acid construct may be a viral vector (eg, a lentiviral vector). A CAR can target a tumor-associated antigen (eg, CD 19). The insertion step may include transduction.

In another aspect, this document relates to methods for obtaining a CAR T cell having a reduced level of cytokine polypeptides. The methods may include, or essentially consist of, ex vivo administering to a T cell a complex that includes: a) gRNA complementary to a cytokine mRNA; and b) Cas nuclease; and introducing into the T cell ex vivo a nucleic acid encoding a CAR. Cytokine polypeptides may include GM-CSF polypeptides and/or IL-6 polypeptides. The cytokine polypeptides may be GM-CSF polypeptides and the gRNA may include the nucleic acid sequence shown in SEQ ID NO: 1. The Cas nuclease may be a Cas9 nuclease. The nucleic acid encoding a CAR may include the nucleic acid sequence shown in SEQ ID NO: 2. The complex may be a ribonucleoprotein (RNP). A CAR can target a tumor-associated antigen (eg, CD19). The steps of administration may include electroporation.

In another aspect, this document relates to methods for generating a CAR T cell having a reduced level of GM-CSF polypeptides. The methods may comprise, or essentially consist of, ex vivo introducing into a T cell a nucleic acid construct comprising: a) a nucleic acid encoding an gRNA complementary to GM-CSF mRNA; b) a nucleic acid encoding a Cas nuclease; and c) a nucleic acid encoding a CAR. The gRNA may include the nucleic acid sequence shown in SEQ ID NO: 1. The Cas nuclease may be a Cas9 nuclease. The nucleic acid encoding a CAR may include the nucleic acid sequence shown in SEQ ID NO: 2. The nucleic acid construct may be a viral vector (eg, a lentiviral vector). A CAR can target a tumor-associated antigen (eg, CD19). The insertion step may include transduction.

In another aspect, this document relates to methods for generating a CAR T cell having reduced levels of GM-CSF polypeptides. The methods may include, or essentially consist of, ex vivo administering to a T cell a complex comprising: a) gRNA complementary to GM-CSF mRNA; and b) Cas nuclease; and introducing into the T cell ex vivo a nucleic acid encoding a CAR. The gRNA may include the nucleic acid sequence shown in SEQ ID NO: 1. The Cas nuclease may be a Cas9 nuclease. The nucleic acid encoding a CAR may include the nucleic acid sequence shown in SEQ ID NO: 2. The complex may be a RNP. A CAR can target a tumor-associated antigen (eg, CD19). The steps of administration may include electroporation.

In another aspect, this document relates to methods of treating a mammal suffering from cancer. The methods may include or essentially consist of administering to a cancerous mammal CAR T cells having reduced levels of cytokine polypeptides. Cytokine polypeptides may include GM-CSF polypeptides and/or IL-6 polypeptides. The cytokine polypeptides may be GM-CSF polypeptides and the gRNA may include the nucleic acid sequence shown in SEQ ID NO: 1. The mammal may be a human. The cancer may be a lymphoma (eg, DLBCL). The cancer may be leukemia (eg, ALL). A CAR can target a tumor-associated antigen (eg, CD19).

In another aspect, this document relates to methods of treating a mammal suffering from cancer. The methods may include or essentially consist of administering to a cancerous mammal CAR T cells having reduced levels of GM-CSF polypeptides. The mammal may be human. The cancer may be a lymphoma (eg, DLBCL). The cancer may be leukemia (eg, ALL). A CAR can target a tumor-associated antigen (eg, CD19).

Unless otherwise indicated, all technical and scientific terms used in the present description have the same meaning in which they are usually understood by experts in the field to which the present invention relates. While methods and materials similar or equivalent to those disclosed herein may be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned in the present description are incorporated into the description in its entirety by reference. In case of conflict, the present description, including definitions, shall prevail. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

A more complete description of one or more embodiments of the invention is given below with reference to the accompanying drawings. Other features, objects and advantages of the invention will be apparent from the description and drawings, as well as from the claims.

DESCRIPTION OF THE DRAWINGS

In FIG. 1 is a diagram of an exemplary method for using CRISPR to generate a GM-CSF knockout (KO) cell. A guide RNA (GACCTGCCTACAGACCCGCC; SEQ ID NO: 1) targeting exon 3 of GM-CSF (also known as colony stimulating factor 2 (CSF2)) was synthesized and cloned into a lentiviral plasmid (LV). This LV plasmid was used to transduce 293T cells and lentivirus particles were harvested at 24 hours and 48 hours and concentrated. To generate GM-CSF knockout CART cells, T cells were stimulated with CD3/CD28 beads on day 0. On day 1, T cells were transduced with CAR19 lentiviral particles and simultaneously with GMCSF knockout CRISPR/Cas9 lentiviral particles. T cells were expanded for 8 days and then harvested.

In FIG. 2A and 2B show the efficiency of CAR transduction and GM-CSF knockout. In FIG. 2A is a graph showing that the knockout efficiency using CRISPR/Cas9 lentivirus with guide RNA directed to exon 3 of GM-CSF is 24.1%. After propagation, CART cells were collected, DNA was isolated, which was sent for sequencing for comparison with control sequences. The resulting knockout efficiency was 24.1%. In FIG. 2B shows the results of flow cytometry analysis showing that the CAR transduction efficiency after lentivirus transduction was 73%. Analysis by flow cytometry was performed on the 6th day after transduction with lentivirus.

In FIG. 3 shows that GM-CSF KO CART19 cells produce less GM-CSF compared to CART cells, and GM-CSF knockout control T cells produce less GM-CSF than control non-transduced T cells (UTD). CART19, GM-CSF KO CART19, UTD or GM-CSF KO UTD were cultured together with the CD19-positive NALM6 cell line at a ratio of 1:5, after 4 hours the cells were harvested, permeabilized and fixed; and performed intracellular staining for cytokines.

In FIG. 4 shows that GM-CSF KO CART19 cells proliferate more stably compared to CART19. After transduction of T cells with the virus, the kinetics of their reproduction was observed. Reproduction of GM-CSF KO CART19 was more stable than CART19 alone.

In FIG. 5 shows an exemplary nucleic acid sequence (SEQ ID NO: 2) encoding a CAR targeting CD19 (CAR19).

In FIG. 6A-6D show that neutralization of GM-CSF in vitro enhances CAR-T cell proliferation in the presence of monocytes and does not impair CAR T cell effector function. In FIG. 6A is a graph showing that lenzilumab neutralizes GM-CSF produced by CAR T cells in vitro compared to isotype control treatment, as evidenced by multiplex analysis performed after 3 days of culture with only CART19 in media or with CART19 cultured. shared with NALM6, n=2 experiments, 2 replicates per experiment; representative experiment shown, ***p<0.001 between lenzilumab and isotype control treatment, t-test, mean±SEM. In FIG. 6B is a graph showing that GM-CSF treatment with neutralizing antibodies did not suppress the proliferating ability of CAR T cells, as determined by flow cytometry analysis of proliferation of live CD3 cells labeled with CSFE, n=2 experiments, 2 replicates per experiment; shows representative experiment at 3 days, ns (non-significant) p>0.05 between lenzilumab and isotype control treatment, t-test, mean±SEM. Only: CART19 in medium only, MOLM13: CART19+MOLM13, PMA/ION: CART19+5 ng/ml PMA/0.1 µg/ml ION, NALM6: CART19+NALM6. In FIG. 6C is a graph showing that lenzilumab increased CART19 proliferation compared to CART19-treated isotype control when co-cultured with monocytes, n=3 biological replicates (replicates) after 3 days, 2 replicates per biological replica, ****p <0.0001, mean ±SEM. In FIG. 6D is a graph showing that lenzilumab treatment did not suppress the cytotoxicity of CART19 or non-transduced T cells (UTD) when cultured with NALM6, n=2 experiments, 2 replicates per experiment, shows a representative experiment at 48 hours, ns p>0.05 between lenzilumab and isotype control treatment, t-test, mean ±SEM.

In FIG. 7A-7E show that in vivo neutralization of GM-CSF enhances the antitumor activity of CAR-T cells in xenograft models. In FIG. 7A is an experimental scheme showing that NSG mice were injected with the CD19+luciferase+ NALM6 cell line (1 x 10 ⁶ cells per IV mouse). After 4-6 days, mice were imaged, randomized, and the next day received 1-1.5×10 ⁶ CAR-T19 or equivalent total control UTD cells with either lenzilumab or control IgG (10 mg/kg, administered IP daily in within 10 days from the day of the CAR-T injection). Mice were monitored with serial bioluminescent imaging to assess the severity (burden) of the disease, starting from day 7 after injection of CAR T cells, and observed for overall survival. Blood sampling from the tail vein was performed 7-8 days after the injection of CAR T cells. In FIG. 7B is a graph showing that lenzilumab neutralizes in vivo serum GM-CSF produced by CAR-T compared to isotype control treatment, as measured by single duplex GM-CSF assay, n=2 experiments, 7-8 mice per group, representative experiment , serum day 8 post CAR T cell/UTD injection, ***p<0.001 between lenzilumab and isotype control treatment, t-test, mean ±SEM. In FIG. 7C is a graph showing that in vivo lenzilumab-treated CAR-Ts are equally effective in controlling tumor burden compared to isotype control-treated CAR-Ts in an ALL high tumor burden xenograft model, day 7 post-CAR injection. -T, n=2 experiments, 7-8 mice per group, representative experiment shown, ***p<0.001, *p<0.05, ns p>0.05, t-test, mean ±SEM. In FIG. 7D is an experimental scheme showing that NSG mice were injected with blasts derived from ALL patients (1 x 10 ⁶ cells per mouse intravenously). Mice were bled continuously and when CD19+ cells became >1/µl mice were randomized to receive 5×10 ⁶ CART19 (transduction efficiency was approximately 50%) or UTD cells with either lenzilumab or control IgG (10 mg/kg , administered IP daily for 10 days starting from the day of CAR-T injection). Mice were constantly bled from the tail vein to assess the severity of the disease, starting from the 14th day after the injection of CAR-T cells, and observed for overall survival. In FIG. 7E is a graph showing that lenzilumab treatment with CAR T cell therapy results in more robust control of tumor burden over time in a primary acute lymphoblastic leukemia (ALL) xenograft model compared with isotype control treatment with CAR T cell therapy, 6 mice. per group, **p<0.01, *p<0.05, ns p>0.05, t-test, mean ±SEM.

In FIG. 8 is a graph showing that mice treated with lenzilumab+CAR T cells have comparable survival compared to mice treated with isotype control+CAR T cells in a high ALL tumor burden xenograft model. n=2 experiments, 7-8 mice per group, representative experiment depicted, ****p<0.0001, ***p<0.001, *p<0.05, logarithmic rank.

Figure 9 is a graph showing a representative TIDE sequence to test for genome change in GM-CSF knockout CAR T cells by the CRISPR-Cas9 method. n=2 experiments, a representative experiment is shown.

In FIG. 10A-10E show that GM-CSF knockout CAR T cells by CRISPR exhibit reduced GM-CSF expression similar to levels of key cytokines and increased antitumor activity. In FIG. 10A is a graph showing that CRISPR Cas9 GMCSF ^k/o CAR-T show reduced GMCSF production compared to wild-type CART19, but production of other cytokines and degranulation is not inhibited by disruption of the GM-CSF gene, n=3 experiments, 2 repeats per experiment, ***p<0.001, *p<0.05, ns p>0.05 comparing GM-CSFk/o CAR-T and CAR-T, t-test, mean ±SEM. In FIG. 10B is a graph showing that GM-CSFk/o CAR-Ts have reduced serum levels of human GM-CSF in vivo compared to CAR-T treatment by multiple regression analysis, 5-6 mice per group (4- 6 times blood sampling, 8 days after CART administration), ****p<0.0001, ***p<0.001 between GM-CSFk/o CAR-T cells and wild-type CAR-T cells, t-test, mean ±SEM. In FIG. 10C is a graph showing that GM-CSF ^k/o CART19 increases in vivo overall survival compared to wild-type CART19 in an ALL high tumor burden xenograft model, 5-6 mice per group, **p<0.01, log rank. In FIG. 10D and 10E are heat maps showing human (D) and mouse (E) cytokines from the serum multiplex test other than human GM-CSF; shows no statistical difference between GM-CSFk/o CAR T cells and wild-type CAR T cells; reduction in GM-CSF expression does not adversely affect important T-cell cytokines, 5-6 mice per group (4-6 blood samples taken), ****p<0.0001, t-test.

11 is a graph showing that GM-CSF knockout CAR T cells in vivo exhibit slightly improved tumor load control by ALL compared to CAR-T in a high tumor burden xenograft model with tumor recurrence. Days after CAR-T injection are plotted on the x-axis, 5-6 mice per group (UTD group remained 2 at day 13), representative experiment depicted, ****p<0.0001, *p<0.05, two-way ANOVA, mean ±SEM.

