CD33 specific chimeric antigen receptors

Abstract

To provide chimeric antigen receptors (CARs) for cancer therapy.SOLUTION: The present invention provides an isolated nucleic acid encoding a chimeric antigen receptor (CAR), where the CAR comprises: (a) a CD33 antigen binding domain; (b) a stalk domain; (c) a transmembrane domain; (d) a costimulatory signaling domain comprising 4-1BB or CD28, or both; and (e) a CD3 zeta signaling domain.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application was filed on June 8, 2016, the entire contents of which are incorporated herein by reference. This application claims the benefit of U.S. Provisional Patent Application No. 62 / 347,503, filed on 2013-02-13.

[0002] Reference to sequence listing This application has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The present application contains a sequence listing, which is incorporated herein by reference in its entirety. The ASCII copy is named "50471_709_601_SL.txt" It is 103,120 bytes in size. [Background technology]

[0003] Presented herein are compositions and methods for treating cancer in humans. The invention further relates to a specific kinase for treating myeloid malignancies, such as acute myeloid leukemia. The present invention also relates to mela antigen receptors and vectors.

[0004] Acute myeloid leukemia (AML) is a type of cancer in which the bone marrow produces abnormal myeloblasts. AML is the most common form of acute leukemia in adults (Siegel, R., et al. L., Cancer Statistics, CA Cancer J Clin, 64(1):9-29 (2014)). AML is It is a rapidly progressive disease with a median age of 65-70 years. AML is an acute myeloid leukemia. acute myelocytic leukemia, acute myelogenous leukemia It is known by many names, including acute granulocytic leukemia, and acute nonlymphocytic leukemia. "Acute" means that the condition can progress rapidly and, if untreated, may progress within months of diagnosis. This indicates the aggressive nature of the disease, which can be fatal in a short time. The disease spreads rapidly through the bloodstream to other anatomical sites. It is more common in older people, although it can also be seen in older adults. The prevalence of AML increases with age, but people of all ages can develop AML. Approximately 8 in 10 people have AML, and approximately 1 in 6 children with leukemia have AML. In the United States, an average of 12,000 new cases of AML are currently expected each year. Approximately 30,000 patients are alive or experiencing remission. do.

[0005] Elderly AML patients (≥65 years) have a shorter median survival (65-74 years) Treatment options for patients with AML are limited. The outcome is usually poor, with an average 5-year survival rate of 20% and a median survival rate of 20% for those over 65 years of age. The 5-year survival rate for patients with glaucoma is less than 5% (Thein, M. et al., Outcome of older patients. nts with acute myeloid leukemia: an analysis of SEER data over 3 decades, Cancer , 119(15):2720-7 (2013)). A specific subgroup of AML is characterized by abnormalities within chromosome 7, complex nuclei, and type, arising from a previous myelodysplastic syndrome (MDS) or myeloproliferative neoplasm (MPN), recurrent In particular, patients with recurrent and / or refractory AML and AML have poorer outcomes. Patients aged 65 years or older with AML have a higher risk of developing unfavorable cell deaths than younger patients. These cytogenetic factors are likely to be related to resistance to chemotherapy. In addition to response rates, elderly patients with AML show significantly lower response rates to treatment. was considered a candidate for standard induction chemotherapy due to poor tolerability and procedural outcomes. Induction chemotherapy is often not given or is not chosen to be given to patients with this condition. Associated with high rates of treatment-related mortality and low rates of complete response (CR) in a subset of patients Currently, there are no approved therapies for patients with AML who are not treated with standard induction chemotherapy. Due to lack of treatment and cytogenetics associated with poor response rates, AML remains a popular Identify the medical need for new agents that demonstrate clinical benefit with a favorable safety profile. It is not fulfilled.

[0006] Hematopoiesis is a tissue-specific, hierarchical process that develops from pluripotent stem cells to more mature differentiated cell phenotypes. Similar to homeostatic hematopoiesis, AML accumulates within this dormant stem cell population. AML is thought to arise from mutations that result in leukemic stem cells (LSCs). The inability to eliminate the LSC population results in relapse and treatment failure. will be done.

[0007] Most patients with AML achieve remission with induction chemotherapy, but many experience post-remission Relapses occur despite intensive therapy. Relapses can occur anywhere from weeks to years later. Up to 10% of patients become resistant to induction chemotherapy. Both relapsed and refractory disease constitute a particularly poor risk group. Allogeneic stem cell transplantation is Although this is considered the recommended approach for the majority of these patients, it is feasible in only a minority of patients. and is associated with significant morbidity and mortality (Hamadani, M., Awan, F. & Copelan, E., Hematopoietic stem cell transplantation in adults with acute myeloid leukemi a, Biol Blood Marrow Transplant, 5:556-67 (2008)). In addition, The outcomes of transplanted patients are also poor (Duval, M. et al., Hematopoietic stem-cell transplantation splation for acute leukemia in relapse or primary induction failure, J Clin O ncol, 28(23):3730-8 (2010)), and nearly half of patients with recurrent disease develop chemotherapy resistance. and therefore unsuitable for transplantation (Hamadani, M., Awan, F. & Copelan, E., Hematopoietic stem cell transplantation in adults with acute myeloid leukemia Bi ol Blood Marrow Transplant, 5:556-67 (2008), Estey, E., 2013. Acute myeloid leuk emia: 2013 update on risk-stratification and management. Am J Hematol, 88(4), pp 318-27). Many new drugs and approaches are being explored for this group of patients. However, the CR rate is generally less than 30% (Litzow, M. et al., Failure of three no vel regimens to improve outcome for patients with relapsed or refractory acute m yeloid leukaemia: a report from the Eastern Cooperative Oncology Group, Br J Hae motol, 148:217-25 (2010), Cortes, J. et al., Phase 2 randomized study of p53 ant isense oligonucleotide (cenersen) plus idarubicin with or without cytarabine in refractory and relapsed acute myeloid leukemia, Cancer, 118(2):418-27 (2012), Ki rschbaum, M. et al., A phase 1 trial dose-escalation study of tipifarnib on a we ek-on, week-off schedule in relapsed, refractory or high-risk myeloid leukemia, Leukemia, 25(10):1543-7 (2011)).

[0008] In patients with AML, cytogenetics is an important prognostic factor in predicting response to treatment. (Grimwade, D. et al., The importance of diagnostic cytogenetics on outcome in AML: analysis of 1,612 patients entered into the MRC AML 10 trial. The Medica l Research Council Adult and Children's Leukaemia Working Parties, Blood, 92(7) :2322-33 (1998)). AML is associated with leukemic cells with t(8;21), t(15; Patients with t(17), t(16;16), or inv(16) translocations were treated with induction chemotherapy. However, chromosome 5 or Abnormalities of chromosome 7, 11q23, or complex karyotypes are not treated with currently available induction chemotherapy and Normal karyotype or trisomy 8 Patients with t(9;22) or t( 4;11) carries a very poor prognosis. Patients with AML due to t(9;22) However, chemotherapy alone rarely, if ever, produces a cure. Determination of antigen immunophenotype may aid in diagnosis and may be useful in the diagnosis of myeloid leukemia, T-cell leukemia, and This has important implications for the treatment and prognosis of leukemia and B-cell leukemia. Given the limited success of long-term survival in AML with current therapies, There is a need for treatment strategies to Summary of the Invention

[0009] One aspect of the present disclosure is an isolated nucleic acid encoding a chimeric antigen receptor (CAR), comprising: R consists of (a) the CD33 antigen-binding domain, (b) the stalk domain, and (c) the transmembrane domain. (d) at least one of 4-1BB and CD28, or a fragment thereof. (e) a CD3 zeta signaling domain, and Optionally, (f) a truncated epidermal growth factor receptor (HER1t or HER1t-1)-containing , relates to isolated nucleic acids.

[0010] As used herein, a scaffold and (a) a CD33 antigen-binding domain; and (b) a stalk domain. (c) a transmembrane domain; and (d) 4-1BB or CD28, or both. and (e) a CD3 zeta signaling domain. A vector comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR) is provided. In certain embodiments, truncated epidermal growth factor receptor (HER1t or HER1t-1), Alternatively, a vector further comprising a nucleic acid encoding full-length or truncated CD20 is provided. As shown.

[0011] As used herein, the scaffold and the polypeptide of the present invention comprise: (1) a truncated epidermal growth factor receptor or a functional variant thereof; (2) a CD33 antigen-binding domain, (b) a stalk domain, (c ) a transmembrane domain; (d) a co-stimulatory protein comprising 4-1BB or CD28, or both; (e) a chimera comprising a CD3 zeta signaling domain; and (f) a chimera comprising a CD3 zeta signaling domain. A vector comprising a nucleic acid sequence encoding an antigen receptor (CAR) is provided.

[0012] In certain embodiments, the scaffold and (1) full-length CD20 and its functional variants, and (2) (a) CD33 antigen-binding domain, (b) stalk domain, (c) transmembrane domain. (d) a costimulatory signaling domain that includes 4-1BB or CD28, or both; (e) a chimeric antigen receptor comprising a CD3 zeta signaling domain; A vector comprising a nucleic acid sequence encoding a receptor (CAR) is provided.

[0013] In certain embodiments, the scaffold comprises: (a) a CD33 antigen-binding domain; and (b) a stoichiometric antibody. (c) a transmembrane domain; (d) 4-1BB or CD28, or any of these. (e) a costimulatory signaling domain that contains both a CD3 zeta signaling domain and a CD4+ signaling domain; and (f) truncated epidermal growth factor receptor (HER1t or HER1t-1), full length C D20, and a chimera comprising at least one of a truncated form of CD20 (CD20t-1). and a nucleic acid sequence encoding a CAR. ed cells), e.g., immune effector cells, are presented.

[0014] In certain embodiments, (1) a truncated epidermal growth factor receptor (HER1t or HER 1t-1), and (2) (a) CD33 antigen-binding domain, (b) stalk domain (c) a transmembrane domain; and (d) 4-1BB or CD28, or both. and (e) a costimulatory signaling domain comprising a CD3 zeta signaling domain. Engineered cells, e.g., immune effector cells, containing a chimeric antigen receptor (CAR) such as can be.

[0015] In certain embodiments, (1) a kill switch, a selectable marker, a biomarker, or (2)(a) cell tags for use in combination with these, and (2)(b) CD33 antigen binding (b) synthase domain, (b) stalk domain, (c) transmembrane domain, (d) costimulatory signal (e) a chimeric antigen receptor comprising a CD3 zeta signaling domain. Modified cells, such as immune effector cells, containing a CAR are presented. In embodiments, the costimulatory signaling domain is at least one of 4-1BB and CD28. In some embodiments, the cell tag includes any one of HER1t, HER1t-1, CD 20, or CD20t-1.