In FIG. 12A-12D show a patient-derived xenograft model for assessing neurotoxicity and cytokine release syndrome. In FIG. 12A is an experimental design showing that mice were injected with 1-3x10 ⁶ primary blasts derived from the peripheral blood of patients with primary ALL. Graft engraftment in mice was observed for 10-13 weeks by taking blood from the tail vein. When the number of CD19+ cells in serum reached >10 cells/μl, mice were injected with CART19 (2-5×10 ⁶ cells) and began 10 days of antibody therapy as indicated. Mice were weighed daily as an indicator of physical condition. Mouse brain MRI was performed 5-6 days after CART19 injection, blood samples were taken from the tail vein for cytokine levels, and T cell analysis was performed 4-11 days after CART19 injection, 2 independent experiments. In FIG. 12B is a graph showing that the combination of GM-CSF neutralization with CART19 is as effective in controlling ALL CD19+ cell load as isotype control antibodies in combination with CART19, representative experiment, 3 mice per group, 11 days post CART19 injection, *p <0.05 between GM-CSF+CART19 neutralization and isotype control+CART19, t-test, mean ±SEM. In FIG. 12C shows that MRI of the brain with CART19 therapy shows an increase in T1, indicating a violation of the blood-brain barrier and possible edema. 3 mice per group, 5-6 days after CART19 injection, representative image. In FIG. 12D are graphs showing that high tumor burden primary ALL xenografts treated with CART19 show brain infiltration with human CD3 cells compared to untreated PDX controls. 3 mice per group, representative image.

In FIG. 13A-13D show that in vivo neutralization of GM-CSF reduces cytokine release syndrome after CART19 therapy in a xenograft model. In FIG. 13A is a graph showing that lenzilumab and anti-mouse GM-CSF antibody prevented CRS-induced weight loss compared to mice treated with CART19 and isotype control antibodies, 3 mice per group, two-way ANOVA, mean±SEM. In FIG. 13B is a graph showing that human GM-CSF was neutralized in xenografts from patients treated with lenzilumab and mouse GM-CSF neutralizing antibody, 3 mice per group, ***p<0.001, *p<0.05, t-test, mean ±SEM. In FIG. 13C is a heat map showing that human cytokines (serum collected 11 days after CART19 injection) show an increase in cytokines typical of CRS after CART19 treatment. Neutralization of GM-CSF results in a significant decrease in the levels of several cytokines compared to mice treated with CART19 and isotype control antibodies, including several myeloid-associated cytokines as indicated in the panel, 3 mice per group, serum 11 days post CART19 injection, ** *p<0.001, **p<0.01, *p<0.05, comparison of GM-CSF neutralizing antibody treated mice with isotype control treated mice that received CAR T cell therapy, t-test. In FIG. 13D is a heat map showing that mouse cytokines (serum collected 11 days after CART 19 injection) show an increase typical of CRS after CART19 treatment. Neutralization of GM-CSF results in a significant decrease in several cytokines compared to treatment with CART19 with control antibodies, including several myeloid-differentiating cytokines as indicated in the panel, 3 mice per group, serum 11 days post CART19 injection, *p<0.05 comparing GM-CSF neutralizing antibody treated mice with isotype control treated mice treated with CAR T cells, t-test.

In FIG. 14A-14D show that in vivo neutralization of GM-CSF reduces neurotoxicity after CART19 therapy in a xenograft model. In FIG. 14A and 14B show that gadolinium-enhanced hyperintense signal at T1 (cc mm) MRI showed that GM-CSF neutralization helped reduce brain inflammation, blood-brain barrier disruption, and possible edema compared to representative isotype control images (A), (B ) 3 mice per group, **p<0.01, *p<0.05, one-way ANOVA, mean±SD. In FIG. 14C is a graph showing that human CD3 T cells were present in the brain after treatment with CART19 therapy. Neutralization of GM-CSF resulted in a reduction in brain CD3 infiltration as determined by flow cytometry in the cerebral hemispheres, 3 mice per group, mean±SEM. In FIG. 14D is a graph showing that according to flow cytometric analysis of the cerebral hemispheres, the number of bright CD11b+ macrophages is reduced in the brains of mice treated with GM-CSF neutralization during CAR-T therapy compared with isotype control during CAR-T therapy, 3 mice per group, mean ± SEM.

In FIG. 15A-15B show exemplary generation of GM-CSFk/o CART19 cells. The scheme of the experiment is shown. In FIG. 15A shows the targeting site of the gRNA sequence in CSF2 (GACCTGCCTACAGACCCGCC; SEQ ID NO: 11) to generate GM-CSFk/o CART19. In FIG. 15B shows the primer sequences (TGACTACAGAGAGGCACAGA (SEQ ID NO: 12) and TCACCTCTGACCTCATTAACC (SEQ ID NO: 13)) and the gRNA sequence (GACCTGCCTACAGACCCGCC; SEQ ID NO: 7) used to generate GM-CSFk/o CART19. To create GM-CSFk/o CART19 cells, gRNA was cloned into a Cas9 lentiviral vector under the control of the U6 promoter and used to produce lentivirus. T cells from normal donors were stimulated with CD3/CD28 beads and 24 hours later transduced twice with CAR19 virus and CRISPR/Cas9 virus. Magnetic CD3/CD28 beads were removed on day +6 and GM-CSFk/o CART19 cells or control CART19 cells were cryopreserved on day 8.

In FIG. 16 is a flow chart of the procedures used in RNA sequencing. The binary representation of the data was converted to fastq format using the Illumina bcl2fastq software. Adapter sequences were removed with a Trimmomatic and FastQC was used for quality control. The latest reference human (GRCh38) and mouse (GRCm38) genomes were downloaded from NCBI. Genome index files were generated with STAR30 and paired end reads were mapped to the genome for each condition. HTSeq was used to calculate the number of expression events for each gene, and DeSeq2 was used to calculate differential expression. Gene ontology was evaluated using Enrichr.

In FIG. 17A-17B show that GM-CSF receptors are activated upon stimulation on T cells and CART cells. In FIG. 17A shows measurements of CSF2RA (CD116) and CSF2RB (CD131) on T cells compared to T cells at rest (negative control) during an 8 day T cell expansion protocol using CD3/CD28 beads. Expression of CSF2RA and CSF2RB increased after initial stimulation, peaked on day 3, and decreased slightly after weight loss on day 6. In FIG. 17B shows measurements of CSF2RA (CD 116) and CSF2RB (CD131) on CART19 and UTD cells compared to controls during 8 days of CART production. Expression decreased slightly on day 1 and peaked on day 3.

In FIG. 18 shows that the interaction of GM-CSF with the CSF2 receptor is dependent on the beta chain (CSF2RB). Expression of the phosphorylated Stat5 protein increased in the presence of irradiated Nalm6 and CSF2RA blockade, but decreased in the presence of GM-CSF and CSF2RB blockade. FAS is downstream of the CSF2 receptor (see, e.g., Takesono et al., Journal of Cell Science 115:3039-3048 (2002)), and is slightly downregulated in the presence of GM-CSF blockade along with irradiated Nalm6, but not CSF2RA blockade. or CSF2RB.

In FIG. 19A-19C show transcriptome differences between GM-CSF ^k/o CART19 and CART19 on day 8 of CART production. In FIG. 19A shows 236 genes whose expression varied greatly (Benjamini-Hochberg adjusted p<0.05). In FIG. 19B shows genes that were highly downregulated in GM-CSF ^k/o CART19 compared to CART19. The volcano plot (volkano) shows an increase in highly downregulated genes between GM-CSF ^k/o CART19 and CART19. In FIG. 19C shows that GM-CSF knockout in CART19 normalized gene expression.

In FIG. 20A-20C show an exemplary method for precise editing of the CSF2 gene using the CRISPR-Cas9 system. In FIG. 20A shows the expected cut site in CSF2 CRISPR gRNA 1 (SEQ ID NO: 7) (3 bp upstream of PAM) compared to the actual cut site (6 bp upstream of PAM) (top panel). Shown (lower panel) are the reference sequence (SEQ ID NO: 1), deletion pattern (SEQ ID NO: 8), and insertion pattern (SEQ ID NO: 9), and the frequency of deletion and insertion at base 132074828 of chromosome 5 in each biological replica. CART19. In FIG. 20B shows single nucleotide variants (SNV) and insertion/deletion (indel) counts under CRISPR knockout conditions compared to their controls (T cells and CART19 cells). In FIG. 20C shows the total variants found in CRISPR-edited cells compared to their respective controls (CART19 or T cells). The single SNP at the intersection is a deletion in the CSF2 gene (FIG. 20A, bottom panel).

In FIG. 21 is a graph showing that GM-CSF blockade in the presence of M2 macrophages significantly enhances CART19 proliferation upon CD19 stimulation compared to isotype control treatment. **p<0.005.

DETAILED DESCRIPTION

Methods and materials are provided for generating T cells (eg, chimeric antigen receptor (CAR) (CART) T cells) having a reduced level of expression of one or more cytokine polypeptides (eg, GM-CSF polypeptides). In some cases, a T cell (e.g., CART) can be engineered to knock out (KO) a nucleic acid encoding a GM-CSF polypeptide in order to reduce expression of the GM-CSF polypeptide in that T cell (e.g., compared to a T cell, which is not engineered for a KO nucleic acid encoding a GM-CSF polypeptide). A T cell engineered for a KO nucleic acid encoding a GM-CSF polypeptide may also be referred to as a GM-CSF KO T cell. In some instances, the methods and materials provided herein can be used to modulate myeloid cells. In some instances, the methods and materials provided herein can be used to deplete myeloid cells. In some instances, the methods and materials provided herein can be used to improve the performance of T cells (eg, CART).

T cells (eg, CART) provided herein can be engineered to have a reduced level of expression of any suitable cytokine polypeptide or combination of cytokine polypeptides. For example, a T cell (eg, CART) provided herein can be engineered to have a reduced level of expression of a GM-CSF polypeptide, an interleukin 6 (IL-6) polypeptide, a G-CSF, an interferon gamma polypeptide (IFN-g), IL-1B polypeptide, IL-10 polypeptide, monocyte chemoattractant protein 1 (MCP-1) polypeptide, interferon gamma-induced monokine (MIG) polypeptide, macrophage inflammatory protein (MIP) polypeptide (e.g., MIP-1β polypeptide ), a tumor necrosis factor alpha (TNF-α) polypeptide, an IL-2 polypeptide, a perforin polypeptide, or any combination thereof. For example, a T cell can be engineered to have a reduced level of expression of GM-CSF and IL-6 polypeptides.

The term "low level" as used herein in relation to the level of expression of a cytokine (eg GM-CSF) refers to any level that is lower than the reference level of expression of that cytokine (eg GM-CSF). The term "reference level" as used herein in relation to a cytokine (e.g., GM-CSF) refers to the level of that cytokine (e.g., GM-CSF) normally observed in a sample (e.g., control) obtained from one or more mammalian (eg, humans) that is not engineered to have reduced expression of that cytokine (eg, GM-CSF polypeptides) as described herein. Control samples may include, without limitation, T cells that are wild-type T cells (eg, T cells that are not GM-SCF KO T cells). In some instances, a reduced level of expression of a cytokine polypeptide (eg, GM-CSF polypeptide) may represent an undetectable level of that cytokine (eg, GM-CSF). In some cases, the reduced level of expression of GM-CSF polypeptides may be downregulated by the level of GM-CSF.

In some instances, a T cell that has (e.g., engineered to have) reduced expression of one or more cytokine polypeptides, such as a GM-CSF KO T cell, may retain normal T cell functions, such as T cell degranulation. -cells and release of cytokines (eg, compared to CART, which is not designed to reduce the level of expression of this cytokine (eg, GM-CSF polypeptides) according to the present description).

In some instances, a T cell having (e.g., engineered to have) a reduced level of GM-CSF polypeptides (e.g., a GM-CSF KO T cell) may have enhanced CART function such as antitumor activity, proliferation, killing (destruction) of cells, cytokine production, susceptibility to depletion, antigen-specific effector functions, resistance and differentiation (eg, compared to CART, which is not designed to have a reduced level of GM-CSF polypeptides as described herein).

In some instances, a T cell having (e.g., engineered to have) a reduced level of GM-CSF polypeptides (e.g., a GM-CSF KO T cell) may have an improved ability to proliferate (e.g., compared to CART, which is not designed to have a reduced level of GM-CSF polypeptides as described herein).