[0016] As used herein, T cell-mediated immunity to a target cell population or target tissue in humans is A method for stimulating a response, comprising administering to a human an effective amount of a gene encoding a CAR. The method includes administering modified cells, the CAR comprising: (a) a CD33 antigen-binding domain; (b) the stalk domain, (c) the transmembrane domain, and (d) 4-1BB or CD2 8, or a costimulatory signaling domain that includes both of these; (e) CD3 zeta signaling domain (f) a truncated epidermal growth factor receptor (HER1t or HER1t A method is presented, including:

[0017] Provided herein is an isolated nucleic acid encoding a chimeric antigen receptor (CAR), wherein the CAR (a) CD33 antigen-binding domain, (b) stalk domain, (c) transmembrane domain (d) a costimulatory signaling domain comprising 4-1BB or CD28, or both; Main, (e) An isolated nucleic acid is presented that includes a CD3 zeta signaling domain.

[0018] In some embodiments, the CD33 antigen-binding domain has the amino acid sequence of: (a) SEQ ID NO:8 With row (hM195scFv), at least 90%, 91%, 92%, 93%, 94%, Polypeptides with 95%, 96%, 97%, 98%, 99% or 100% identity (b) at least 90% identical to the amino acid sequence of SEQ ID NOs: 9 and 10 (M2H12); , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or (c) a polypeptide having 100% identity to the amino acid sequences of SEQ ID NOs: 11 and 12. (DRB2) with at least 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, 99% or 100% identity to the polypeptide of (d) at least 90% identity with the amino acid sequence of SEQ ID NOs: 14 and 15 (My9-6); 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 1 The polypeptides include at least one of the polypeptides having 0.00% identity to the sequence of the polypeptide of the present invention.

[0019] Optionally, the CD33 antigen-binding domain comprises the amino acid sequence of SEQ ID NO:8 (hM19 5scFv) at least 90%, 91%, 92%, 93%, 94%, 95%, 96% %, 97%, 98%, 99%, or 100% identity to the polypeptide. In this case, the stalk domain has the amino acid sequence of SEQ ID NO: 22 (CD8 alpha hinge ) with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99%, or 100% identity to the polypeptide.

[0020] In some embodiments, the costimulatory signaling domain comprises 4-1BB. The costimulatory signaling domain of 4-1BB has at least one amino acid sequence with the amino acid sequence of SEQ ID NO:24. At least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity. In some embodiments, the signal transduction domain comprises CD28. The transduction domain has at least 90%, 91%, 92%, or 100% identity with the amino acid sequence of SEQ ID NO:28. %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical The present invention includes polypeptides having the following properties:

[0021] Optionally, the CD3 zeta signaling domain has the amino acid sequence of SEQ ID NO:26. of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9 In some embodiments, the polypeptides have 8%, 99%, or 100% identity to the polypeptide. Optionally, the isolated nucleic acid further comprises a truncated epidermal growth factor receptor. The growth factor receptor is HER1t, and has at least 90% affinity to the amino acid sequence of SEQ ID NO:32. %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and The polypeptides include those having 100% identity. The homologue is HER1t-1 and has at least 90% identity with the amino acid sequence of SEQ ID NO:54. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 1 00% identity to the polypeptide.

[0022] In some embodiments, the CAR is selected from the group consisting of SEQ ID NOs: 39, 41, 43, 45, 47, 49, 51 , 53, or 55. %, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity The present invention includes polypeptides comprising the above-mentioned

[0023] As used herein, a scaffold and (1) HER1t, HER1t-1, or a functional equivalent thereof (2)(a) a truncated epidermal growth factor receptor comprising at least one of the mutants; and CD33 antigen-binding domain, (b) stalk domain, (c) transmembrane domain, (d) a costimulatory signaling domain comprising 4-1BB or CD28, or both; and (e) a chimeric antigen receptor (CAR) comprising a CD3 zeta signaling domain. A vector comprising the nucleic acid sequence encoding the ribozyme is provided.

[0024] As used herein, the framework and (1) full-length CD20, truncated CD20 (CD20t-1), or or functional variants thereof, and (2) (a) a CD33 antigen-binding domain, (b ) the stalk domain, (c) the transmembrane domain, (d) 4-1BB or CD28, or (c) CD3 zeta signaling domains, which contain both of these co-stimulatory domains; and (d) CD3 zeta signaling domains, which contain both of these co-stimulatory domains. A vector comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising a transduction domain. is presented.

[0025] In some embodiments, the vector is a lentiviral vector, a retroviral vector. Optionally, the truncated epidermal growth factor receptor is a vector having the sequence At least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity Optionally, the vector comprises a polypeptide having a truncated CD20 (CD20t- 1), or a nucleotide sequence encoding this functional variant, -1 is a sequence having at least 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity Contains a polypeptide.

[0026] In some embodiments, the full length CD20 has at least one amino acid sequence identical to that of SEQ ID NO:36. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or a polypeptide having 100% identity. A nucleic acid encoding a truncated Thosea asigna virus (T2A) peptide Optionally, the vector backbone further comprises a nucleotide sequence encoding the Sleeping B The plasmid pFUGW is a DNA plasmid carrying the beauty transposon.

[0027] Optionally, the vector further comprises a promoter. is hEF1a1. Optionally, the CD33 antigen binding domain comprises (a) SEQ ID NO: At least 90%, 91%, 92%, or more of the amino acid sequence of No. 8 (hM195scFv) 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity (b) a polypeptide having the amino acid sequence of SEQ ID NO: 9 and SEQ ID NO: 10 (M2H12); At least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99%, or 100% identity to SEQ ID NOs: 11 and 1; 2 amino acid sequence (DRB2) at least 90%, 91%, 92%, 93%, 94% %, 95%, 96%, 97%, 98%, 99%, or 100% identity to and (d) a peptide having the amino acid sequence of SEQ ID NOs: 14 and 15 (My9-6). At least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, At least one of the polypeptides has 99% or 100% identity.

[0028] In some embodiments, the CD33 antigen-binding domain has the amino acid sequence of SEQ ID NO:8. , at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% %, 99%, or 100% identity to the polypeptide. The cDNA domain has at least one amino acid sequence of SEQ ID NO: 22 (CD8 alpha hinge). Also 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% , or a polypeptide having 100% identity. The transduction domain comprises 4-1BB. In some embodiments, the costimulatory signal of 4-1BB The transduction domain has at least 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity The nucleic acid sequence includes a nucleic acid sequence having the formula:

[0029] Optionally, the costimulatory signaling domain of the vector comprises CD28. Thus, the costimulatory signaling domain of CD28 has at least one amino acid sequence with the amino acid sequence of SEQ ID NO:28. At least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a nucleic acid sequence of the CD3 zeta The signaling domain has at least 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% The present invention includes nucleic acid sequences having an identity to the above-mentioned sequence.

[0030] In some embodiments, the vectors comprise plasmids. The vector comprises an expression plasmid. g Beauty transposon. In some embodiments, the immune effector cells are , may comprise the nucleotides disclosed herein.

[0031] As used herein, the present invention relates to a scaffold and a method for the preparation of a medicament for the treatment ... 2) (a) CD33 antigen-binding domain, (b) stalk domain, (c) transmembrane domain (d) costimulatory signaling involving 4-1BB or CD28, or both domain, and (e) a chimeric antigen receptor (C) containing the CD3 zeta signaling domain. and a nucleic acid sequence encoding the AR. .

[0032] The present specification further provides: (1) a kill switch, a selectable marker, a biomarker, or (2)(a) CD33 antigen-binding protein; and (2)(b) a CD33 antigen-binding protein. domain, (b) the stalk domain, (c) the transmembrane domain, (d) 4-1BB or (e) CD3 Immune effectors including chimeric antigen receptors (CARs) containing the zeta signaling domain Cells are also presented.

[0033] In some embodiments, the cell tag is HER1t, HER1t-1, CD20t-1, or or CD20. Optionally, the cell tag comprises HER1t, said HER1t being Optionally, the cell tag comprises the polypeptide sequence of SEQ ID NO: 32. wherein the HER1t-1 comprises the polypeptide sequence of SEQ ID NO:54. In some embodiments, the immune effector cells comprise a vector disclosed herein. The cells include T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), or a regulatory T cell. Optionally, the cell is a T cell, wherein the CD33 antigen-binding domain is a C Binding to D33 confers anti-tumor immunity.

[0034] As used herein, the present invention relates to a method for the treatment of a target cell population or a target tissue in a human subject in need thereof. A method for stimulating a T cell mediated immune response in a human subject, comprising administering to the human subject an effective amount of Administering a cell genetically modified to express a CAR, wherein the CAR is a) CD33 antigen-binding domain, (b) stalk domain, (c) transmembrane domain, ( d) a costimulatory signaling domain that contains 4-1BB or CD28, or both; (e) CD3 zeta signaling domain, and (f) truncated epidermal growth factor receptor. (HER1t), a method is presented.

[0035] In some embodiments, the human has been diagnosed with acute myeloid leukemia (AML). Thus, acute myeloid leukemia is relapsed or refractory AML.

[0036] An isolated nucleic acid encoding a chimeric antigen receptor (CAR), wherein the CAR comprises (a) SEQ ID NO: (b) a CD33 antigen-binding domain having the amino acid sequence of SEQ ID NO: 8; (c) a stalk domain having the amino acid sequence of SEQ ID NO: 28 (d) a HER1t having the amino acid sequence of SEQ ID NO: 32; and HER1t-1 having the amino acid sequence of SEQ ID NO: 54. (e) CD3 zeta signaling domain with the amino acid sequence of SEQ ID NO: 26; An isolated nucleic acid is provided, comprising:

[0037] Further provided herein is an isolated nucleic acid encoding a chimeric antigen receptor (CAR), comprising: AR comprises (a) a CD33 antigen-binding domain having the amino acid sequence of SEQ ID NO: 8; (c) a stalk domain with the amino acid sequence of SEQ ID NO: 24; (d) a costimulatory signaling domain comprising 4-1BB with the amino acid sequence of SEQ ID NO: 32; and a small portion of HER1t-1 having the amino acid sequence of SEQ ID NO:54. (e) a CD3 gene having the amino acid sequence of SEQ ID NO: 26; Also provided is an isolated nucleic acid comprising a signaling domain.