A T cell having (eg, engineered to have) reduced expression of one or more cytokines (eg, a GM-CSF polypeptide), such as a GM-CSF KO T cell, may be any suitable T cell. The T cell may be a naive T cell. Examples of T cells that can be engineered to have reduced expression of one or more cytokines as described herein include, without limitation, cytotoxic T cells (eg, CD4+ CTL and/or CD8+ CTL). For example, a T cell that can be engineered to have a reduced level of GM-CSF polypeptides as described herein may be a CART. In some cases, one or more T cells may be obtained from a mammal (eg, a mammal suffering from cancer). For example, T cells can be obtained from a mammal to be treated using the materials and methods disclosed herein.

A T cell having (eg, engineered to have) reduced expression of one or more cytokine polypeptides (eg, a GM-CSF polypeptide), such as a GM-CSF KO T cell, can be obtained by any suitable method. In some cases, a T cell (eg, CART) can be engineered to KO a nucleic acid encoding a GM-CSF polypeptide to reduce expression of the GM-CSF polypeptide in that T cell.

In some instances where a T cell (eg, CART) is engineered for a KO nucleic acid encoding a cytokine (eg, a GM-CSF polypeptide), any suitable method for KO nucleic acid can be used to reduce the expression of that cytokine polypeptide in that T cell. acid encoding this cytokine. Examples of knockout techniques that can be applied to a nucleic acid sequence encoding a cytokine polypeptide (e.g., a GM-CSF polypeptide) include, without limitation, gene editing, homologous recombination, non-homologous end joining, and microhomology based end joining. For example, for a KO of a nucleic acid encoding a GM-CSF polypeptide, gene editing (eg, with engineered nucleases) can be used. Nucleases suitable for genome editing include, but are not limited to, CRISPR-associated (Cas) nucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALEs), and homing endonucleases (HE; also referred to as meganucleases).

In some instances, for a KO nucleic acid encoding a cytokine polypeptide (e.g., a GM-CSF polypeptide), Clustered Regularly Spaced Short Palindromic Repeats (CRISPR)/Cas (see ., for example, Fig. 1 and example 1). The CRISPR/Cas system used for the KO of a nucleic acid encoding a cytokine polypeptide (eg, a GM-CSF polypeptide) may include any suitable guide RNA (gRNA). In some instances, the gRNA may be complementary to a nucleic acid encoding a GM-CSF polypeptide (eg, GM-CSF mRNA). Examples of gRNAs specific for a nucleic acid encoding a GM-CSF polypeptide include, without limitation,

GACCTGCCTACAGACCCGCC (SEQ ID NO: 1), GCAGTGCTGCTTGTAGTGGC (SEQ ID NO: 10), TCAGGAGACGCCGGGCCTCC (SEQ ID NO: 3), CAGCAGCAGTGTCTCTACTC (SEQ ID NO: 4), CTCAGAAATGTTTGACCTCC (SEQ ID NO: 5) and GGCCGGTCTCACTCCTGGAC (SEQ ID NO : 6). In some cases, the gRNA component of the CRISPR/Cas system designed for the KO nucleic acid encoding the GM-CSF polypeptide may include the nucleic acid sequence shown in SEQ ID NO: 1.

The CRISPR/Cas system used for KO of a nucleic acid encoding a cytokine polypeptide (eg, a GM-CSF polypeptide) may include any suitable Cas nuclease. Examples of Cas nucleases include, without limitation, Cas1, Cas2, Cas3, Cas9, Cas10, and Cpf1. In some instances, the Cas component of the CRISPR/Cas system for the KO of a nucleic acid encoding a cytokine polypeptide (eg, a GM-CSF polypeptide) may be a Cas9 nuclease. For example, the Cas9 nuclease of the CRISPR/Cas9 system disclosed herein may be lentiCRISPRv2 (see e.g. Shalem et al., 2014 Science 343:84-87; and Sanjana et al., 2014 Nature methods 11:783-784) .

Components of the CRISPR/Cas system (e.g., gRNA and Cas nuclease) used for KO of a nucleic acid encoding a cytokine polypeptide (e.g., a GM-CSF polypeptide) can be introduced into one or more T cells (e.g., CART) at any suitable format. In some instances, a component of the CRISPR/Cas system may be introduced into one or more T cells as a nucleic acid encoding a gRNA and/or a nucleic acid encoding a Cas nuclease. For example, a nucleic acid encoding at least one gRNA (e.g., an gRNA sequence specific for a nucleic acid encoding a GM-CSF polypeptide) and a nucleic acid of at least one Cas nuclease (e.g., Cas9 nuclease) can be introduced into one or more T cells. In some cases, a component of the CRISPR/Cas system may be introduced into one or more T cells as gRNA and/or as Cas nuclease. For example, at least one gRNA (eg, an gRNA sequence specific for a nucleic acid encoding a GM-CSF polypeptide) and at least one Cas nuclease (eg, a Cas9 nuclease) can be introduced into one or more T cells.

In some cases, when components of the CRISPR/Cas system (e.g., gRNA and Cas nuclease) are introduced into one or more T cells as a nucleic acid encoding those components (e.g., nucleic acid encoding gRNA and nucleic acid encoding Cas nuclease ), the nucleic acid may be in any suitable form. For example, the nucleic acid may be a construct (eg, an expression construct). The nucleic acid encoding at least one gRNA and the nucleic acid encoding at least one Cas nuclease may be in separate nucleic acid constructs or in the same nucleic acid construct. In some cases, a nucleic acid encoding at least one gRNA and a nucleic acid encoding at least one Cas nuclease may be in the same nucleic acid construct. The nucleic acid construct may be any suitable type of nucleic acid construct. Examples of nucleic acid constructs that can be used to express at least one gRNA and/or at least one Cas nuclease include, without limitation, expression plasmids and viral vectors (eg, lentiviral vectors). In cases where a nucleic acid encoding at least one gRNA and a nucleic acid encoding at least one Cas nuclease are in different nucleic acid constructs, the nucleic acid constructs may be of the same type or of different types. In some instances, a nucleic acid encoding at least one gRNA sequence specific for a nucleic acid encoding a cytokine polypeptide (e.g., a GM-CSF polypeptide) and a nucleic acid encoding at least one Cas nuclease may reside in the same lentiviral vector. . For example, a lentiviral vector encoding at least one gRNA sequence specific for a nucleic acid encoding a cytokine polypeptide (e.g., a GM-CSF polypeptide) encoding at least one gRNA comprising the sequence shown in SEQ ID NO: 1, and encoding at least one Cas9 nuclease can be used to construct ex vivo T cells with reduced expression of this cytokine (eg, GM-CSF polypeptide).

In some instances, components of the CRISPR/Cas system (eg, gRNA and Cas nuclease) can be introduced directly into one or more T cells (eg, as gRNA and/or Cas nuclease). gRNA and Cas nuclease can be introduced into one or more T cells separately or together. In cases where gRNA and Cas nuclease are introduced into one or more T cells together, the gRNA and Cas nuclease may be in a complex. When gRNA and Cas nuclease are in a complex, the gRNA and Cas nuclease can be attached covalently or non-covalently. In some cases, the complex comprising gRNA and Cas nuclease may also include one or more additional components. Examples of complexes that may include components of the CRISPR/Cas system (eg, gRNA and Cas nuclease) include, without limitation, ribonucleoproteins (RNPs) and effector complexes (eg, containing CRISPR RNA (crRNA, crRNA) and Cas nuclease). For example, at least one gRNA and at least one Cas nuclease may be included in the RNP. In some instances, the RNP may include gRNA and Cas nucleases in a ratio of about 1:1 to about 10:1 (e.g., about 1:1 to about 10:1, about 2:1 to about 10:1, about 3 :1 to about 10:1, about 5:1 to about 10:1, about 8:1 to about 10:1, about 1:1 to about 9:1, about 1:1 to about 7: 1, from about 1:1 to about 5:1, from about 1:1 to about 4:1, from about 1:1 to about 3:1, from about 1:1 to about 2:1, from about 2: 1 to about 8:1, about 3:1 to about 6:1, about 4:1 to about 5:1, or about 5:1 to about 7:1). For example, RNP may include gRNA and Cas nucleases in a ratio of about 1:1. For example, RNP may include gRNA and Cas nucleases in a ratio of about 2:1. In some cases, an RNP containing at least one gRNA sequence specific for a nucleic acid encoding a GM-CSF polypeptide (e.g., encoding at least one gRNA, including the sequence shown in SEQ ID NO: 1), and at least one Cas9 nuclease.

The components of the CRISPR/Cas system (e.g., gRNA and Cas nuclease) used for KO of a nucleic acid encoding a cytokine polypeptide (e.g., a GM-CSF polypeptide) can be introduced into one or more T cells (e.g., CART) by any suitable way. The method of introducing components of the CRISPR/Cas system into a T cell can be a physical method. The method of introducing the components of the CRISPR/Cas system into a T cell may be a chemical method. The method for introducing components of the CRISPR/Cas system into a T cell may be a particle-based method. Examples of methods that can be used to introduce components of the CRISPR/Cas system into one or more T cells include, without limitation, electroporation, transfection (eg, lipofection), transduction (eg, viral vector-mediated transduction), microinjection, and nucleofection. In some cases, when components of the CRISPR/Cas system are introduced into one or more T cells as a nucleic acid encoding the components, such nucleic acid encoding the components can be transduced into one or more T cells. For example, T cells (e.g., ex vivo T cells) can be transduced with a lentiviral vector encoding at least one gRNA sequence specific for a nucleic acid encoding a GM-CSF polypeptide (e.g., encoding at least one gRNA, including the sequence shown in SEQ ID NO: 1), and at least one Cas9 nuclease. In some cases, when components of the CRISPR/Cas system are introduced directly into one or more T cells, such components can be electroporated into one or more T cells. For example, an RNP comprising at least one gRNA sequence specific for a nucleic acid encoding a GM-CSF polypeptide (eg, encoding at least one gRNA including the sequence shown in SEQ ID NO: 1), and at least one Cas9 nuclease. In some cases, components of the CRISPR/Cas system can be introduced ex vivo into one or more T cells. For example, ex vivo construction of T cells with reduced levels of GM-CSF polypeptides may involve transducing the isolated T cells with a lentiviral vector encoding components of the CRISPR/Cas system. For example, ex vivo construction of T cells having reduced levels of GM-CSF polypeptides may involve electroporation of a complex comprising components of the CRISPR/Cas system into isolated T cells. In cases where T cells are engineered ex vivo to produce reduced levels of GM-CSF polypeptides, T cells can be obtained from any suitable source (e.g., a mammal, such as a mammal, to be treated, or a mammalian donor or cell line) .

In some instances, a T cell (e.g., CART) may be treated with one or more inhibitors of GM-CSF polypeptide expression or GM-CSF polypeptide activity to reduce expression of the GM-CSF polypeptide in that T cell (e.g., compared to a T cell that has not been treated with one or more inhibitors of GM-CSF polypeptide expression or GM-CSF polypeptide activity). An inhibitor of GM-CSF polypeptide expression or GM-CSF polypeptide activity may be any suitable inhibitor. Examples of inhibitors of GM-CSF polypeptide expression or GM-CSF polypeptide activity include, but are not limited to, nucleic acid molecules designed to induce RNA interference (e.g., siRNA molecule or shRNA molecule), antisense molecules, miRNAs, receptor blockade, and antibodies (e.g., antagonistic antibodies and neutralizing antibodies).

A T cell having (e.g., engineered to have) a reduced level of expression of one or more cytokines (e.g., a GM-CSF KO T cell) may express (e.g., be engineered to express) any suitable antigen receptor. In some cases, the antigen receptor may be a heterologous antigen receptor. In some cases, the antigen receptor may be a CAR. In some instances, the antigen receptor may be a tumor antigen (eg, tumor specific antigen) receptor. For example, a T cell can be engineered to express a tumor specific antigen receptor that targets a tumor specific antigen (eg, cell surface tumor specific antigen) expressed by a cancer cell in a mammal with cancer. Examples of antigens that can be recognized by an antigen receptor expressed on a T cell having reduced expression of a cytokine polypeptide (e.g., a GM-CSF polypeptide) as described herein include, but are not limited to, cluster of differentiation 19 (CD19), mucin 1 (MUC -1), human epidermal growth factor receptor 2 (HER-2), estrogen receptor (ER), epidermal growth factor receptor (EGFR), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), CA-125, epithelial tumor antigen (ETA), melanoma-associated antigen (MAGE), CD33, CD 123, CLL-1, E-cadherin, folic acid receptor alpha, folic acid receptor beta, IL13R, EGFRviii, CD22, CD20, kappa light chain, lambda light chain , desmopressin, CD44v, CD45, CD30, CD5, CD7, CD2, CD38, BCMA, CD138, FAP, CS-1 and C-met. For example, a T cell having a reduced level of GM-CSF polypeptides can be engineered to express an antigen receptor targeting CD19. An exemplary nucleic acid sequence encoding a CAR targeting CD19 (CAR19) is shown in FIG. five.

Any suitable method can be used to express an antigen receptor on a T cell having (eg, engineered to have) reduced expression of one or more cytokine polypeptides (eg, a GM-CSF KO T cell). For example, a nucleic acid encoding an antigen receptor can be introduced into one or more T cells. In some cases, viral transduction into a non-dividing cell may be used to introduce a nucleic acid encoding an antigen receptor. The nucleic acid encoding the antigen receptor can be introduced into the T cell by any suitable method. In some cases, a nucleic acid encoding an antigen receptor may be introduced into a T cell by transduction (eg, viral transduction using a retroviral vector, such as a lentiviral vector) or transfection. In some instances, a nucleic acid encoding an antigen receptor may be introduced ex vivo into one or more T cells. For example, ex vivo construction of T cells expressing an antigen receptor may involve transducing the isolated T cells with a lentiviral vector encoding the antigen receptor. In cases where T cells are engineered ex vivo to express an antigen receptor, T cells can be obtained from any suitable source (eg, from a mammal, such as a mammal, to be treated, or from a mammalian donor, or from a cell line).

In some cases, when a T cell that has (e.g., engineered to have) a reduced level of expression of one or more cytokine polypeptides (e.g., a GM-CSF KO T cell) also expresses (e.g., engineered to express) antigen receptor, such a T cell can be engineered to have a reduced level of expression of that cytokine and can be engineered to express the antigen receptor by any suitable method. In some cases, a T cell may be engineered first to have a reduced level of expression of a cytokine polypeptide (eg, a GM-CSF polypeptide) and then engineered to express an antigen receptor, or vice versa. In some instances, a T cell may be simultaneously engineered to have a reduced level of expression of one or more cytokine polypeptides (eg, a GM-CSF polypeptide) and to express an antigen receptor. For example, one or more T cells can be simultaneously introduced with one or more nucleic acids used to reduce the expression level of a cytokine polypeptide, such as a GM-CSF polypeptide (e.g., a lentiviral vector encoding at least one gRNA sequence specific for nucleic acid encoding this cytokine and at least one Cas9 nuclease or nucleic acid encoding at least one oligonucleotide complementary to the mRNA of this cytokine), and one or more nucleic acids encoding an antigen receptor (eg, CAR). One or more nucleic acids used to reduce the expression level of a cytokine polypeptide (e.g., a GM-CSF polypeptide) and one or more nucleic acids encoding an antigen receptor can be introduced into one or more T cells in separate nucleic acid constructs or in one nucleic acid construct. In some instances, one or more nucleic acids used to reduce the expression level of a cytokine polypeptide (e.g., a GM-CSF polypeptide) and one or more nucleic acids encoding an antigen receptor may be introduced into one or more T cells in a single construct. nucleic acid. In some cases, one or more nucleic acids used to reduce the expression level of a cytokine polypeptide (eg, a GM-CSF polypeptide) and one or more nucleic acids encoding an antigen receptor can be introduced ex vivo into one or more T cells. In cases where T cells are engineered ex vivo to reduce the expression levels of one or more cytokine polypeptides (e.g., a GM-CSF polypeptide) and to express an antigen receptor, the T cells can be derived from any suitable source (e.g., a mammal, such as the mammal being treated or the donor mammal or cell line).

In some instances, a T cell having (eg, engineered to have) reduced expression of one or more cytokine polypeptides (eg, a GM-CSF KO T cell) may be stimulated. The T cell can be stimulated concurrently with the production of a reduced level of one or more cytokine polypeptides, or independently of the production of a reduced level of one or more cytokine polypeptides. For example, one or more T cells having reduced levels of GM-CSF polypeptides used in adoptive cell therapy may be first stimulated and then engineered to have a reduced level of expression of GM-CSF polypeptides, or vice versa. In some instances, one or more T cells having reduced expression of a cytokine polypeptide (e.g., a GM-CSF polypeptide) used in adoptive cell therapy may first be stimulated and then engineered to have a reduced level of that polypeptide. -cytokine. T-lymphocytes can be stimulated by any suitable method. For example, a T cell can be stimulated by contacting the T cell with one or more CD polypeptides. Examples of CD polypeptides that can be used to stimulate T cells include, without limitation, CD3, CD28, inducible T cell costimulator (ICOS), CD137, CD2, OX40, and CD27. In some instances, the T cell may be stimulated with CD3 and CD28 prior to introducing into the T cell components of the CRISPR/Cas system (e.g., gRNA and/or Cas nuclease) for a KO nucleic acid encoding one or more cytokine polypeptides (e.g., a GM polypeptide -CSF).

Methods and materials used for cancer treatment are also provided. For example, one or more T cells having (e.g., engineered to have) reduced expression of a cytokine polypeptide (e.g., GM-CSF KO T cells) can be administered (e.g., in adoptive cell therapy such as CART therapy) to a mammal (eg, human) suffering from cancer to treat the mammal. In some instances, the methods of treating a mammal suffering from cancer as described herein can reduce the number of cancer cells (eg, cancer cells expressing a tumor antigen) in the mammal. In some instances, the methods of treating a mammal suffering from cancer disclosed herein can reduce the size of one or more tumors (eg, tumor antigen-expressing tumors) in the mammal.

In some instances, administration to a mammal of T cells having (eg, engineered to have) reduced expression of a cytokine polypeptide (eg, GM-CSF KO T cells) does not result in CRS. For example, administration of T cells having reduced levels of GM-CSF polypeptides to a mammal does not result in the release of CRS-associated cytokines (eg, CRS-critical cytokines). Examples of cytokines associated with CRS include, without limitation, IL-6, G-CSF, IFN-g, IL-1B, IL-10, MCP-1, MIG, MIP, MIP 1b, TNF-a, IL-2 and perforin.

In some instances, administration to a mammal of T cells having (eg, engineered to have) reduced expression of a cytokine polypeptide (eg, GM-CSF KO T cells) does not result in neurotoxicity. For example, administration of T cells having reduced levels of GM-CSF polypeptides to a mammal does not result in differentiation and/or activation of leukocytes whose differentiation and/or activation is associated with neurotoxicity. Examples of leukocytes whose differentiation and/or activation is associated with neurotoxicity include, without limitation, monocytes, macrophages, T cells, dendritic cells, microglia, astrocytes, and neutrophils.

Any suitable mammal (eg, human) suffering from cancer may be treated as described herein. Examples of mammals that can be treated as described herein include, without limitation, humans, primates (such as monkeys), dogs, cats, horses, cows, pigs, sheep, mice, and rats. For example, a person suffering from cancer can be treated with one or more T cells having (e.g., engineered to have) reduced expression of a cytokine polypeptide (e.g., a GM-CSF polypeptide), such as adoptive T cell therapy, such as CART cell therapy using the described methods and materials.

When treating a mammal (eg, human) suffering from cancer as described herein, the cancer may be any suitable cancer. In some cases, the cancer being treated as described herein may be a solid tumor. In some instances, the cancer being treated as described herein may be a hematologic cancer. In some cases, the cancer being treated as described herein may be a primary cancer. In some cases, the cancer being treated as described herein may be metastatic cancer. In some cases, the cancer being treated as described herein may be a refractory cancer. In some cases, the cancer being treated as described herein may be recurrent cancer. In some instances, a cancer treated as described herein may express a tumor-associated antigen (eg, an antigenic substance produced by a cancer cell). Examples of cancers that can be treated as described herein include, but are not limited to, B cell cancer (e.g. diffuse large B cell lymphoma (DLBCL) and B cell leukemia), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL ), follicular lymphoma, mantle cell lymphoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, acute myeloid leukemia (AML), multiple myeloma, head and neck cancer, sarcomas, breast cancer, gastrointestinal malignancies, bladder cancer, urothelial cancer , kidney cancer, lung cancer, prostate cancer, ovarian cancer, cervical cancer, cancer of the genital organs (eg, cancer of the male genital organs and cancer of the female genital organs), and bone cancer. For example, one or more T cells having (eg, engineered to have) reduced levels of GM-CSF polypeptides (eg, GM-CSF KO T cells) can be used to treat a mammal having DLBCL. For example, one or more T cells having (eg, engineered to have) reduced levels of GM-CSF polypeptides (eg, GM-CSF KO T cells) can be used to treat a mammal having ALL.

Any suitable method can be used to identify a mammal suffering from cancer. For example, imaging and biopsy techniques can be used to identify mammals (eg, humans) with cancer.

Once a cancer (eg, DLBCL or ALL) has been identified, one or more T cells having (eg, engineered to have) reduced expression of a cytokine polypeptide (eg, GM-CSF KO T cells) can be administered to the mammal according to the present invention. description.

For example, one or more T cells having (e.g., engineered to have) reduced expression of a cytokine polypeptide (e.g., GM-CSF KO T cells) can be used in adoptive T cell therapy (e.g., CART cell therapy) for the treatment of a mammal suffering from cancer. For example, one or more T cells having reduced levels of GM-CSF polypeptides can be used in adoptive T cell therapy (e.g., CART cell therapy) targeting any suitable antigen in a mammal (e.g., a mammal suffering from cancer) . In some cases, the antigen may be a tumor-associated antigen (eg, an antigenic substance produced by a cancer cell). Examples of tumor-associated antigens that can be targeted by adoptive T cell therapy provided herein include, without limitation, CD19 (associated with DLBCL, ALL and CLL), AFP (associated with germ cell tumors and/or hepatocellular carcinoma ), CEA (associated with bowel cancer, lung cancer and/or breast cancer), CA-125 (associated with ovarian cancer), MUC-1 (associated with breast cancer), ETA (associated with breast cancer), MAGE (associated with malignant melanoma), CD33 (AML associated), CD123 (AML associated), CLL-1 (AML associated), E-cadherin (epithelial tumor associated), folic acid receptor alpha (ovarian cancer associated), beta folic acid receptor (associated with ovarian cancer and AML), IL13R (associated with brain cancer), EGFRviii (associated with brain cancer), CD22 (associated with B-cell cancer), CD20 (associated with B-cell and cancers), kappa light chain (associated with B cell cancer), lambda light chain (associated with B cell cancer), CD44v (associated with AML), CD45 (associated with hematologic cancer), CD30 (associated with Hodgkin's and T-cell lymphomas), CD5 (associated with T-cell lymphomas), CD7 (associated with T-cell lymphomas), CD2 (associated with T-cell lymphomas), CD38 (associated with multiple myeloma and AML), BCMA (associated with multiple myeloma), CD138 (associated with multiple myeloma and AML), FAP (associated with solid tumors), CS-1 (associated with multiple myeloma) and c-Met (associated with breast cancer). For example, one or more T cells having reduced levels of GM-CSF polypeptides can be used in a CD19-targeting CART cell therapy (eg, CART19 cell therapy) to treat cancer as described herein.

In some instances, one or more T cells having (e.g., engineered to have) reduced expression of a cytokine polypeptide (e.g., GM-CSF KO T cells) may be used in adoptive T cell therapy (e.g., , CART cell therapy) for the treatment of a mammal suffering from a disease or disorder other than cancer. For example, one or more T cells having reduced levels of GM-CSF polypeptides can be used in adoptive T cell therapy (e.g., CART cell therapy) targeting any suitable disease-associated antigen (e.g., an antigenic substance produced by a cell afflicted with a certain disease) in the body of a mammal. Examples of disease-associated antigens that can be targeted by adoptive T cell therapy provided herein include, without limitation, desmopressin (associated with autoimmune skin diseases).

In some instances, one or more T cells having (eg, engineered to have) reduced expression of a cytokine polypeptide (eg, GM-CSF KO T cells) used in adoptive T cell therapy (eg, CART cell therapy) can be administered to a mammal suffering from cancer as a combination therapy with one or more additional agents used to treat cancer. For example, one or more T cells having a reduced level of GM-CSF polypeptides used in adoptive cell therapy may be administered to a mammal in combination with one or more anti-cancer treatments (eg, surgery, radiation therapy, chemotherapy (eg, with alkylating agents such as busulfan), targeted therapy (eg with GM-CSF inhibitory agents such as lenzilumab), hormonal therapy, angiogenesis inhibitors, immunosuppressants (eg with interleukin-6 inhibitory agents such as tocilizumab)) and/ or one or more treatments for CRS (eg, with ruxolitinib and ibrutinib). In cases where one or more T cells having a reduced level of GM-CSF polypeptides used in adoptive cell therapy are used with additional agents for cancer treatment, one or more additional agents may be administered simultaneously or independently. In some cases, one or more T cells having reduced levels of GM-CSF polypeptides used in adoptive cell therapy may be administered first and one or more additional agents second, or vice versa.

Further, the invention is described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES

Example 1 Production of Cytokine Deficient CART Cells to Increase The Therapeutic Index of CART Cell Therapy

This example describes the generation of GM-CSF knockout (GM-CSF KO) CART19 cells and shows that the resulting GM-CSF KO CART19 cells function normally and have increased levels of reproduction.