[0038] In some embodiments, the vector comprises one of the polynucleotides disclosed herein. Optionally, the vector is a lentiviral vector, a retroviral vector, or a Optionally, the vector is a viral vector or a non-viral vector. is a Sleeping Beauty transposon. The vector is a plurality of vectors.

[0039] A system for expressing a CAR in immune effector cells, comprising: In some embodiments, the system includes one or more vectors encoding an isolated nucleic acid that In some embodiments, the immune effector cell is a T cell or a NK cell. The system further comprises a nucleic acid encoding at least one additional gene. In some embodiments, the additional gene comprises a cytokine. IL-2, IL-15, IL-12, IL-21, and IL-15 and IL-15R Optionally, the cytokine comprises at least one of a fusion with In some embodiments, the cytokine is in a membrane-bound form.

[0040] Optionally, the system comprises one vector. The vector can be a lentiviral vector, a retroviral vector, or a non-viral vector. In some embodiments, the non-viral vector is a Sleeping Beauty vector. ty transposon. Optionally, the system is Optionally, the Sleeping Beauty transposase is Optionally, the sposase is SB11, SB100X, or SB110. The immune effector cell is a mammalian cell. In some embodiments, the immune effector cell is a The method of expressing a CAR in a cell comprises the step of: The method includes contacting the system with a

[0041] Provided herein is a method for stimulating the proliferation and / or survival of modified T cells, comprising: (a) Obtaining a sample of cells from a subject, the sample comprising T cells or precursors of T cells. (b) administering to the cell a gene encoding a gene encoding a nucleotide sequence according to any one of claims 1 to 13 and 44 to 45. One or more vectors encoding the isolated nucleic acid described herein, and a vector encoding a transposase. The mice were transfected with a vector that encodes the engineered CD33 CAR to generate a population of T cells expressing the engineered CD33 CAR. and (c) optionally, culturing the population of CD33 CAR T cells ex vivo. and culturing the cells for 2 days or less in step o. .

[0042] In some embodiments, the method includes transfecting a cell with a vector encoding a cytokine. Optionally, the cytokines include IL-15 and Optionally, the one or more vectors are fusion proteins comprising IL-15Rα. The vectors can be lentiviral, retroviral, or non-viral. In some embodiments, the non-viral vector is a Sleeping Beauty vector. Optionally, the method comprises: Optionally, the Sleeping Beauty transposase The enzyme is SB11, SB100X, or SB110.

[0043] The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the present invention and the advantages thereof will be readily apparent from the following detailed description of exemplary embodiments in which the principles of the present disclosure are employed. The foregoing can be obtained by reference to the following detailed description and accompanying drawings, which set forth: [Brief description of the drawings]