Experiment plan:

CAR19 has been used in B-cell leukemia xenografts. These plasmids were used to package and produce lentiviruses as described herein. Two models were used as a mouse model:

1. Xenograft Models: NSG mice were subcutaneously inoculated with CD19-positive, luciferase-positive NALM6 cells. Engraftment was confirmed by bioluminescent imaging. Mice were injected with human PBMC intravenously and lentiviral particles were injected into the tumor. The generation of CART cells was measured by flow cytometry. The movement of CART to tumor sites was assessed, and the antitumor response was measured using bioluminescent imaging as a measure of the severity (burden) of the disease.

2. Humanized Immune System (HIS) mice were obtained from Jackson's laboratory. These mice were injected with fetal CD34+ cells when they were newborns and consequently developed human hematopoiesis. These mice were inoculated with the CD19+ NALM6 cell line as previously described. Similarly, CART19 was generated in vivo by intratumoral injection of lentiviral particles. The NALM6 killing activity of CART19 cells was then measured and compared with ex vivo lentivirus transduced CART19 cells (currently used in the clinic).

Materials and methods:

Creation of the CAR plasmid:

Anti-CD19 clone FMC63 was synthesized de novo in a CAR framework using 4IBB and CD3-zeta and then cloned into a third-generation lentivirus framework.

To generate control CART19 cells, normal donor T cells were negative-selected with a T cell kit and expanded ex vivo using anti-CD3/CD28 Dynabeads (Invitrogen, which was added on the first day of culture). T cells were transduced with lentiviral supernatant one day after challenge with a MOI = 3. Anti-CD3/CD28 Dynabead was removed on day 6 and T cells were grown in T cell medium (X-vivo medium 15.5% human serum, penicillin, streptomycin, and glutamine) for 15 days and then cryopreserved for future experiments. Before all experiments, T cells were thawed and left overnight at 37°C.

Creation of CART cells with GM-CSF knockout:

GM-CSF CART knockout cells were generated using the CRISR-Cas9 system using two methods:

1. Created gRNA and cloned into a lentiviral vector encoding Cas9 and gRNA. During expansion of T cells, these cells were transduced with the above lentivirus on day 1, the same day, simultaneously with CAR19 lentiviral particles. Cells were expanded for 8 days and then harvested, DNA isolated and sequenced to assess knockout efficiency. These cells were frozen and used for future in vitro or in vivo experiments. The nucleic acid coding sequence is shown in FIG. five.

2. mRNA was generated from gRNA and used to knock out GM-CSF. For this, gRNA was mixed with RNP in a ratio of 1:1, and then T cells were subjected to electroporation on day 3 after stimulation with CD3/CD28 beads. Cells were expanded for 8 days and then harvested, DNA isolated and sequenced to assess knockout efficiency. These cells were cryopreserved and used for future in vitro or in vivo experiments.

Cells

The NALM6 cell line was derived from ATCC and maintained in RIO medium (RPMI medium, 10% fetal calf serum, penicillin and streptomycin). As indicated, NALM6 cells transduced with luciferase-GFP under the control of the EFla promoter were used in some experiments. Deidentified primary human ALL samples were obtained from the Mayo Clinic biobank. All samples were obtained after informed written consent. For all functional studies, cells were thawed at least 12 hours before analysis and left overnight at 37°C.

Analysis by flow cytometry

Anti-human antibodies were purchased from BioLegend, eBioscience or BD Biosciences. Cells were isolated from in vitro culture or obtained from animals, washed once with PBS supplemented with 2% fetal calf serum, and stained at 4°C after blockade of Fc receptors. Countbright beads (Invitrogen) were used for cell quantification according to the manufacturer's instructions (Invitrogen). In all assays, the population of interest was recorded based on forward and side scatter characteristics followed by singlet particle gating, and live cells were recorded using Live Dead Aqua (Invitrogen). Surface expression of anti-CD19 CAR was determined by staining with Alexa Fluor 647 conjugated goat anti-mouse F(ab') ₂ antibody from Jackson Immunoresearch.

T cell function assays:

Degranulation of T cells and analysis of intracellular cytokines:

Briefly, T cells were incubated with target cells in a ratio of 1:5. After staining for CAR expression; CD107a, CD28, CD49d and monensin were added during incubation. After 4 hours, cells were harvested and stained for CAR expression, CD3 and Live Dead staining kit (Invitrogen). Cells were fixed and permeabilized (FIX&PERM® Cell Fixation&Cell Permeabilization Kit, Life technologies), and then intracellular cytokine staining was performed.

Proliferation Assays:

T cells were washed and resuspended at a density of 1×10 ⁷ /ml in 100 μl PBS and labeled with 100 μl CFSE 2.5 μm (Life Technologies) for 5 minutes at 37°C. The reaction was then quenched with cold R10 and the cells were washed three times. The targets were irradiated at a dose of 100 Gy. T cells were incubated in a 1:1 ratio with irradiated target cells for 120 hours. Cells were then harvested, stained for CD3, CAR and Live Dead aqua kit (Invitrogen), and Countbright beads (Invitrogen) were added prior to flow cytometric analysis.

Cytotoxicity assays:

For cytotoxicity analysis, NALM6-Luc cells or CFSE-labeled primary ALL samples (Invitrogen) were used. Briefly, targets were incubated at the indicated ratios with effector T cells for 4, 16, 24, 48 and/or 72 hours. Killing was calculated either by bioluminescence imaging on a Xenogen IVIS-200 Spectrum camera or by flow cytometry. In the latter case, the cells were harvested; Countbright beads and 7-AAD (Invitrogen) were added prior to analysis. The remaining live target cells were CFSE+ 7-AAD-.

Measurement of secreted cytokines:

Effector and target cells were incubated at a 1:1 ratio in T cell medium for 24 or 72 hours as indicated. The supernatant was collected and analyzed with a 30-plex Luminex array according to the manufacturer's protocol (Invitrogen).

results

GM-CSF KO CART cells were created using the CRISR-Cas9 system. During T cell expansion, these cells were transduced (Day 1) with a lentivirus encoding gRNA and Cas9 and a lentivirus encoding CAR19. Cells were propagated for 8 days. After 8 days, T cells were harvested, DNA was isolated, and the isolated DNA was sequenced to assess knockout efficiency. See, for example, FIG. 1. T cells showed a knockout efficiency of 24.1% (FIG. 2A) and a CAR transduction efficiency of 73% (FIG. 2B).

To assess the cellular effector functions of GM-CSF KO CART CART19 cells, GM-CSF KO CART 19, UTD or GM-CSF KO UTD cells were co-cultured with the CD19-positive NALM6 cell line at a ratio of 1:5. After 4 hours, cells were harvested, permeabilized, fixed and stained for cytokines (FIG. 3).

To assess the proliferation of GM-CSF KO CART cells, the kinetics of expansion after transduction of T cells was observed. GM-CSF KO CART cells proliferated better than cells transduced with CART19 alone (FIG. 4).

These results demonstrate that GM-CSF knockout CART can enhance CART cell function and antitumor activity. These results also demonstrate that GMCSF blockade in combination with CART19 does not affect the effector functions of CART cells.

Example 2: GM-CSF Depletion During CART Therapy Reduces Cytokine Release Syndrome and Neurotoxicity and May Enhance CART Cell Function

In this example, the depletion of granulocyte and macrophage colony stimulating factor (GM-CSF) and myeloid cells was investigated as a potential strategy for managing toxicity associated with CART cells. Blockade of GM-CSF with a neutralizing antibody has not been found to suppress CART function in vitro or in vivo . CART cell proliferation was enhanced in vitro and CART cells provided more effective control of leukemia in patient-derived xenografts after GM-CSF depletion. Furthermore, in a primary acute lymphoblastic leukemia xenograft model for assessing CRS and NT, GM-CSF blockade resulted in reduced infiltration of myeloid cells and T cells in the brain and reduced the development of CRS and NT. Finally, GM-CSF knockout CART cells were generated by damaging the GM-CSF gene with the CRISPR/cas9 system during CART cell generation. GM-CSF ^k/o CART cells continued to function normally and resulted in enhanced antitumor activity in vivo . This suggests that GM-CSF neutralization can eliminate neurotoxicity and CRS and may also improve CART cell function.

Materials and methods

Cell lines and primary cells

NALM6 and MOLM13 were purchased from ATCC, Manassa, VA, USA, transduced with lentivirus luciferase-ZsGreen (addgene) and sorted to 100% purity. The isolated cells were cultured in RIO (RPMI, 10% FCS v/v, 1% penicillin and streptavidin v/v). Primary cells were obtained from the Mayo Clinic biobank for patients with acute leukemia in accordance with the protocol approved by the institutional review board. The use of recombinant DNA in the laboratory has been approved by the Institutional Biosafety Committee (IBC).

Primary T cells and CART cells

Peripheral blood mononuclear cells (PBMCs) were isolated from de-identified donated blood apheresis containers according to the FICOLL protocol (see, for example, Dietz et al, 2006 Transfusion 46:2083-2089). T cells were subjected to negative selection with magnetic beads (Stemcell technologies), and monocytes were subjected to positive selection with CD14+ magnetic beads (Stemcell technologies). Primary cells were cultured in X-Vivo 15 medium with 5% human serum, penicillin, streptomycin, and glutamax. CD19-targeted CART cells were generated by transduction of normal donor T cells with lentivirus as described below. The second generation CAR19 constructs were synthesized in do novo (IDT) and cloned into a third generation lentivirus under the control of the EF-1α promoter. The CD19 directed single chain variable fragment was generated from clone FMC63. A CAR construct co-stimulated with second generation 4IBB (FMC63-41BBz) was synthesized and used in these experiments. Lentiviral particles were obtained by transient transfection of the plasmid into 293T virus producing cells in the presence of Lipofectamine 3000, VSV-G and packaging plasmids. T cells derived from normal donors were stimulated with CD3/CD28 stimulating beads (StemCell) at a ratio of 1:3, and then transduced with lentiviral particles 24 hours after stimulation with an MOI of 3.0. The magnetic beads were removed on day 6 and CART cells were harvested and cryopreserved on day 8 for future experiments. CART cells were thawed and left in T cell medium for 12 hours prior to their use in experiments.

GM-CSF generation ^k/o CART cells:

Guide RNA (gRNA) targeting exon 3 of human GM-CSF was selected by screening for gRNAs previously reported to be highly effective against human GM-CSF.25. This gRNA was introduced into a third generation CAS9 lentiviral construct (lentiCRISPRv2) controlled by the U6 promoter (GenScript, Township, NJ, USA). Lentiviral particles encoding this construct were prepared as described above. T cells were double transduced with CAR19 with lentiviruses and GM-CSFgRNA-lentiCRISPRv2 24 hours after stimulation with CD3/CD28 beads. Then, the expansion of CAR-T cells was continued as described above. To analyze GM-CSF targeting efficiency, genomic DNA was extracted from GM-CSF ^k/o CART19 cells using the PureLink Genomic DNA Mini kit (Invitrogen, Carlsbad, CA, USA). DNA of interest was amplified by PCR using a Choice Taq Blue Mastermix (Thomas Scientific, Minneapolis, MN, USA) and extracted from the gel using a QIAquick gel extraction kit (Qiagen, Germantown, MD, USA) to determine editing. PCR amplicons were sent to Eurofins (Louisville, KY, USA) for sequencing and allele modification rates were calculated using the TIDE (Tracking Mutation Indel Digest) software available at tide.nki.nl. In FIG. 15 shows the gRNA sequence, primer sequences, and generation scheme for GM-CSF ^k/o CART19.

GM-CSF neutralizing antibodies and isotype controls

Lenzilumab (Humanigen, Brisbane, CA) is a humanized antibody that neutralizes human GM-CSF. For in vitro experiments , lenzilumab or an isotype control was used at 10 µg/mL. For in vivo experiments, 10 mg/kg of lenzilumab or an isotype control was administered, and the schedule, route, and frequency of administration are indicated in a separate experimental design. In some experiments, a neutralizing antibody to mouse GM-CSF (10 mg/kg) was also used, as indicated in the experimental design.

T cell function experiments

Cytokine assays were performed 24 or 72 hours after co-cultivation of CART cells with targets in a 1:1 ratio as indicated. GM-CSF analysis was performed in the mode of one human GM-CSF product per reaction (singleplex) (Millipore), 30 human GM-CSF products (Millipore), or 30 mouse GM-CSF products (Millipore) per reaction in the supernatant collected in these experiments. , as stated. Analysis was performed using a flow cytometry bead test or Luminex; intracellular cytokine assays and T cell degranulation assays were performed after incubation of CART cells with targets at a ratio of 1:5 for 4 hours at 37° C., in the presence of monensin, hCD49d and hCD28. After 4 hours, cells were harvested and intracellular stained after surface staining followed by fixation and permealization (FIX & PERM Cell Fixation & Cell Permeabilization Kit, Life Technologies). For proliferation assays, CFSE-labeled (Life Technologies) effector cells (CART19) and irradiated target cells were co-cultured in a 1:1 ratio. In some experiments, CD14+ monocytes were added to co-culture in a 1:1:1 ratio as indicated. The cells were co-cultured for 3-5 days as indicated in the specific experiment, and then the cells were harvested and surface stained with anti-hCD3 and Live/Dead Aqua. PMA/ionomycin was used as a positive non-specific T cell stimulator at various concentrations as indicated in specific experiments. For the CD19 ⁺ luciferase ⁺ ALL killing assay, the NALM6 cell line or CD19 ^- luciferase ⁺ MOLM13 control cells were incubated at the indicated ratios with effector T cells for 24 or 48 hours as specified in the specific experiment. Killing value was calculated using bioluminescence imaging with a Xenogen IVIS-200 Spectrum camera (PerkinElmer, Hopkinton, MA, USA) as a measure of remaining living cells. Samples were treated with 1 μl of D-luciferin (30 μg/ml) per 100 μl of sample 10 minutes prior to imaging.

Multivariable flow cytometry

Anti-human antibodies were purchased from Biolegend, eBioscience or BD Biosciences. Cells were isolated from in vitro culture or from animal peripheral blood (after ACK lysis), washed twice with phosphate-buffered saline supplemented with 2% fetal calf serum, and stained at 4°C. Countbright beads (Invitrogen) were used for cell quantification according to the manufacturer's instructions (Invitrogen). In all assays, the population of interest was recorded based on forward and side scatter characteristics followed by singlet particle gating, and live cells were recorded using Live Dead Aqua (Invitrogen). Surface expression of CAR was determined by goat anti-mouse F(ab') ₂ staining. Flow cytometry was performed on a four-laser Canto II analyzer (BD Biosciences). All analyzes were performed using FlowJo X10.0.7r2.

Xenogeneic mouse models

Male and female NOD-SCID-IL2rγ-/- (NSG) mice aged 8-12 weeks were bred and cared for in the Department of Comparative Medicine at the Mayo Clinic following a breeding protocol approved by the Institutional Animal Care and Use Committee (IACUC) . Mice were kept in animal enclosures approved by the institutional biosafety committee for experiments at the BSL2+ level.

NALM6 cell line xenografts

The CD19 ⁺ luciferase ⁺ ALL cell line NALM6 was used to create ALL xenografts. These xenograft experiments were approved by another IACUC protocol. In this case, 1×10 ⁶ cells were administered intravenously by injection into the tail vein. Six days post-injection, mice were subjected to bioluminescence imaging with a Xenogen IVIS-200 Spectrum camera to confirm engraftment. Imaging was performed after an intraperitoneal injection of 10 μl/g D-luciferin (15 mg/ml). Mice were then randomized based on the bioluminescent image to receive different treatment regimens as indicated in specific experiments. Typically, 1-2×10 ⁶ CART or UTD cells were administered, and the exact doses are indicated in the detailed description of the experiment. Weekly imaging was performed to assess and monitor disease severity. Blood samples from the tail vein were taken 7-10 days after the injection of CART cells to assess the expansion of T cells, and thereafter as needed. Mouse peripheral blood was lysed using ACK lysis buffer (Thermofisher), which was then used in flow cytometry analysis. Bioluminescent images were acquired with a Xenogen IVIS-200 Spectrum camera (PerkinElmer, Hopkinton, MA, USA) and analyzed with Living Image software version 4.4 (Caliper LifeSciences, PerkinElmer). Therapy for mice treated with antibodies included the administration of antibodies (10 mg/kg lenzilumab or isotype control) intraperitoneally for 10 days.

Patient-derived primary ALL xenografts

To create primary ALL xenografts, NSG mice were first injected with 30 mg/kg busulfan ip. The next day, mice were injected with 2×10 ⁶ primary blasts obtained from the peripheral blood of patients with relapsed refractory ALL. Graft engraftment in mice was followed for 4-6 weeks, and with continuous monitoring of blood levels of CD19+ cells (>1 cell/µl), mice were randomized to receive different courses of treatment with CART19 or UTD (1×10 ⁶ cells) in combination with therapy with or without antibodies (10 mg/kg of lenzilumab or IP isotype control for 10 days from the day of receiving CART cell therapy). Mice were monitored periodically for leukemic load by taking blood samples from the tail vein.

Patient-derived primary ALL xenografts for CRS/NT

As in the experiments described above, mice were intraperitoneally injected with 30 mg/kg of busulfan. The next day they were injected with 1-2×10 ⁶ primary blasts obtained from the peripheral blood of patients with relapsed refractory ALL. Graft engraftment in mice was observed for 4-6 weeks, and when CD19+ cell levels became high (>10 cells/µl), they were injected with CART19 (2-5×10 ⁶ cells) and started antibody therapy for 10 days, as indicated when describing specific experiments. Mice were weighed daily to assess their physical condition. Bryan MRI of mice was performed 5-6 days after CART injection, and blood samples were taken from the tail vein 4-11 days after CART injection. MRI images of the brain were analyzed using Azalyze.

Getting an MRI

A Bruker Avance II 7 Tesla Small Animal Vertical Hole MRI System (Bruker Biospin) was used to obtain images to assess central nervous system (CNS) vascular permeability. Anesthesia was performed by inhalation, which was maintained using 3-4% isoflurane. Respiratory rate was monitored during data collection sessions with an MRI compatible vital signs monitoring system (Model 1030; SA Instruments, Stony Brook, NY). Mice were given an intraperitoneal injection of gadolinium at a dose of 100 mg/kg based on weight, and after a standard 15 minute delay, a T1-weighted spin echo sequence was used to collect volumetric data (rep time=150 ms, echo time=8 ms , field of view: 32 mm x 19.2 mm x 19.2 mm, matrix: 160 x 96 x 96, number of averages = 1) to obtain T1-weighted images. Changes in MRI enhanced with gadolinium indicated a violation of the blood-brain barrier. Volumetric analysis was performed using the Analyze software package developed by the Biomedical Imaging Resource department at the Mayo Clinic.

RNA sequencing in mouse brain tissue

RNA was isolated using the miRNeasy Micro kit (Qiagen, Gaithersburg, MD, USA) and treated with an RNase free DNase kit (Qiagen, Gaithersburg, MD, USA). RNA sequencing was performed on an Illumina HTSeq 4000 (Illumina, San Diego, CA, USA) using the Genome Analysis Core at the Mayo Clinic. The binary representation of the data was converted to fastq format using the Illumina bcl2fastq software. Adapter sequences were removed with a Trimmomatic and FastQC was used for quality control. The latest reference human (GRCh38) and mouse (GRCm38) genomes were downloaded from NCBI. Genome index files were generated with STAR30 and paired end reads were mapped to the genome for each condition. HTSeq was used to calculate the number of expression events for each gene, and DeSeq2 was used to calculate differential expression. Gene ontology was evaluated using Enrichr. In FIG. 16 shows the steps described above. RNA sequencing data is available from the Gene Expression Omnibus website under accession number GSE121591.

Statistics

Prism Graph Pad and Microsoft Excel were used for data analysis. High concentrations of cytokines on the heat map were normalized to "1" and low concentrations were normalized to "0" using Prism. Statistical tests are described in the figure captions.

results

Neutralization of GM-CSF in vitro enhances the proliferation of CAR-T cells in the presence of monocytes and does not impair the effector function of CAR-T cells.

When using GM-CSF neutralization after CAR-T cell therapy as a strategy to prevent CRS and NT, it should not suppress the effectiveness of CAR-T cells. Thus, the initial experiments were aimed at studying the effect of GM-CSF neutralization on the effector functions of CAR-T cells. In these experiments, CART19 cells were cultured with or without the CD19 ⁺ ALL cell line NALM6 in the presence of lenzilumab (GM-CSF neutralizing antibody) or an isotype control (IgG). It was found that lenzilumab, but not the control IgG antibody, was indeed able to completely neutralize GM-CSF (FIG. 6A) but not to inhibit specific proliferation of CAR-T cell antigen (FIG. 6B). When CART19 cells were co-cultured with the CD19 ⁺ NALM6 cell line in the presence of monocytes, lenzilumab in combination with CART19 showed an exponential increase in CART19 antigen-specific proliferation compared to CART19 plus isotype control IgG (P<0.0001, Fig. 6C). To examine the specific cytotoxicity of CAR-T, either CART19 or control UTD T cells were cultured with the luciferase ⁺ CD19 ⁺ NALM6 cell line, which were then treated with either an isotype control antibody or a GM-CSF neutralizing antibody (Fig. 1D). Treatment with the GM-CSF neutralizing antibody did not inhibit the ability of CAR-T cells to kill NALM6 target cells (FIG. 6D). Overall, these results indicate that lenzilumab does not inhibit CAR-T cell function in vitro and enhances CART19 cell proliferation in the presence of monocytes, suggesting that GM-CSF neutralization may increase CAR-T cell-mediated efficacy.

Neutralization of GM-CSF in vivo enhances the antitumor activity of CAR-T cells in xenograft models.

To confirm that GM-CSF depletion does not suppress CART19 effector functions, the GM-CSF neutralizing effect of lenzilumab on the antitumor activity of CART19 was studied in xenograft models. First, a relapse model was used to carefully study the effect of GM-CSF neutralization on the antitumor activity of CART19 cells. NSG mice were injected with 1×10 ⁶ luciferase ⁺ NALM6 cells and then imaged 6 days later, allowing enough time for the mice to reach a very high tumor burden. Mice were randomized to receive a single injection of CART19 or UTD cells and isotype control antibody or lenzilumab for 10 days (FIG. 7A). Analysis of GM-CSF in serum collected 8 days after CART19 injection showed that lenzilumab successfully neutralized GM-CSF during CART19 therapy (Fig. 7B). Bioluminescent imaging one week after CART19 injection showed that CART19 in combination with lenzilumab effectively controlled leukemia in this high tumor burden recurrence model and was significantly better than control UTD cells (Fig. 7C). Treatment with CART19 in combination with lenzilumab provided potent antitumor activity and improved overall survival, similar to CART19 with control antibody, despite neutralization of GM-CSF levels, indicating that GM-CSF does not impair in vivo activity of CAR T cells (Fig. 8). Secondly, these experiments were performed on a xenograft model derived from a patient with primary ALL in the presence of human PBMC, since this model is a more suitable heterogeneous model. Following a busulfan chemotherapy conditioning regimen, mice were injected with blasts obtained from patients with relapsed ALL. Mice engraftment was monitored for several weeks by continuous tail vein blood sampling, and when blood levels of CD19 ⁺ blasts became >1/μL, mice were randomized to receive either CART19 or UTD treatment in combination with either PBMC or lenzilumab plus an anti-mouse antibody. neutralizing GM-CSF, or with isotype control IgG antibodies starting from the day of CART19 injection for 10 days (Fig. 7D). In this primary ALL xenograft model, neutralization of GM-CSF in combination with CART19 therapy resulted in a significant improvement in leukemia control, which was maintained for more than 35 days after CART19 administration, compared with CART19 plus isotype control (Fig. 7E). This suggests that GM-CSF neutralization may play a role in reducing relapses and increasing the duration of complete responses after CART19 cell therapy.

CAR-T cells with GM-CSF knockout by CRISPR show reduced GM-CSF expression, similar levels of key cytokines, and increased antitumor activity.

To safely rule out any critical role of GM-CSF in CAR T cell function, the GM-CSF gene was damaged in the generation of CAR T cells with a reportedly highly efficient gRNA that was cloned into the CRISPR lentiviral backbone. Using this gRNA, approximately 60% knockout efficiency was achieved in CART19 cells (FIG. 9). When CAR T cells were stimulated with CD19+ cell line NALM6, GM-CSF ^k/o CAR T cells produced statistically significantly lower levels of GM-CSF compared to CART19 with the wild-type GM-CSF locus ("wild-type CART19 cells"). Knockout of GM-CSF in CAR T cells did not disrupt the production of other key T cell cytokines, including IFN-γ, IL-2, or antigen-specific degranulation of CAR T cells (CD107a) (Fig. 10A), but resulted in a decrease in expression of GM-CSF (Fig. 10B). To confirm that GM-CSF ^k/o CAR-T cells continue to function normally, their efficacy was tested in vivo in a recurrent ALL xenograft model with a high tumor burden (as described in Fig. 7A). In this xenograft model, the use of GM-CSF ^k/o CART19 instead of wild-type CART19 resulted in a marked decrease in serum levels of human GM-CSF 7 days after CART19 treatment (FIG. 10B). Bioluminescent imaging data suggest that GM-CSF ^k/o CART19 cells provide enhanced leukemic control compared to CART19 in this model (FIG. 11). Importantly, GM-CSF ^k/o CART19 cells showed a significant improvement in overall survival compared to wild-type CART19 cells (FIG. 10). With the exception of GM-CSF, no statistically significant changes were found in either human cytokines (FIG. 10D) or mouse cytokines (FIG. 10E). Together, these results confirm the data shown in FIG. 6 and 7, indicating that GM-CSF depletion does not adversely affect cytokines that are critical to the functional efficiency of CAR-T. In addition, the results in Fig. 10 show that GM-CSF ^k/o CART may represent a therapeutic option for "built-in" control of GM-CSF as a modification during CAR-T cell production.