[0044] [Figure 1] FIG. 1 is a schematic diagram depicting the structure of CD33 isoforms. [Diagram 2] FIG. 1 depicts the CD33 CAR lentiviral vector map. [Diagram 3] FIG. 1 is an exemplary schematic diagram for the Sleeping Beauty transposon system. [Figure 4]FIG. 1 is an exemplary schematic diagram for lentiviral transduction to generate CAR-T cells. [Diagram 5] FIG. 1 depicts the expression of CD33 CAR and HER1t on adoptively transferred T cells. [Figure 6] FIG. 14 confirms Western blot confirmation of CD33 CAR expression from lentiviral transduced cells. [Figure 7] Figure 1 shows the kinetics of absolute number expansion of in vitro cultured CD33 CAR-T cells and fold increase of total T cell population from untransduced T cells (UNT) and CD33 CAR-T cells (CD33 CARLV) after stimulation with CD3 / CD28 beads. [Figure 8] FIG. 1 provides an assessment of CD33 CAR-T cell copy number after lentiviral transduction. [Figure 9-1] Figures 9A, 9B, and 9C: Figures supporting the target-specific in vitro cytotoxicity of CD33 CAR-T cells. CD33-specific cytotoxicity was measured by europium-labeled target cells (EL4, EL4 / CD33, and MOLM-13) (Figure 9A). Surface expression levels of CD33 on the tested cell lines were measured by flow cytometry. Bold shaded lines indicate staining with an isotype control antibody, and light shaded lines indicate staining with an anti-human CD33 antibody (Figures 9B and 9C). [Figure 9-2] Figures 9A, 9B, and 9C: Figures supporting the target-specific in vitro cytotoxicity of CD33 CAR-T cells. CD33-specific cytotoxicity was measured by europium-labeled target cells (EL4, EL4 / CD33, and MOLM-13) (Figure 9A). Surface expression levels of CD33 on the tested cell lines were measured by flow cytometry. Bold shaded lines indicate staining with an isotype control antibody, and light shaded lines indicate staining with an anti-human CD33 antibody (Figures 9B and 9C). [Figure 10A]Figures confirm the production of cytokines (IFNγ (Figure 10A), IL-2 (Figure 10B), and TNF (Figure 10C)) from ex vivo expanded CD33 CAR-T cells. [Figure 10B] Figures confirm the production of cytokines (IFNγ (Figure 10A), IL-2 (Figure 10B), and TNF (Figure 10C)) from ex vivo expanded CD33 CAR-T cells. [Figure 10C] Figures confirm the production of cytokines (IFNγ (Figure 10A), IL-2 (Figure 10B), and TNF (Figure 10C)) from ex vivo expanded CD33 CAR-T cells. [Figure 11] FIG. 1 shows cetuximab-induced antibody-dependent cell-mediated cytotoxicity (ADCC) of HER1t-expressing CD33 CAR-T cells. [Figure 12] FIG. 14 supports survival curves of NSG mice by treatment group in an AML tumor model. [Figure 13] Figures 13A and 13B: Quantitative analysis of tumor burden as measured by fLUC bioluminescence is presented: Figure 13A: ventral view and Figure 13B: dorsal view. [Figure 14A] FIG. 13 presents flow cytometry analysis of blood, bone marrow, and spleen samples from NSG mice treated with CD33 CAR-T cells. [Figure 14B] FIG. 13 presents flow cytometry analysis of blood, bone marrow, and spleen samples from NSG mice treated with CD33 CAR-T cells. [Figure 15] Figures 15A and 15B: Figures demonstrating the expression of CD33 CAR and HER1t on diverse human T cells transduced with LV. [Figure 16A]16A depicts a graph depicting flow cytometry results for CD33 expression on various AML cell lines (FIG. 16A). FIG. 16B depicts a graph depicting the results of a 2-hour europium release assay for various LV CD33 CARs on various CD33-expressing AML cell lines (FIG. 16B). FIG. 16C depicts the results of expression of cytokines released by T cells upon recognition of various CD33-expressing AML cell lines by LV CD33 CARs (FIG. 16C). [Figure 16B] 16A depicts a graph depicting flow cytometry results for CD33 expression on various AML cell lines (FIG. 16A). FIG. 16B depicts a graph depicting the results of a 2-hour europium release assay for various LV CD33 CARs on various CD33-expressing AML cell lines (FIG. 16B). FIG. 16C depicts the results of expression of cytokines released by T cells upon recognition of various CD33-expressing AML cell lines by LV CD33 CARs (FIG. 16C). [Figure 16C] 16A depicts a graph depicting flow cytometry results for CD33 expression on various AML cell lines (FIG. 16A). FIG. 16B depicts a graph depicting the results of a 2-hour europium release assay for various LV CD33 CARs on various CD33-expressing AML cell lines (FIG. 16B). FIG. 16C depicts the results of expression of cytokines released by T cells upon recognition of various CD33-expressing AML cell lines by LV CD33 CARs (FIG. 16C). [Figure 17] FIG. 1 presents a graph depicting lysis of CD33-expressing leukemia cell lines by CAR33-CD8a-CD28m-Z (Sleeping Beauty) T cells. [Figure 18] FIG. 13 presents graphs depicting flow cytometry results demonstrating the effect of CAR33-CD8a-CD28m-Z (Sleeping Beauty) T cells on CD107a degranulation and IFNγ production in the presence of CD33-expressing target cells. [Figure 19] FIG. 1 presents graphs depicting flow cytometry results supporting expression of CD33 CAR (Sleeping Beauty) and HER1t on genetically modified T cells. [Figure 20A] 20A depicts the effect of the CAR described herein in CD33-expressing AML cells. Figure 20A presents a graph depicting the flow cytometry results for CD33 expression on various AML cell lines. Figure 20B presents a graph depicting the results of a 2-hour europium release assay for CAR33-CD8a-CD28m-Z (Sleeping Beauty) on various CD33-expressing AML cell lines. [Figure 20B] 20A depicts the effect of the CAR described herein in CD33-expressing AML cells. Figure 20A presents a graph depicting the flow cytometry results for CD33 expression on various AML cell lines. Figure 20B presents a graph depicting the results of a 2-hour europium release assay for CAR33-CD8a-CD28m-Z (Sleeping Beauty) on various CD33-expressing AML cell lines. [Figure 21-1] FIG. 1 provides a sequence listing illustrating nucleic acid and polypeptide sequences for the methods and compositions described herein. [Figure 21-2] FIG. 1 provides a sequence listing illustrating nucleic acid and polypeptide sequences for the methods and compositions described herein. [Figure 21-3] FIG. 1 provides a sequence listing illustrating nucleic acid and polypeptide sequences for the methods and compositions described herein. [Figure 21-4] FIG. 1 provides a sequence listing illustrating nucleic acid and polypeptide sequences for the methods and compositions described herein. [Figure 21-5] FIG. 1 provides a sequence listing illustrating nucleic acid and polypeptide sequences for the methods and compositions described herein. [Figure 21-6] FIG. 1 provides a sequence listing illustrating nucleic acid and polypeptide sequences for the methods and compositions described herein. [Figure 21-7] FIG. 1 provides a sequence listing illustrating nucleic acid and polypeptide sequences for the methods and compositions described herein. [Figure 21-8] FIG. 1 provides a sequence listing illustrating nucleic acid and polypeptide sequences for the methods and compositions described herein. [Figure 21-9] FIG. 1 provides a sequence listing illustrating nucleic acid and polypeptide sequences for the methods and compositions described herein. [Figure 21-10] FIG. 1 provides a sequence listing illustrating nucleic acid and polypeptide sequences for the methods and compositions described herein. [Figure 21-11] FIG. 1 provides a sequence listing illustrating nucleic acid and polypeptide sequences for the methods and compositions described herein. [Figure 21-12] FIG. 1 provides a sequence listing illustrating nucleic acid and polypeptide sequences for the methods and compositions described herein. [Figure 21-13] FIG. 1 provides a sequence listing illustrating nucleic acid and polypeptide sequences for the methods and compositions described herein. [Figure 21-14] FIG. 1 provides a sequence listing illustrating nucleic acid and polypeptide sequences for the methods and compositions described herein. [Figure 21-15] FIG. 1 provides a sequence listing illustrating nucleic acid and polypeptide sequences for the methods and compositions described herein. [Figure 22A-1]Figures 22A and 22B present the effect of compositions described herein when contacted with tumor cells, including in vivo results in mice. Figure 22A presents a graph depicting cytokine analysis from plasma of mice administered CAR-T cells on day 11 of the study (3 days after CAR-T cell administration). Plasma from mice was collected and human cytokines were assessed by a multiplex analyte platform. Mean values ​​± SEM are shown for several analytes measured (GM-CSF, IFNγ, TNFα, IL-10, IL-18, and IP-10). N=11-15 mice per group, including mice that were part of satellite groups for tissue collection at the specified time points. *p<0.05; **p<0.01; ***p<0.001; One-way analysis of variance (ANOVA) with Tukey's multiple comparison test for saline, non-transduced T cells, and CD19-CAR-T cells for each treatment group. FIG. 22B presents a graph depicting a representative sampling of blood samples taken from AML tumor-bearing mice after treatment with CAR T cells. AML tumor-bearing mice were treated with saline, non-transduced T cells, a single dose of CD19-CAR-T cells, or CD33-CAR-T cells on day 8. The group noted as dose 2 received a second dose of equal doses of CD33-CAR-T cells alone on day 15 of the study. Blood samples were obtained from mice to assess the presence and persistence of CAR T cells, as well as the presence of MOLM-13 tumor cells (based on expression of CD123). (A) On day 16, when the mice became moribund, blood samples were taken from mice treated with saline alone, non-transduced T cells, or CD19-CAR-T cells. (B) On day 18, blood samples were taken from the remaining mice treated with CD33-CAR-T cells. Data shown was gated on FSC / SSC / human CD45+ cells. Figure 22C: Graph depicting cytotoxic activity against ALM tumor cell lines (THP1 and HL60). [Figure 22A-2]Figures 22A and 22B present the effect of compositions described herein when contacted with tumor cells, including in vivo results in mice. Figure 22A presents a graph depicting cytokine analysis from plasma of mice administered CAR-T cells on day 11 of the study (3 days after CAR-T cell administration). Plasma from mice was collected and human cytokines were assessed by a multiplex analyte platform. Mean values ​​± SEM are shown for several analytes measured (GM-CSF, IFNγ, TNFα, IL-10, IL-18, and IP-10). N=11-15 mice per group, including mice that were part of satellite groups for tissue collection at the specified time points. *p<0.05; **p<0.01; ***p<0.001; One-way analysis of variance (ANOVA) with Tukey's multiple comparison test for saline, non-transduced T cells, and CD19-CAR-T cells for each treatment group. FIG. 22B presents a graph depicting a representative sampling of blood samples taken from AML tumor-bearing mice after treatment with CAR T cells. AML tumor-bearing mice were treated with saline, non-transduced T cells, a single dose of CD19-CAR-T cells, or CD33-CAR-T cells on day 8. The group noted as dose 2 received a second dose of equal doses of CD33-CAR-T cells alone on day 15 of the study. Blood samples were obtained from mice to assess the presence and persistence of CAR T cells, as well as the presence of MOLM-13 tumor cells (based on expression of CD123). (A) On day 16, when the mice became moribund, blood samples were taken from mice treated with saline alone, non-transduced T cells, or CD19-CAR-T cells. (B) On day 18, blood samples were taken from the remaining mice treated with CD33-CAR-T cells. Data shown was gated on FSC / SSC / human CD45+ cells. Figure 22C: Graph depicting cytotoxic activity against ALM tumor cell lines (THP1 and HL60). [Figure 22B-1]Figures 22A and 22B present the effect of compositions described herein when contacted with tumor cells, including in vivo results in mice. Figure 22A presents a graph depicting cytokine analysis from plasma of mice administered CAR-T cells on day 11 of the study (3 days after CAR-T cell administration). Plasma from mice was collected and human cytokines were assessed by a multiplex analyte platform. Mean values ​​± SEM are shown for several analytes measured (GM-CSF, IFNγ, TNFα, IL-10, IL-18, and IP-10). N=11-15 mice per group, including mice that were part of satellite groups for tissue collection at the specified time points. *p<0.05; **p<0.01; ***p<0.001; One-way analysis of variance (ANOVA) with Tukey's multiple comparison test for saline, non-transduced T cells, and CD19-CAR-T cells for each treatment group. FIG. 22B presents a graph depicting a representative sampling of blood samples taken from AML tumor-bearing mice after treatment with CAR T cells. AML tumor-bearing mice were treated with saline, non-transduced T cells, a single dose of CD19-CAR-T cells, or CD33-CAR-T cells on day 8. The group noted as dose 2 received a second dose of equal doses of CD33-CAR-T cells alone on day 15 of the study. Blood samples were obtained from mice to assess the presence and persistence of CAR T cells, as well as the presence of MOLM-13 tumor cells (based on expression of CD123). (A) On day 16, when the mice became moribund, blood samples were taken from mice treated with saline alone, non-transduced T cells, or CD19-CAR-T cells. (B) On day 18, blood samples were taken from the remaining mice treated with CD33-CAR-T cells. Data shown was gated on FSC / SSC / human CD45+ cells. Figure 22C: Graph depicting cytotoxic activity against ALM tumor cell lines (THP1 and HL60). [Figure 22B-2]Figures 22A and 22B present the effect of compositions described herein when contacted with tumor cells, including in vivo results in mice. Figure 22A presents a graph depicting cytokine analysis from plasma of mice administered CAR-T cells on day 11 of the study (3 days after CAR-T cell administration). Plasma from mice was collected and human cytokines were assessed by a multiplex analyte platform. Mean values ​​± SEM are shown for several analytes measured (GM-CSF, IFNγ, TNFα, IL-10, IL-18, and IP-10). N=11-15 mice per group, including mice that were part of satellite groups for tissue collection at the specified time points. *p<0.05; **p<0.01; ***p<0.001; One-way analysis of variance (ANOVA) with Tukey's multiple comparison test for saline, non-transduced T cells, and CD19-CAR-T cells for each treatment group. FIG. 22B presents a graph depicting a representative sampling of blood samples taken from AML tumor-bearing mice after treatment with CAR T cells. AML tumor-bearing mice were treated with saline, non-transduced T cells, a single dose of CD19-CAR-T cells, or CD33-CAR-T cells on day 8. The group noted as dose 2 received a second dose of equal doses of CD33-CAR-T cells alone on day 15 of the study. Blood samples were obtained from mice to assess the presence and persistence of CAR T cells, as well as the presence of MOLM-13 tumor cells (based on expression of CD123). (A) On day 16, when the mice became moribund, blood samples were taken from mice treated with saline alone, non-transduced T cells, or CD19-CAR-T cells. (B) On day 18, blood samples were taken from the remaining mice treated with CD33-CAR-T cells. Data shown was gated on FSC / SSC / human CD45+ cells. Figure 22C: Graph depicting cytotoxic activity against ALM tumor cell lines (THP1 and HL60). [Figure 22C]Figures 22A and 22B present the effect of compositions described herein when contacted with tumor cells, including in vivo results in mice. Figure 22A presents a graph depicting cytokine analysis from plasma of mice administered CAR-T cells on day 11 of the study (3 days after CAR-T cell administration). Plasma from mice was collected and human cytokines were assessed by a multiplex analyte platform. Mean values ​​± SEM are shown for several analytes measured (GM-CSF, IFNγ, TNFα, IL-10, IL-18, and IP-10). N=11-15 mice per group, including mice that were part of satellite groups for tissue collection at the specified time points. *p<0.05; **p<0.01; ***p<0.001; One-way analysis of variance (ANOVA) with Tukey's multiple comparison test for saline, non-transduced T cells, and CD19-CAR-T cells for each treatment group. FIG. 22B presents a graph depicting a representative sampling of blood samples taken from AML tumor-bearing mice after treatment with CAR T cells. AML tumor-bearing mice were treated with saline, non-transduced T cells, a single dose of CD19-CAR-T cells, or CD33-CAR-T cells on day 8. The group noted as dose 2 received a second dose of equal doses of CD33-CAR-T cells alone on day 15 of the study. Blood samples were obtained from mice to assess the presence and persistence of CAR T cells, as well as the presence of MOLM-13 tumor cells (based on expression of CD123). (A) On day 16, when the mice became moribund, blood samples were taken from mice treated with saline alone, non-transduced T cells, or CD19-CAR-T cells. (B) On day 18, blood samples were taken from the remaining mice treated with CD33-CAR-T cells. Data shown was gated on FSC / SSC / human CD45+ cells. Figure 22C: Graph depicting cytotoxic activity against ALM tumor cell lines (THP1 and HL60). [Figure 23-1]Figures 23A-23E: Describe the generation of CD33-CAR-T cells from T cells of an AML patient and support the cytotoxic activity against AML tumor cells. Figure 23A: PBMCs from an AML donor were obtained and phenotypically characterized for expression of CD33 and CD3. Figure 23B: T cells were isolated from the PBMC fraction using a bead-based method and the unlabeled fraction was characterized for CD33+ tumor cells. Figure 23C: T cells were transduced with LV-CD33-CAR construct and show expression of the CAR at day 14 of culture. Figure 23D: Shows co-culture of CD33-CAR-T cells from an AML donor with MOLM-13 tumor cells or AML donor tumor cells at day 0 (when the assay was ready) and day 3. All cells were gated for FSC / SSC / viable cells. Figure 23E: Cytokine expression of CD33-CAR-T cells from an AML patient after co-culture with CD33-expressing tumor cells. Intracellular cytokine staining was performed on CD33-CAR-T cells (derived from AML donor E261) 18 hours after co-culture with the indicated tumor cells. T cells were cell surface stained and then stained for the cytokines IFNγ and IL-2 following fixation / permeabilization. Cells were gated based on FSC / SSC / Live / CD3 T cells. AML E261 cells refer to the patient's autologous CD33 tumor cells present in the sample. [Figure 23-2]Figures 23A-23E: Describe the generation of CD33-CAR-T cells from T cells of an AML patient and support the cytotoxic activity against AML tumor cells. Figure 23A: PBMCs from an AML donor were obtained and phenotypically characterized for expression of CD33 and CD3. Figure 23B: T cells were isolated from the PBMC fraction using a bead-based method and the unlabeled fraction was characterized for CD33+ tumor cells. Figure 23C: T cells were transduced with LV-CD33-CAR construct and show expression of the CAR at day 14 of culture. Figure 23D: Shows co-culture of CD33-CAR-T cells from an AML donor with MOLM-13 tumor cells or AML donor tumor cells at day 0 (when the assay was ready) and day 3. All cells were gated for FSC / SSC / viable cells. Figure 23E: Cytokine expression of CD33-CAR-T cells from an AML patient after co-culture with CD33-expressing tumor cells. Intracellular cytokine staining was performed on CD33-CAR-T cells (derived from AML donor E261) 18 hours after co-culture with the indicated tumor cells. T cells were cell surface stained and then stained for the cytokines IFNγ and IL-2 following fixation / permeabilization. Cells were gated based on FSC / SSC / Live / CD3 T cells. AML E261 cells refer to the patient's autologous CD33 tumor cells present in the sample. [Figure 23-3]Figures 23A-23E: Describe the generation of CD33-CAR-T cells from T cells of an AML patient and support the cytotoxic activity against AML tumor cells. Figure 23A: PBMCs from an AML donor were obtained and phenotypically characterized for expression of CD33 and CD3. Figure 23B: T cells were isolated from the PBMC fraction using a bead-based method and the unlabeled fraction was characterized for CD33+ tumor cells. Figure 23C: T cells were transduced with LV-CD33-CAR construct and show expression of the CAR at day 14 of culture. Figure 23D: Shows co-culture of CD33-CAR-T cells from an AML donor with MOLM-13 tumor cells or AML donor tumor cells at day 0 (when the assay was ready) and day 3. All cells were gated for FSC / SSC / viable cells. Figure 23E: Cytokine expression of CD33-CAR-T cells from an AML patient after co-culture with CD33-expressing tumor cells. Intracellular cytokine staining was performed on CD33-CAR-T cells (derived from AML donor E261) 18 hours after co-culture with the indicated tumor cells. T cells were cell surface stained and then stained for the cytokines IFNγ and IL-2 following fixation / permeabilization. Cells were gated based on FSC / SSC / Live / CD3 T cells. AML E261 cells refer to the patient's autologous CD33 tumor cells present in the sample. [Figure 24] FIG. 1 describes the expression of CD33 CAR and HER1t on NK cells. [Diagram 25] FIG. 1 describes the expression of HER1t and HER1t-1 tags on CD33 CAR NK cells. [Figure 26] FIG. 1 discloses the expression of CD20t-1 tag on CD33 CAR NK cells. [Figure 27] FIG. 14 demonstrates that CD33-CAR NK cells efficiently lyse NK-resistant CD33+ AML cells (ML-2, KG-1). [Figure 28]FIG. 14 demonstrates that CD33-CAR NK cells efficiently lyse an NK-resistant AML cell line (KG-1) at low E:T ratios. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] Unless otherwise specified, all technical terms used herein are and scientific terms have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In describing and claiming the invention, the following terminology is used:

[0046] definition In the claims and / or this specification, the term "comprising" may be used in conjunction with When used, the use of the words "a" or "an" means "one" can mean "one or more," "at least one," and "one or This also coincides with the meaning of "is more than one."

[0047] Throughout this application, the term "about" is used to refer to the device used to determine a value. This refers to the inherent variation in method error or the variation that exists between study subjects. Typically, the term is used to refer to approximately 1%, 2%, 3%, 4%, 5%, or %, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16 %, 17%, 18%, 19%, or 20% variation or less It is intended that.

[0048] The term "or" in the claims refers only to the alternative or to the alternative. "And / or" is used unless it is expressly indicated that items are mutually exclusive. ", however, this disclosure includes definitions that refer only to the alternatives, and "and / or We support the definition that refers to "or".

[0049] As used in this specification and claims, "comprising" means "comprise" and "comprises" are used to express the idea of ​​"including" something. g)"), "having" ("have" and "having "has" or any form of "having"); "including" ("include" and "includes" are examples of "including" "containing" (any form of "containing") or "containing " and "contains", any form of "containing" The term "list" may be inclusive or open-ended and may include additional, unrecited elements or methods. Any embodiment discussed herein does not exclude any method steps of the present invention. Alternatively, it is contemplated that the present invention may be practiced with respect to compositions and vice versa. Additionally, the compositions of the present invention can be used to accomplish the methods of the present invention.

[0050] As used herein, "synthetic" or "modified" refers to a chemical compound, as opposed to a naturally occurring compound. It refers to a compound formed or expressed through biological processes and / or human intervention.

[0051] "Isolation" refers to the removal of a nucleic acid from its natural environment. "Purification" refers to the removal of a given nucleic acid from its natural environment. may be isolated from nature (including genomic DNA and mRNA) or synthesized. Purity, whether pure (including cDNA) and / or amplified under laboratory conditions In this case, "purity" is a relative term and not "absolute purity." On the other hand, nucleic acids and proteins are formulated with a diluent or adjuvant. It is understood that, although it may be assumed that for practical purposes, it may be considered isolated. For example, the nucleic acid may be used in an acceptable carrier or diluent for introduction into a cell. Typically, it is mixed with

[0052] As used herein, a "polynucleotide" or "oligonucleotide" refers to a ribonucleotide. A polynucleotide of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Double-stranded and single-stranded DNA, triplex DNA, as well as double-stranded and single-stranded RNA The term also includes, for example, methylation and / or capping of polynucleotides. The term may also include modified forms of polynucleotides, such as those modified by ligation, as well as unmodified forms of polynucleotides. Unnatural or synthetic nucleotides, as well as nucleotide analogues It is also intended to include molecules that contain

[0053] "Polypeptide" refers to the terms "polypeptide", "peptide", and "protein". The term is used interchangeably with the term ribozyme and refers to a polymer of amino acid residues. A long protein, optionally glycosylated or within a given intracellular environment. It is a protein containing other modifications typical of.

[0054] Nucleic acids and / or nucleic acid sequences may be derived from natural or artificial common ancestral nucleic acids or ancestral nucleic acid sequences. A protein and / or protein sequence is "homologous" if it is derived from the sequence. their coding DNA is derived, naturally or artificially, from a common ancestral nucleic acid or ancestral nucleic acid sequence Homologous molecules are sometimes referred to as homologs. For example, Any naturally occurring protein may be modified by any available mutagenesis method. When expressed, this mutant nucleic acid produces the protein encoded by the original nucleic acid. Homology generally refers to the sequence of two or more nucleic acids or is inferred from the sequence identity between proteins (or their sequences). The exact percentage of identity between sequences is useful for determining the identity of the nucleic acid and protein of interest. Although varying, sequence identity of at least 25% is used to establish homology. Sequence identity, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% %, or 99% or more sequence identity may also be used to establish homology. Methods for determining percent sequence identity (e.g., data The following describes the default parameters for BLASTP and BLASTN: , which are publicly available.

[0055] "Identical" or "similar" in the context of two nucleic acid or amino acid sequences of a polypeptide The term "sequence identity" refers to the alignment of sequences over a specified comparison window for maximum similarity. As used herein, "a" refers to residues in two sequences that are the same when aligned together. A "comparison range" is at least about 20, typically about 50 to about 200, more typically about 100 to about 200. A segment of about 150 consecutive positions, in which the sequences are separated into two sequences, After proper alignment, segments that can be compared to the reference sequence by the same number of consecutive positions are generated. Methods of sequence alignment for comparison are well known in the art. The optimal sequence alignment for this purpose is described by Smith and Waterman, Adv. Appl. Math., 2:482 (1981) local homology algorithm; Needleman and Wunsch, J. Mol. Biol., 48:443 (1970) Alignment algorithm; Pearson and Lipman, Proc. Nat. Acad. Sci USA, 85:2444 (1988) similarity search methods; computerized implementations of these algorithms (International PC / Gen by lligentics, Mountain View Calif. CLUSTAL in the e-program, Wisconsin Genetics Software are Package, Genetics Computer Group (GCG) , 575 Science Dr., Madison, Wis., USA. , GAP, BESTFIT, BLAST, FASTA, and TFASTA. The CLUSTAL program can be performed by any of the following methods: Ins and Sharp, Gene, 73:237-244 (1988) and Higgins and Sharp, CABIOS, 5:151-153 (1989), Corpet et al., Nucleic Acids Res., 16:10881-10890 (1988), Huang et al., Computer Applications in the Biosciences, 8:155-165 (1992), and Pearson et al. al., Methods in Molecular Biology, 24:307-331 (1994). Alignment is also often performed by visual and manual alignment.

[0056] In one class of embodiments, the polypeptides herein are, e.g., Use BLASTP (or CLUSTAL, or any other available and a reference polypeptide or a fragment thereof, as determined by a sequence alignment software. At least 80%, 85%, 90%, 98% 99%, or 100% identical. In addition, nucleic acids can also be described in terms of the starting nucleic acid, e.g., they can be, e.g., Use default parameters, BLASTN (or CLUSTAL, or other As measured by any available alignment software, the reference nucleic acid or Fragments of 50%, 60%, 70%, 75%, 80%, 85%, 90%, 98%, 99%, Or they can be 100% identical. A molecule can have a certain percentage of identity to a larger molecule. This means that when two molecules are optimally aligned, The percentage of residues in the small molecule are arranged in the order in which the two molecules are optimally aligned. This means finding matching residues within a larger molecule.

[0057] The term "substantially identical" as applied to a nucleic acid sequence or an amino acid sequence means The acid or amino acid sequence is analyzed using the programs described above, e.g., using standard parameters. At least 90% sequence identity compared to a reference sequence using BLAST, or greater sequence identity, at least 95%, at least 98%, and For example, the BLASTN program The gram (for nucleotide sequences) has a default word length (W) of 11, a word length of 10 Use the expectation value (E) of 1, M=5, N=-4, and a comparison of both strands. For this purpose, the BLASTP program uses a word length (W) of 3, an expectation of 10, and value (E), and using the BLOSUM62 scoring matrix (Henikoff & H (See Enikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992)). The percentage is calculated by comparing two optimally aligned sequences over a comparison window. A portion of the polynucleotide sequence within the comparison region is determined to be the optimal alignment of the two sequences. To do this, the addition or deletion (i.e. , gaps). The percentage is the number of identical nucleic acid bases or amino acid residues in both sequences. Determine the number of positions that occur in the This is then divided by the total number of positions in the comparison region and the result multiplied by 100 to obtain the percentage sequence identity. In some embodiments, substantial identity is calculated by: The region of the sequence is at least about 100 residues long, and in some embodiments, In some embodiments, the sequences are substantially identical over at least about 150 residues. In some embodiments, the sequences are substantially identical over the entire length of the coding regions.

[0058] A "functional variant" of a protein disclosed herein is, for example, a variant having at least one, two, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 19, 20, or 30 conservative amino acid substitutions, or approximately these numbers of conservative amino acid substitutions. The term "conservative amino acid substitutions" or "conservative amino acid substitutions" may include the amino acid sequence of a reference protein with an amino acid substitution. The phrase "conservative mutation" refers to a substitution of one amino acid with another amino acid that shares a common property. The functional scheme that defines the common properties between individual amino acids is: By analyzing the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz, GE and Schirmer, RH, Principles of Protein Structure, Spring According to such an analysis, amino acids within a group have preferential interactions with each other. are actively exchanged and are therefore the most important in their influence on the overall protein structure. It is possible to define groups of amino acids that are similar to each other (Schulz, GE and Schi, supra). rmer, RH). Examples of conservative mutations are amino acid substitutions of amino acids within the above subgroups, e.g. For example, substitution of arginine with lysine, and vice versa, may be used to maintain a positive charge. replacement of aspartic acid with glutamic acid so as to maintain a negative charge; and The reverse substitution; replacement of threonine with serine, which would maintain a free -OH; and substitution of asparagine with glutamine such that a free -NH2 can be maintained.

[0059] Alternatively, or in addition, a functional variant contains at least one non-conservative amino acid substitution. "Non-conservative mutations" can include mutations that are related to the amino acid sequence of a reference protein. Amino acid substitutions between, for example, tryptophan for lysine, or phenylalanine In this case, non-conservative amino acid substitutions are required to produce functional mutants. Preferably, non-conservative amino acid substitutions do not interfere with or inhibit the biological activity of the polypeptide. The functional variant is expressed as a CAR such that the biological activity of the functional variant is increased compared to the parent CAR. The biological activity of the compound may be enhanced.

[0060] The proteins disclosed herein (including functional portions and functional variants thereof) , may contain synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known, for example, aminocyclohexane carboxylic acid, Leucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine in, trans-3-hydroxyproline and trans-4-hydroxyproline , 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine alanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylalanine, Xylglycine, Indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline Phosphorus-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl- N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ol Nitin, α-aminocyclopentane carboxylic acid, α-aminocyclohexane carboxylic acid, α-Aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carbo α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[0061] "CD33" is a 67 kDa single-pass transmembrane glycoprotein that binds sialic acid. It is a member of the globulin-like lectin (Siglecs) superfamily. CD3 3 contains the V-set Ig-like domain, which is responsible for binding to sialic acid, and its extracellular domain. It is characterized by a C2 set of Ig-like domains in the nucleus. As shown in Figure 1, lysing is a process in which the Ig-like domains of the V set, as well as the Ig short chain, which also lacks the disulfide bonds linking the Ig-like domains of the C2 set In healthy subjects, CD33 is primarily involved in normal multipotency. It is found on myeloid progenitor cells, unipotent colony forming cells, monocytes, and mature granulocytes. CD33 is expressed as a myeloid differentiation antigen on more than 80% of myeloid leukemia cells. It is expressed on normal hematopoietic stem cells or mature granulocytes (Andrews, R. et al. al., The L4F3 antigen is expressed by unipotent and multipotent colony-forming CD33 is a marker for the expression of leukocytes in the inflammatory process. It has been reported to be expressed on myeloid cells, activated T cells, and activated NK cells. and is found in at least a subset of blast cells in the majority of patients with AML (Pollar d, J. et al., Correlation of CD33 expression level with disease characteristics and response to gemtuzumab ozogamicin containing chemotherapy in childhood AML, Blood, 119(16):3705-11 (2012)). In addition to its widespread expression on AML blasts, CD33 is It can also be expressed on the stem cells that underlie AML.

[0062] The term "substantially purified" refers to a nucleic acid, polypeptide, protein, or other The compounds may be polynucleotides, proteins, polypeptides, and other molecules with which they are naturally associated. Essentially free, i.e., not more than about 50%, more than about 70%, of the molecule Contains no, or more than about 90% free of, any nucleic acid sequence, polypeptide, protein or other chemical It refers to a mixture.

[0063] As used herein, a "coding sequence" refers to a polynucleotide that encodes a protein. A region or sequence includes a sequence that is adjacent to the 5' end and bounded by an initiation codon. Near the 3' end, a stop codon is bounded. A coding sequence may also be an open reading frame. It may also be called a frame.

[0064] As used herein, "operably linked" refers to a DNA segment that is in communication with another DNA segment. A segment, the physical and / or functional linkage to the A segment, The term refers to a linkage that enables the nucleic acid to function in its intended manner. The DNA sequence may be, for example, a promoter, enhancer, and / or silencer. Regulatory sequences, such as sensors, directly or indirectly modulate the transcription of DNA sequences. A regulatory sequence is operably linked when it is linked in a manner that allows for the expression of the regulatory sequence. For example, a DNA sequence is operably linked to a promoter. A transcription start site is located downstream of the correct reading frame. When ligated to a promoter within the genome, the DNA sequence is used to regulate transcription. An enhancer or silencer is a gene that allows the elongation of a gene to proceed. Ligation to a DNA sequence increases or decreases transcription of that DNA sequence, respectively. A gene product is operably linked to a DNA sequence encoding a gene product when the gene is Enhancers and silencers can also be located upstream of the coding region of a DNA sequence. , which may be located downstream or embedded within it. When expressed as a preprotein that participates in the secretion of a polypeptide, the signal sequence The DNA is operably linked to the DNA encoding the polypeptide. Linkage to regulatory sequences is typically by ligation at appropriate restriction sites, and is inserted into the sequence using restriction endonucleases known to those skilled in the art, Or via a linker.

[0065] The term "transcriptional regulator" refers to a promoter-driven DNA Biochemical elements that act to prevent or inhibit transcription of a sequence (e.g., replicase or nuclear inhibitory proteins), or under certain environmental conditions, A biosynthetic molecule that acts to enable or stimulate the transcription of a motor-driven DNA sequence. "Enhanced" refers to a genetic element (e.g., an inducer or enhancer) that is capable of regulating a gene expression.

[0066] The term "induction" refers to the transcription, promotion, or other action of a nucleic acid sequence, which is brought about by a transcriptional regulator. activity of the target gene and / or expression of the promoter relative to some basal level of transcription. Refers to the rise.

[0067] "Promoter" refers to a region of a polynucleotide that induces transcription of a coding sequence. A promoter is a molecule that is located near the transcription start site of a gene, on the same strand of DNA, and A promoter is located upstream (towards the 5' region of the sense strand). Since they are active under all circumstances, they are constitutive, whereas other promoters, e.g. Inducible promoters are regulated and active in response to a specific stimulus.

[0068] The term "promoter activity" refers to an operably linked promoter whose activity is being measured. It refers to the degree of expression of the nucleotide sequence to which it is linked. Direct analysis was performed by determining the amount of RNA transcripts produced by immunoblot analysis. Alternatively, the expression level can be measured by using a linked reporter nucleic acid sequence, such as a reporter nucleic acid sequence linked to a promoter. It can also be measured indirectly by determining the amount of a product encoded by a nucleic acid sequence. Cut.

[0069] As used herein, an "inducible promoter" refers to a promoter that is regulated by a transcriptional regulator, e.g., a biological factor. Or it refers to a promoter that is induced to activity by the presence or absence of an abiotic factor. Inducible promoters are promoters that regulate the expression of genes operably linked to them in an organism or in particular in a This is useful because it can be turned on or off at certain stages of tissue development. Examples of inducible promoters include alcohol-regulated promoters, tetracycline-regulated promoters, promoter, steroid-regulated promoter, metal-regulated promoter, pathogenesis-regulated promoter promoters, temperature-regulated promoters, and light-regulated promoters. In some embodiments, the inducible promoter is part of a gene switch.

[0070] As used herein, the term "enhancer" refers to, for example, a An enhancer is a DNA sequence that increases the transcription of a nucleic acid sequence encoded by the They can be located many kilobases away from the target region, and are involved in the binding of regulatory factors and DNA In the art, various different Many enhancers are known and have been cloned from various sources. Available as a nucleic acid fragment or within a cloned polynucleotide (e.g., (e.g., from depositories such as the ATCC, as well as other commercial or private sources). Many polynucleotides, including promoters (such as the commonly used CMV promoter), are It also includes enhancer sequences. Enhancers may be located upstream of a coding sequence. It may be located within or downstream of this. The term "enhancer" refers to an enhancer region that maps within the immunoglobulin (Ig) locus. Enhancer elements derived from (such enhancers include, for example, the heavy chain (mu) 5' enhancer, light chain (kappa) 5' enhancer, kappa intron enhancer This refers to enhancers that are either - or mu intronic, as well as 3' enhancers. In general, see Paul WE (ed), Fundamental Immunology, 3rd Edition, Raven Press, New York. See, e.g., 1993, pages 353-363, and U.S. Pat. No. 5,885,827. sea ​​bream).

[0071] An "expression vector" or "vector" refers to an autonomous vector that mediates replication of a polynucleotide within a cell. behaves as a unit (i.e., capable of replicating under its own control), or is any genetic element capable of replication upon insertion into a host cell chromosome, e.g. , plasmids, chromosomes, viruses, transposons, etc. and / or by incorporating another polynucleotide segment into said Suitable vectors include plasmids, transposons, Vectors include, but are not limited to, bacteriophages and cosmids. Ligation or insertion of the vector into a host cell of the joined segments is performed. The host cell may contain polynucleotide sequences necessary to effect expression of the host cell. Such sequences may be Varies depending on the host organism; promoter sequences that effect transcription, enhancers that increase transcription The sequence includes a sequence for ribosome binding sites, and a transcription / translation termination sequence. The vector involves ligation or integration of the vector into the DNA sequence of the host cell. It may be possible to directly express the product of a nucleic acid sequence encoded therein without the use of a recombinant vector.