Patient Tumor Xenograft Model for Evaluation of Neurotoxicity and Cytokine Release Syndrome

In this model, conditioned NSG mice were inoculated with primary ALL blasts and observed for engraftment for several weeks until a high tumor burden was reached (FIG. 12A). When peripheral blood levels of CD19 ⁺ blasts reached >10/µl, mice were randomized to different therapies as indicated (FIG. 12A). Treatment with CART19 (with IgG control antibodies or anti-GM-CSF neutralizing antibodies) successfully eradicated the disease (FIG. 12B). Within 4-6 days after CART19 treatment, mice began to develop motor weakness, stoop and progressive weight loss: symptoms consistent with CRS and NT. This was because serum levels of key cytokines increased 4-11 days after CART19 injection, similar to that seen in human CRS following CAR T cell therapy (including human GM-CSF, TNF-α, IFN- γ, IL-10, IL-12, IL-13, IL-2, IL-3, IP-10, MDC, MCP-1, MIP-1α, MIP-1β and mouse IL-6, GM-CSF, IL -4, IL-9, IP-10, MCP-1 and MIG). These CART19-treated mice also developed NT, as evidenced by brain MRI analyzes showing an abnormal increase in T1, indicative of a breach in the blood-brain barrier and possibly cerebral edema (Fig. 12D), together with flow cytometry analysis of obtained brain samples, showing infiltration of human CART19 cells (FIG. 12E). In addition, RNA sequencing analysis of brain sections of mice that developed these features of NT showed significant upregulation of genes regulating the T cell receptor, cytokine receptors, T cell immune system activation, T cell transport, and T cell and myeloid cell differentiation. cells (table 1).

Table 1. Table of canonical pathways altered in the brain of mice with patient tumor xenografts after treatment with CART19 cells.

Canonical Path Speed p-value Genes Regulation of the immune response (GO:0050776) 9.45Е-14 IFITM1, ITGB2, TRAC, ICAM3, CD3G, PTPN22, CD3E, ITGAL, SAMHD1, SLA2, CD3D, ITGB7, SLAMF6, B2M, NPDC1, CD96, BTN3A1, ITGA4, SH2D1A, HLA-B, HLA-C, BTN3A2, HLA- A, CD8B, SELL, CD8A, CD226, CD247, CLEC2D, HOST, BIRC3 Cytokine-mediated signaling pathway (GO:0019221) 1.36T-12 IFITM1, SP100, TRADD, ITGB2, IL2RG, SAMHD1, IL27RA, OASL, CNN2, IL18RAP, RIPK1, CCR5, IL12RB1, B2M, GBP1, IL6R, JAK3, CCR2, IL32, ANXA1, IL4R, TGFB1, IL10RB, IL10RA, STAT2, PRKCD, HLA-B, HLA-C, IL16, HLA-A, TNFRSF1B, CD4, IRF3, OAS2, IL2RB, FAS, TNFRSF25, LCP1, P4HB, IL7R, MAP3K14, CD44, IL18R1, IRF9, MYD88, BIRC3 T cell receptor complex (GO:0042101) 1.30E-11 ZAP70, CD4, CD6, CD8B, CD8A, CD3G, CD247, CD3E, CD3D, CARD11 T cell activation (GO:0042110) 2.07E-11 ITK, RHOH, CD3G, NLRC3, PTPN22, CD3E, SLA2, CD3D, CD2, ZAP70, CD4, PTPRC, CD8B, CD8A, LCK, CD28, LCP1, LAT Regulation of T cell activation (GO:0050863) 2.46Е-10 PTPN22, LAX1, CCDC88B, CD2, CD4, LCK, SIT1, TBX21, TIGIT, JAK3, LAT, PAG1, CCR2 T cell receptor signaling (GO:0050852) 4.35E-08 ITK, BTN3A1, TRAC, WAS, CD3G, PTPN22, BTN3A2, CD3E, CD3D, ZAP70, CD4, PTPRC, LCK, GRAP2, LCP2, CD247, CARD11, LAT, PAG1 Upregulation of cytokine production (GO:0001819) 1.57502E-07 GBP5, ANXA1, TGFB1, CYBA, PTPN22, PARK7, TMEM173, CCDC88B, MAVS, CD6, IRF3, CD28, RIPK1, SLAMF6, CD46, IL12RB1, TIGIT, IL6R, CARD11, MYD88, CCR2 T cell differentiation (GO:0030217) 2.36E-07 ZAP70, CD4, ANXA1, PTPRC, CD8A, LCK, CD28, RHOH, PTPN22, CD3D Activation of the cytokine receptor (GO:0004896) 2.43E-07 IL4R, IL10RB, IL10RA, IL2RG, CD4, CXCR3, IL2RB, CCR5, IL12RB1, IL7R, IL6R, CD44, CCR2 Type I interferon signaling pathway (GO:0060337) 3.27E-07 IFITM1, SP100, IRF3, OAS2, STAT2, HLA-B, HLA-C, HLA-A, SAMHD1, IRF9, MYD88, OASL Cytokine response (GO:0034097) 0.0004679 SIGIRR, IFITM1, SP100, HCLS1, RIPK1, PTPN7, IKBKE, IL6R, JAK3, IL18R1, MYD88, AES Regulation of the innate immune response (GO:0045088) 0.001452 GBP5, GFI1, STAT2, ADAM8, NLRC3, PTPN22, SAMHD1, BIRC3 Regulation of necrosis factor production (GO:0032680) 0.003843 CD2, MAVS, CYBA, NLRC3, PTPN22, RIPK1, SLAMF1 T cell receptor binding (GO:0042608) 0.0102397 LCK, CD3G, CD3E Regulation of tumor necrosis factor-mediated signaling (GO:0010803) 0.0124059 SHARPIN, TRADD, CASP4, RIPK1, TRAF1, BIRC3 Positive myeloid differentiation (GO:0002763) 0.0376647 CD4, HCLS1, RIPK1, EVI2B

Neutralization of GM-CSF in vivo alleviates cytokine release syndrome and neurotoxicity after CART19 therapy in a xenograft model.

Using the patient-derived xenograft model for NT and CRS assessment shown in FIG. 4A examined the effect of GM-CSF neutralization on CART19 toxicity. To eliminate the concomitant effect of mouse GM-CSF, mice were injected with CART19 cells in combination with 10 days of anti-GM-CSF antibody therapy (10 mg/kg lenzilumab and 10 mg/kg mouse GM-CSF neutralizing antibody) or isotype control antibodies. Treatment with GM-CSF neutralizing antibodies prevented CRS-induced weight loss after CART19 therapy (FIG. 13A). Cytokine analysis 11 days after CART19 cell therapy showed that human GM-CSF was neutralized by the antibody (FIG. 13B). In addition, GM-CSF neutralization resulted in a significant reduction in several human (IP-10, IL-3, IL-2, IL-IRa, IL-12p40, VEGF, GM-CSF) (Fig. 5C) and murine (MIG , MCP-1, KC, IP-10) (Fig. 13D) cytokines. Interferon-gamma-induced protein (IP-10, CXCL10), among other cell types, is produced by monocytes and serves as a chemoattractant for many cell types, including monocytes, macrophages, and T cells. IL-3 plays a role in the differentiation of myeloid progenitors. IL-2 is a key cytokine in T cells. The interleukin-1 receptor antagonist (IL-IRa) inhibits IL-1. (IL-1 is produced by macrophages and is a family of critical inflammatory cytokines.) IL-12p40 is a subunit of IL-12 that is produced by macrophages, among other cell types, and may promote Th1 differentiation. Vascular endothelial growth factor (VEGF) promotes the formation of blood vessels. Monokine induced by interferon gamma (MIG, CXCL9) is a T cell chemoattractant. Monocytic chemoattractant protein 1 (MCP-1, CCL2) attracts monocytes, T cells, and dendritic cells. KC (CXCL1) is produced by macrophages, among other cell types, and attracts myeloid cells such as neutrophils. After neutralization of GM-CSF, a decrease in a number of other human and mouse cytokines was also observed. This suggests that GM-CSF plays a role in the downstream activity of several cytokines that play an important role in the cascade leading to CRS and NT.

Brain MRI 5 days after CAR19 treatment showed that GM-CSF neutralization reduced T1 elevation, measures of brain inflammation, blood-brain barrier disruption, and possibly edema, compared with CART19 plus control antibodies. MRI images after GM-CSF neutralization (with lenzilumab and anti-mouse GM-CSF antibody) were similar to pre-treatment scans, suggesting that GM-CSF neutralization contributed to the effective elimination of NT associated with CART19 therapy (Fig. 14A, B). When using human ALL blasts and human CART19 in this patient-derived xenograft model, neutralization of GM-CSF after CART19 provided a 59% reduction in neuroinflammation compared to CART19 plus isotype control (Fig. 14B). This is an important finding, and it has been demonstrated for the first time in vivo that NT caused by CART19 can be effectively eliminated. After CART19 therapy, human CD3 T cells were present in the brain, as determined by flow cytometry, and upon neutralization of GM-CSF, there was a trend towards a decrease in the number of CD3 T cells in the brain (Fig. 14C). Finally, in the brains of mice that received GM-CSF neutralization during CAR T cell therapy, there was a trend towards a decrease in CD11b+ bright macrophages compared to isotype control administered during CAR-T therapy (Fig. 14D), suggesting that neutralization of GM-CSF helps to reduce the number of macrophages in the brain.

Example 3 Interaction of GM-CSF with GM-CSF Receptors

Materials and methods

GM-CSF receptor (CSF2R) assay

Reproduction of T-cells. Isolated T cells were stimulated with CD3/CD28 beads. Expression of the CSF2 receptors CSF2RA (CD116) and CSF2RB (CD 131) was measured by flow cytometry on days 0, 1, 3, 6 and 8. Resting T cells were used as a negative control.

Getting CART. CART19 was generated and expression of CSF2RA (CD116) and CSF2RB (CD131) was measured by flow cytometry on days 0, 1, 3 and 6. Resting T cells were used as a negative control and the Nalm6 cell line was used as a positive control.

Western blotting

CART19/UTD cells and irradiated Nalm6 cells were co-cultured in a 1:1 ratio. Antibodies were added to the cells at a dose of 10 μg/ml. Culture conditions were as follows: UTD, CART19, CART19+GM-CSF blockade, CART19+CSF2RA blockade, and CART19+CSF2RB blockade. These conditions were tested with control medium for Nalm6 stimulation.

After culturing cells with antibodies for 24 hours, CART19 was isolated using microbeads. The purity of CART19 (98-100%) was confirmed by flow cytometry. Cells were harvested by centrifugation and polypeptides were isolated from the cells for use in Western blotting.

Results

Upon stimulation, the expression of GM-CSF receptors increased on T-cells and CART cells. Levels of GM-CSF receptors CSF2RA (CD116) and CSF2RB (CD131) on T cells were measured and compared with levels of CSF2RA (CD116) and CSF2RB (CD131) on resting T cells (negative control) according to the 8 day T cell expansion protocol . Expression of CSF2RA and CSF2RB increased after initial stimulation, peaked on day 3, and decreased slightly after a decrease in the number of granules on day 6 (Fig. 17A). The levels of CSF2RA (CD116) and CSF2RB (CD131) on CART19 and UTD cells were also measured and compared with levels of CSF2RA (CD116) and CSF2RB (CD131) on control cells during 8 days of CART production. Expression decreased slightly on day 1 but peaked on day 3 (FIG. 17B).

The interaction of GM-CSF with the CSF2 receptor is dependent on the beta chain (CSF2RB). The expression of phosphorylated Stat5 and phosphorylated Jak2 protein increased in the presence of irradiated Nalm6 and CSF2RA blockade, but decreased in the presence of GM-CSF and CSF2RB blockade (FIG. 18). FAS is downstream of the CSF2 receptor and is slightly downregulated in the presence of Nalm6 with GM-CSF blockade, but not in the presence of CSF2RA or CSF2RB blockade (FIG. 18).

These results demonstrate that CSF2R can be expressed on activated T cells and CART cells. These results also demonstrate that GM-CSF neutralization can inhibit p-STAT5. Similar inhibition was observed when the GM-CSF beta receptor was blocked, suggesting that signaling is driven by an interaction between GM-CSF and the GM-CSF beta receptor on activated CART cells.