[0072] A vector may also contain a "selectable marker gene." The term "selectable marker gene" refers to a gene that identifies cells expressing a nucleic acid sequence in the presence of a corresponding selectable agent. The term refers to a nucleic acid sequence that allows specific selection in the forward or reverse direction of the nucleic acid sequence in the presence of the nucleic acid. Suitable selectable marker genes are known in the art and can be found, for example, in International Patent Application Publication No. Publication No. 1992 / 08796 and No. 1994 / 28143 Wigler et al., Proc. Natl. Acad. Sci. USA, 77: 3567 (1980), O'Hare et al. , Proc. Natl. Acad. Sci. USA, 78: 1527 (1981), Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78: 2072 (1981), Colberre-Garapin et al., J. Mol. Biol., 150:1 (1981 ), Santerre et al., Gene, 30: 147 (1984), Kent et al., Science, 237: 901-903 (19 87), Wigler et al., Cell, 11: 223 (1977), Szybalska & Szybalski, Proc. Natl. Aca d. Sci. USA, 48: 2026 (1962); Lowy et al., Cell, 22: 817 (1980); and U.S. Pat. The invention described in U.S. Pat. Nos. 5,122,464 and 5,770,359 It is being done.

[0073] In some embodiments, the vector is capable of replicating in a host cell and can be appropriately selected. Under selective pressure, they persist as extrachromosomal segments of DNA within the host cell, known as "episodes." A gene expression vector or an episome (e.g., Conese et al., Gene Therapy, 2003). (See, e.g., J. Immunol., 11:1735-1742 (2004)). Representative, commercially available episomal expression vectors include , Epstein-Barr nuclear antigen 1 (EBNA1) and Epstein-Barr virus (EB V) Episomal plasmids that use an origin of replication (oriP), including but not limited to: The vectors pREP4, pCEP4, and pREP7, as well as the Invitrogen pcDNA3.1 and Stratagen from N (Carlsbad, Calif.) pBK-CMV from Eppendorf (LaJolla, Calif.) contains EBNA1 and oriP Non-limiting examples of episomal vectors using the T antigen and SV40 origin of replication instead of This shows a typical example.

[0074] A "transposon" or "transposable element" (TE) is a gene that determines its location in the genome. Change, possibly creating or storing mutations, and altering the genome size of the cell , the vector DNA sequence. Transposition often results in duplication of the TE. Class I TEs are copied in two steps: first, class I TEs are copied from DNA to RNA. The resulting RNA is then reverse transcribed into DNA. The replicated DNA is inserted into a new location in the genome. The transcription factor is catalyzed by the reverse transcriptase enzyme, which can be encoded by the genome. , as well as retroviruses such as HIV. The transposition mechanism does not involve an RNA intermediate. Transposition is mediated by several transposase enzymes. Some transposases bind nonspecifically to any target site in DNA. whereas other transposases bind to specific DNA sequence targets. The enzyme produces staggered cleavages at the target site, resulting in 5 This results in single-stranded DNA overhangs (sticky ends) at the ' or 3' ends. The first step is to excise the DNA transposon, which then transforms it into a new The gap-filling DNA polymerase then ligates to the new target site. The activity of the merase and the activity of the DNA ligase that joins the sugar-phosphate backbone together (close) This results in duplication of the target site. are created by staggered cuts in the target DNA and filling in by DNA polymerase. Short direct repeats that can be inserted into the TE and subsequent excision of the TE by transposase During the S phase of the cell cycle, the donor Metastasis occurs when the site has already replicated but the target site has not yet replicated. The cut-and-paste TE is overlapped. The metastasis is classified as Class I TE and Class II TE. In either case, they can be classified as "autonomous" or "non-autonomous." Autonomous TEs can move on their own, whereas non-autonomous TEs require the help of another TE to move. This is because non-autonomous TEs require the presence of a transposase (in the case of class II) or or (in the case of class I) lack of reverse transcriptase.

[0075] "Transposase" refers to a gene that binds to the ends of transposons and functions as a cut-and-paste enzyme. Catalyzes the movement of a transposon to another part of the genome by a replicative or replicative transfer mechanism. This refers to an enzyme that

[0076] "Sleeping Beauty (SB) transposon system" refers to a DNA sequence that is It refers to a synthetic DNA transposon system for introduction into vertebrate chromosomes. For some exemplary embodiments, see, for example, U.S. Pat. No. 6,489,458. , 8,227,432, 9,228,180 and International Publication No. It is described in the brochure No. 2016 / 145146. Sleeping B The beauty transposon system is a Sleeping Beauty (SB) transposon. In some embodiments, the Sleeping Beauty transposon system, SB11 transposon system, SB100X transposon system The nucleic acid may include a nucleotide sequence of the SB110 transposon system, or a SB110 transposon system.

[0077] The nucleic acid sequences and vectors disclosed or contemplated herein can be "transfected" into cells. The introduction can be by "transfection," "transformation," or "transduction." As used in this publication, "transfection," "transformation," or "transduction" refers to the process of The addition of one or more exogenous polynucleotides to the target cell by using physiological or chemical methods. Transfection refers to the introduction of a gene into a host cell. Many transfection methods are known in the art. For example, the calcium phosphate DNA coprecipitation method (see, for example, Murray EJ (ed.), Meth ods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols, Humana Press (1991); DEAE-dextran method; electroporation; cationic Liposome-mediated transfection; tungsten particle-promoted gene gun method (Johnst on, Nature, 346: 776-777 (1990)); and strontium phosphate DNA co-precipitation ( Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)). Phage vectors or Viral vectors are expressed in suitable packaging cells, many of which are commercially available. The infectious particles can then be multiplied and introduced into host cells.

[0078] As used herein, "T cell" or "T lymphocyte" refers to a T cell that is involved in cell-mediated immunity. A "T cell" or "T lymphocyte" is a type of lymphocyte that plays a central role in The presence of T cell receptors (TCR) on the cell surface allows B cells and natural killer cells They can be distinguished from other lymphocytes, such as NK cells.

[0079] "Helper T cells" (TH cells) regulate the development of B cells into plasma cells and memory B cells. in immune processes, including maturation, and activation of cytotoxic T cells and macrophages; These cells also carry the CD4 glycoprotein on their surface. Helper T cells are also known as CD4+ T cells because they express the antigen-presenting Peptide antigens are presented by MHC class II molecules expressed on the surface of antigen-receptor cells (APCs). Once activated, helper T cells divide rapidly and produce active immune cells. Small proteins called cytokines that regulate or support the response These cells are classified as TH1, TH2, TH3, TH17, Th9, or T There are several subtypes, including FH, that secrete different cytokines and induce different types of immunity. They can differentiate into one of several subtypes that facilitate immune responses. Lymphocyte transduction directs T cells towards specific subtypes.

[0080] "Cytotoxic T cells" (TC cells or CTLs) are antiviral agents that inhibit the proliferation of virus-infected and tumor cells. These cells also have a number of markers on their surface that destroy the endothelial cells and are involved in graft rejection. These cells express the CD8 glycoprotein and are therefore known as CD8+ T cells. These cells react with antigens associated with MHC class I molecules, which are present on the surface of all nucleated cells. Regulatory T cells recognize their targets by binding to IL-1, which is secreted by regulatory T cells. Through erythropoietin, adenosine, and other molecules, CD8+ cells are involuntarily put into an anergic state. This may be activated, thereby preventing autoimmune diseases.

[0081] "Memory T cells" are antigen-specific T cells that persist for a long time after an infection has cleared. Memory T cells are a subset of cells that are re-exposed to their cognate antigen. Once the immune system is infected, it rapidly expands into large numbers of effector T cells, providing the immune system with the ability to fight off previous infections. Memory T cells provide "memory" against infection. There are three subtypes of memory T cells: central memory -T cells (TC M cells) and two types of effector memory T cells (T EM Cells and T E MRA Memory cells can be either CD4+ or CD8+ cells. Memory T cells typically express the cell surface proteins CD45RO, CD4 5RA, and / or CCR7.

[0082] "Regulatory T cells" (Treg cells), formerly known as suppressor T cells, ) play a role in maintaining immune tolerance. The primary role of regulatory T cells is to regulate T cell-mediated By blocking the immune response, autoreactive T cells escape the negative selection process in the thymus. The aim is to suppress cells.

[0083] "Natural killer T cells" (NKT cells: often confused with natural killer cells of the innate immune system) The major histocompatibility complex (MCC) mediates the adaptive immune system and the innate immune system. Unlike conventional T cells, which recognize peptide antigens presented by MHC molecules, NKT The cells recognize glycolipid antigens presented by a molecule called CD1d. These cells have functions ascribed to both Th and Tc cells (i.e., cytotoxicity, immune function, immune response ... NKT cells can also play a role in the regulation of immune responses by secreting cytokines and releasing cytolytic / cell-killing molecules. , which recognizes and eliminates some tumor cells and cells infected with herpes viruses It is also possible.

[0084] As used herein, a "tumor antigen" refers to an antigen that is produced or overexpressed in a tumor cell. Tumor antigens refer to any antigenic substance that is capable of eliciting an immune response in a host, e.g. Alternatively, for purposes of this disclosure, a tumor antigen is an antigen that is expressed by both healthy and tumor cells. However, to identify certain tumor types, there may be proteins that are suitable therapeutic targets. In one embodiment, the tumor antigen is a tumor antigen specific for a myeloid malignancy, such as acute myeloid leukemia (AML). and CD33, a target of CAR T cell therapy for treating

[0085] As used herein, "AML" refers to acute myeloid leukemia, acute granulocytic leukemia, and AML refers to acute myeloid leukemia, also known as acute nonlymphocytic leukemia. The other major form of leukemia is leukemia, with eight different subtypes based on the cell type from which the leukemia develops. Therefore, the term "AML" is used to distinguish between myeloblastic and myeloblastic leukemias, which are characterized by specific analyses. (M0), myeloblastic without maturation (M1), myeloblastic with maturation (M2), promyeloblastic Myeloid (M3), myelomonocytic (M4), monocytic (M5), erythroleukemic (M6), and megakaryocytic As used herein, "AML" also refers to all subtypes, including myeloid leukemia (M1), myeloma (M2), myeloma (M3), myeloma (M4), myeloma (M5), myeloma (M6), myeloma (M7), myeloma (M8), myeloma (M9), myeloma (M10), myeloma (M11), myeloma (M12), myeloma (M13), myeloma (M14), myeloma ( AML also refers to acute myeloid leukemia, a cancer that produces abnormal bone marrow blasts. It is the most common form of acute leukemia in the United States (Siegel, R., Ma, J., Zou, Z. & Jemal, A ., Cancer statistics, 2014, CA Cancer J Clin, 64(1):9-29 (2014)). “Recurrent AM "Refractory AML" refers to a subject who has experienced a period of remission from AML. Initial standard induction therapy (e.g., anthracycline- and / or cytarabine-based In some embodiments, "refractory AML" refers to patients who do not respond to the first cycle of treatment with " refers to a disease that has been treated with one or two or more cycles of standard induction therapy. This refers to patients who do not respond to treatment.