Example 4: Transcription of GM-CSF and CART19 Cells

Materials and methods

RNA sequencing

RNA isolation and RNA sequencing. Total RNA was isolated from three biological replicas (normal donors 105, 115 and 116) for non-transduced T cells, CART19 and GM-CSF ^k/o CART19 using the miRNeasy Micro kit (QIAGEN) and treated with the RNase free DNase kit (QIAGEN). Paired-end RNA sequencing was performed on an Illumina HTSeq 4000 using the Genome Analysis Core at the Mayo Clinic.

Quality. The binary representation of the data was converted to fastq format using the Illumina bcl2fastq software. Adapter sequences were removed with Trimmomatic and FastQC was used for quality control.

Alignment. The latest reference human genomes were downloaded from NCBI (HG38). Genome index files were generated with STAR and paired end reads were mapped to the genome for each condition.

differential expression. HTSeq was used to calculate the number of expression events for each gene, and DeSeq2 was used to calculate differential expression. Conditions with less than 10 complete transcripts were filtered out. The adjusted p value between GM-CSF ^k/o CART19 and CART19 was calculated using the Benjamini-Hochberg method.

results

RNA sequencing was used to identify transcriptome differences between GM-CSF ^k/o CART19 cells and CART19 cells on day 8 of CART production. 236 genes were identified with significant differential expression according to a Benjamini-Hochberg adjusted p-value of <0.05 (FIG. 19A). Three different gene expression patterns were identified: genes whose expression was increased in GM-CSF ^k/o CART19 cells compared to CART19 cells and UTD cells (FIG. 19A, top); genes whose expression was downregulated in GM-CSF ^k/o CART19 cells compared to CART19 cells and UTD cells (FIG. 19A, middle); and genes whose expression in GM-CSF ^k/o CART19 cells was normalized to the level of UTD cells compared to CART19 cells (FIG. 19A, bottom). Among the genes with an average profile, FAS was identified, which is part of the Tec kinase pathway and is responsible for the induction of apoptosis. Genes were also identified that were significantly downregulated in GM-CSF ^k/o CART19 cells compared to CART19 cells (FIG. 19B). Notably, GM-CSF knockout in CART19 cells normalized the altered gene expression seen in CART19 cells (Fig. 19C). Principal component analysis showed that the overall gene expression pattern in GM-CSF ^k/o CART19 cells is more similar to that in UTD cells than in CART19 cells.

These results suggest that GM-CSF ^k/o cells may have a distinct gene expression pattern with an increase in repressed genes in GM-CSF ^k/o CART19 cells.

Example 5: Editing GM-CSF with CRISPR-Cas9

Materials and methods

Whole exome sequencing (WES)

Sample processing. DNA was isolated from three biological replicates: non-transduced T cells, GM-CSF ^k/o T cells, CART19 cells, and GM-CSF ^k/o CART19 cells using the PureLink Genomic DNA mini kit. The isolated DNA was sent to the Mayo Clinic Medical Genome Facility Genome Analysis Core for WES. Samples were sequenced on an Illumina HiSeq 4000.

Primary analysis. Primary analysis was performed in Core by converting the binary representation of the data to fastq format using the Illumina bcl2fastq software.

Secondary analysis. The fastq files were aligned with the HG38 human reference genome using the BWA-Mem alignment algorithm. Secondary analyzes were performed at the Department of Biomedical Statistics and Informatics using Genome Analysis ToolKit (GATK) software to call variants and generate raw VCF data.

Untargeted candidates. A specially designed SAS 9.4 code was used to find editing differences between samples. First tested a specific edit in the target gene CSF2. Variants found under GM-CSF ^k/o conditions were then compared, but not in their controls (non-transduced T cells and CART19), with the exception of variants that were inherent in the individual's genome (present in non-transduced T cells). Cross-references were then generated for these lists, and candidate CRISPR/Cas9 edits were classified into: T cells only, CART19 only, or both T cells and CART19. Alternate allele frequencies <10%, SNPs with read depth <10, and samples that did not pass the quality filter (VQSQRT analysis) were excluded. Done with the VennDiagram package in R.

Predictions of non-targeted candidates. Three different tools were used to predict editing of off-target candidates: Cas-OFFinder, CRISTA, and CCTop. The 15632 predictions generated with Cas-OFFinder (sequence query parameters: ≤6 mismatches, loop size DNA/RNA <2) included all predictions generated with CRISTA or CCTop. Thus, only Cas-OFFinder forecasts were used for the analysis. CRISPR/Cas9 editing candidates were compared to non-targeted CAS-OFFinder candidate predictions by matching the position containing the change to the chromosome.

Genomic representation. The edited CRISPR/Cas9 candidate list was compared with previously generated RNA sequencing data in the same biological replicas. The list of candidates was also filtered by genomic representation, according to the 1000 Genomes Project (allele frequencies <1% were considered rare).

Hypothesis testing. The number of single nucleotide variants (substitutions) or indels (inserts or deletions) in non-transduced T cells was compared with knockout T cells, the number in CART19 cells was compared with the number in knockout CART19 cells, and the number in all controls was compared with the number in all knockout cells. Differences in single nucleotide variants and indels were plotted on a violin plot using GraphPad Prism version 8.1.1 for Windows. Since the samples are dependent, the signed Wilcoxon rank test was used.

Results

WES was used to determine the exact locations of CRISPR-Cas9 editing of the CSF2 gene. CSF2 CRISPR gRNA 1 (SEQ ID NO: 7) was expected to have a 3 bp cut. above the PAM section (FIG. 20A, top panel). The actual cutting site was determined to be 6 bp. above the PAM region and can result in insertions and deletions at base 132074828 of chromosome 5 pattern (Fig. 20A, bottom panel). The difference in the Cas9 cleavage site may be due to the adjacent PAM site on the return chain. The frequency of insertions and deletions in each biological copy of CART19 is shown in FIG. 20A, bottom panel.

The number of single nucleotide variants (SNV) and the number of insertions/deletions (indel) were identified under CRISPR knockout conditions compared to their controls (T cells and CART19 cells) (FIG. 20B). No significant differences were found between groups (Wilcoxon signed rank test, p-value=0.16).

A representation of the common variants found in CRISPR-edited cells was also identified, compared to their respective control (CART19 or T cells). SNPs were preliminarily filtered for their presence in the population genome (less than 1% in the 1000 Genomes Project) and for presence in all three biological replicas. 0.004% (4) SNPs were found only in CART19 cells, 0.006% (5) SNPs were found only in T cells, 0.0001% (1) SNPs were found in both CART19 cells and T cells, and 99% (12,439) SNPs were filtered out (FIG. 20C). An SNP present in both T cells and CART19 cells (crossing in FIG. 20C) was identified as a deletion in the CSF2 gene shown in the bottom panel of FIG. 20A.

These results demonstrate that CSF2 editing resulted in only one editing result that matched the predictions of non-targeted CasOFFinder candidates, and that GM-CSF damage in CART19 cells with the CRISPR/Cas9 system is a safe method to knock out GM-CSF.

Example 6: GM-CSF and CART19 Monocyte Differentiation

Materials and methods

PBMCs were isolated and monocytes were isolated using the Classical Monocyte Isolation Kit (Miltenyi Biotec). Monocytes were then differentiated into M1 macrophages or M2 macrophages using the CellXVivo Human M1 or M2 Macrophage Differentiation Kit (R&D Systems).

CART19, Nalm6 cells and M1 macrophages or M2 macrophages were co-cultured at a ratio of 1:1:1. Cells were harvested on day 3 and stained for CD3 and Live Dead aqua (Invitrogen). Countbright beads (Invitrogen) were added prior to flow cytometric analysis for absolute quantitation.

Results

Neutralization of GM-CSF in the presence of M1 macrophages did not have a statistically significant change in CART19 proliferation upon CD19 stimulation; however, there was a trend towards increased proliferation of CAR-T cells with neutralization of GM-CSF. Blockade of GM-CSF statistically significantly increased CART19 proliferation upon CD19 stimulation in the presence of M2 macrophages (FIG. 21).

OTHER IMPLEMENTATION OPTIONS

It should be understood that although the invention has been described in detail, the foregoing description is intended to be illustrative and not to limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

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SEQUENCE LISTS

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<---

Claims (28)

1. A method for producing a T-cell with a chimeric antigen receptor having a reduced level of granulocyte-macrophage colony-stimulating factor (GM-CSF) polypeptides, comprising: introducing a nucleic acid construct into a T cell ex vivo, said nucleic acid construct comprising: a) a nucleic acid encoding a guide RNA, said guide RNA being complementary to a GM-CSF messenger RNA, and said guide RNA containing the nucleic acid sequence shown in SEQ ID NO: 1; b) a nucleic acid encoding the Cas nuclease, and c) a nucleic acid encoding said chimeric antigen receptor, wherein said nucleic acid encoding said chimeric antigen receptor comprises the nucleic acid sequence set forth in SEQ ID NO: 2, and wherein said chimeric antigen receptor targets tumor-associated CD19 antigen. 2. The method of claim 1 wherein said Cas nuclease is a Cas9 nuclease. 3. The method of claim 1 or 2, wherein said nucleic acid construct is a viral vector. 4. The method of claim 3, wherein said viral vector is a lentiviral vector. 5. The method according to any one of paragraphs. 1-4, wherein said administration step comprises transduction. 6. A method for producing a T-cell with a chimeric antigen receptor having a reduced level of granulocyte-macrophage colony-stimulating factor (GM-CSF) polypeptides, comprising: introducing the complex into a T cell ex vivo, wherein said complex comprises: a) a guide RNA, said guide RNA being complementary to a GM-CSF messenger RNA, and said guide RNA containing the nucleic acid sequence shown in SEQ ID NO: 1; and b) Cas nuclease; and introducing into said T cell ex vivo a nucleic acid encoding said chimeric antigen receptor, wherein said nucleic acid encoding said chimeric antigen receptor comprises the nucleic acid sequence set forth in SEQ ID NO: 2, and wherein said chimeric antigen receptor targets tumor-associated CD19 antigen. 7. The method of claim 6 wherein said Cas nuclease is a Cas9 nuclease. 8. The method according to claim 6 or 7, wherein said complex is a ribonucleoprotein. 9. The method according to any one of paragraphs. 6-8, wherein said administration steps include electroporation. 10. A method of treating a mammal suffering from cancer, comprising administering to said mammal T cells with a chimeric antigen receptor having a reduced level of granulocyte-macrophage colony stimulating factor (GM-CSF) polypeptides, wherein the T cells with a chimeric antigen receptor are obtained by introducing a nucleic acid construct into T ex vivo cell, said nucleic acid construct comprising: a) a nucleic acid encoding a guide RNA, said guide RNA being complementary to a GM-CSF messenger RNA, and said guide RNA comprising the nucleic acid sequence set forth in SEQ ID NO: 1; b) a nucleic acid encoding the Cas nuclease, and c) a nucleic acid encoding said chimeric antigen receptor, wherein said nucleic acid encoding said chimeric antigen receptor comprises the nucleic acid sequence set forth in SEQ ID NO: 2, and wherein said chimeric antigen receptor targets tumor-associated CD19 antigen. 11. The method of claim 10 wherein said mammal is a human. 12. The method of claim 10 or 11, wherein said cancer is lymphoma. 13. The method of claim 12 wherein said lymphoma is diffuse large B cell lymphoma. 14. The method of claim 10 or 11, wherein said cancer is leukemia. 15. The method of claim 14 wherein said leukemia is acute lymphoblastic leukemia. 16. A method for improving T cell effector functions of a T cell with a chimeric antigen receptor, comprising: introducing a nucleic acid construct into a T cell ex vivo, said nucleic acid construct comprising: a) a nucleic acid encoding a guide RNA, said guide RNA being complementary to a GM-CSF messenger RNA, and said guide RNA containing the nucleic acid sequence shown in SEQ ID NO: 1; b) a nucleic acid encoding the Cas nuclease, and c) a nucleic acid encoding said chimeric antigen receptor, wherein said nucleic acid encoding said chimeric antigen receptor comprises the nucleic acid sequence set forth in SEQ ID NO: 2, and wherein said chimeric antigen receptor targets tumor-associated CD19 antigen. 17. A method for improving T cell effector functions of a T cell with a chimeric antigen receptor, comprising: introducing the complex into a T cell ex vivo, said complex comprising: a) a guide RNA, said guide RNA being complementary to a GM-CSF messenger RNA, and said guide RNA containing the nucleic acid sequence shown in SEQ ID NO: 1; and b) Cas nuclease; and introducing into said T cell ex vivo a nucleic acid encoding said chimeric antigen receptor, wherein said nucleic acid encoding said chimeric antigen receptor comprises the nucleic acid sequence set forth in SEQ ID NO: 2, and wherein said chimeric antigen receptor targets tumor-associated CD19 antigen.

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