[0086] "Adoptive T cell transfer" refers to the isolation and ex vivo expansion of tumor-specific T cells. proliferation, in the number of cells that could be obtained by vaccination alone or by the patient's natural tumor response. This refers to the expansion of T cells to achieve larger numbers. These cells then have the ability to attack and target the cancer. The aim is to provide the ability to overwhelm remaining tumors through T cells that can attack and kill them. In an attempt to treat cancer, tumor-specific T cells are infused into patients with cancer. In addition, many forms of T cell therapy involve culturing tumor-infiltrating lymphocytes or TILs. In addition, a form of isolating and expanding one specific T cell or clone has been used. and the use of T cells engineered to strongly recognize and attack tumors. The form is also used.

[0087] An "antigen recognition portion or domain" is a molecule or molecule that specifically binds to an antigen. In one embodiment, the antigen recognition moiety is an antibody, an antibody-like molecule, or a combination thereof. The fragment and the antigen are tumor antigens.

[0088] As used herein, "antibody" refers to a monoclonal or polyclonal antibody. As used herein, the term "monoclonal antibody" refers to a single clone of B cells. Polyclonal antibodies are antibodies produced by the same clone and bind to the same epitope. "Individual antibodies" are antibodies produced by different B cells that bind to different epitopes of the same antigen. Refers to a population of antibodies. Whole antibodies typically consist of four polypeptides: heavy (H) chain polypeptides and two identical copies of a light (L) chain polypeptide. Each heavy chain contains one N-terminal variable (VH) region and three C-terminal constant (CH1, CH Each light chain contains an N-terminal variable (VL) region and an N-terminal variable (CH2, CH3, and CH4) region. The variable regions of each pair of light and heavy chains form an antibody. The VH region and the VL region form an antigen-binding site. They share a common general structure, including four framework regions, which are relatively conserved. The framework regions are connected by three complementarity determining regions (CDRs). The three CDRs, known as CDR1, CDR2, and CDR3, are responsible for binding to an antigen. These form the "hypervariable region" of the antibody.

[0089] An "antibody-like molecule" is, for example, an Ig superunit that is capable of selectively binding to a partner. MHC molecules and T cell receptors can be proteins that are members of the In one embodiment, the antibody-like molecule is a TCR. In this study, the TCR has been modified to increase its binding affinity to MHC.

[0090] As used herein, the terms "antibody fragment," "antibody fragment," "functional antibody fragment," and "antigen The term "binding portion" refers to one or more portions of an antibody that retain the ability to specifically bind to an antigen. are used interchangeably to mean a fragment or portion of a number (generally, Holliger et al., N at. Biotech., 23(9):1126-1129 (2005). An antibody fragment may be, for example, or multiple CDRs, a variable region (or a portion thereof), a constant region (or a portion thereof), or It is desirable to include these combinations. Examples of antibody fragments include (i) a VL domain, a VH domain, and Fab fragment, a monovalent fragment consisting of the main, CL and CH1 domains; ii) contains two Fab fragments linked by a disulfide bridge in the stalk region (iii) a bivalent fragment, the VL domain and (iv) an Fv fragment consisting of a VH domain and a VH domain; The two domains of the Fv fragment are joined by a synthetic linker that allows them to be synthesized as a single chain. Single-chain Fv (scFv) ( For example, Bird et al., Science, 242: 423-426 (1988); Huston et al., Proc. Natl. Ac. ad. Sci. USA, 85: 5879-5883 (1988); and Osbourn et al., Nat. Biotechnol., 16: 77 8 (1998); and (v) each polypeptide chain is to the VL via a peptide linker that is too short to allow pairing between the VH and VL of A dimeric molecule comprising linked VHs and thus having two functional antigen-binding sites. The complementary domains on the different VH-VL polypeptide chains are paired to generate a complementary domain. diabodies, which are dimers of polypeptide chains that move in a circular fashion. Antibody fragments are known in the art and are described, for example, in U.S. Patent Application Publication No. 2009 / 003664. This is described in more detail in specification 93024A1.

[0091] "Chimeric antigen receptor" is also used to refer to artificial T cell receptors, chimeric T cell receptors, chimeric immunoreceptors, etc. Chimeric antigen receptors (CARs) are used to express any specificity as an immune effector. CARs are engineered receptors that are grafted onto host cells. CARs typically contain an antigen-binding domain. An extracellular domain (ectodomain) containing the in and stalk regions, a transmembrane domain, and , and an intracellular domain (endodomain).

[0092] The "stalk" region, which encompasses the terms "spacer" region or "hinge" region, is defined as The antigen-binding domain is used to link the transmembrane domain. The term "stalk domain" generally refers to a domain that, within a polypeptide chain, separates a transmembrane domain. Any oligonucleotide that functions to link the extracellular or cytoplasmic domains. In some embodiments, the stalk domain is an antigen. The binding domains are sufficiently oriented to allow for different orientations to facilitate antigen recognition. In one embodiment, the stalk domain is a human IgG1 derived The alternative is a fusion protein that contains the CH2CH3 region of an immunoglobulin and part of CD3. In one embodiment, the stalk region is a region of the CD8 alpha hinge region. (SEQ ID NO: 22). The term "functional portion" when used in reference to CAR Any portion or fragment of a CAR described herein, wherein the functional portion is one of It refers to a portion or fragment that retains the biological activity of the CAR (parent CAR), which is a part of the CAR. With respect to the nucleic acid sequence encoding the parent CAR, the nucleic acid sequence encoding the functional portion of the CAR is For example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95% %, or more, of the parent CAR.

[0093] As used herein, the term "functional variant" refers to a functional variant of a reference polypeptide. Polypeptides or proteins having qualitative or significant sequence identity or similarity A functional variant refers to a polypeptide that retains the biological activity of the reference polypeptide of which it is a variant. A variant is, for example, a mutant of a CAR described herein (a parent CAR), which is capable of infecting a target cell. to a similar, comparable, or higher degree than the parent CAR. The functional alterations of the CAR in relation to the nucleic acid sequence encoding the parent CAR are encompassed by variants that have the same or similar functional alterations as the parent CAR. The nucleic acid sequence encoding the variant is, for example, about 10% identical to the nucleic acid sequence encoding the parent CAR. , about 25% identical, about 30% identical, about 50% identical, about 65% identical, about 70% identical, about 75% Identical, about 80% identical, about 85% identical, about 90% identical, about 95% identical, or about 99% identical It is possible.

[0094] As used herein, an "inducible promoter" refers to a promoter that is regulated by a transcriptional regulator, e.g., a biological factor. Or it refers to a promoter that is induced to activity by the presence or absence of an abiotic factor. Inducible promoters are promoters that regulate the expression of genes operably linked to them in an organism or in particular in a This is useful because it can be turned on or off at certain stages of tissue development. Examples of inducible promoters include alcohol-regulated promoters, tetracycline-regulated promoters, promoter, steroid-regulated promoter, metal-regulated promoter, pathogenesis-regulated promoter promoters, temperature-regulated promoters, and light-regulated promoters. An inducible promoter can be part of a gene switch. Optionally, the inducible promoter is an inducible promoter of a thiol ligand. Two small molecule ligand-inducible gene switches, such as the HEOSWITCH® gene switch, It may be an ecdysone receptor-based gene switch by a polypeptide. Gene switches include, but are not limited to, those disclosed herein, each of which is incorporated by reference in its entirety. No. 2001 / 009050 (Publication No. PCT / US2001 / 009050), incorporated herein by reference. No. 2001 / 070816, U.S. Pat. No. 7,091,038, Specification No. 7,776,587, Specification No. 7,807,417, Specification No. 8,202, 718, PCT / US Application No. 2001 / 030608 (International Publication No. No. 02 / 029075, U.S. Pat. No. 8,105,825, U.S. Pat. ,168,426; PCT / 1J52002 / 005235 (International Publication No. 2002 / 066613, U.S. Application No. 10 / 468,200 (U.S. Publication No. 20120167239), PCT / U.S. Application No. 2002 / No. 005706 (International Publication No. WO 2002 / 066614), U.S. Pat. Specification No. 7,531,326, Specification No. 8,236,556, Specification No. 8,598,4 No. 09, PCT / U52002 / 005090 (International Publication No. 2002 / 0 No. 66612, U.S. Pat. No. 8,715,959 (U.S. Publication No. 20 No. 060100416, PCT / US Application No. 2002 / 005234 Detailed description (International Publication No. WO 2003 / 027266), U.S. Patent No. 7,601,5 Specification No. 08, Specification No. 7,829,676, Specification No. 7,919,269, Specification No. 7,919,269, No. 8,030,067, PCT / U52002 / 005708 (International Publication Patent application Ser. No. 2002 / 066615, U.S. Patent Application No. 10 / 468,192 (U.S. Publication No. 20110212528), PCT / U.S. Application No. 2002 / 005026 (International Publication No. WO 2003 / 027289), Specification No. 7,563,879, Specification No. 8,021,878, No. 8,497, No. 2005 / 015089 (International Publication No. No. 05 / 108617, U.S. Pat. No. 7,935,510, U.S. Pat. No. 8 ,076,454, PCT / U52008 / 011270 (International Publication No. No. 2009 / 045370), U.S. Application No. 12 / 241,018 ( U.S. Publication No. 20090136465, PCT / U.S. Application No. 2008 / 0 No. 11563 (International Publication No. WO 2009 / 048560), U.S. Application No. No. 12 / 247,738 (U.S. Publication No. 20090123441); PCT / US Application No. 2009 / 005510 (International Publication No. WO 2010 / 04218 No. 9, U.S. Application No. 13 / 123,129 (U.S. Publication No. 20110 No. 268766), PCT / US Application No. 2011 / 029682 ( WO 2011 / 119773, U.S. Application Serial No. 13 / 636,473 No. (U.S. Publication No. 20130195800), PCT / U.S. Application No. 012 / 027515 specification (International Publication No. 2012 / 122025 pamphlet), and any of the systems described in U.S. Pat. No. 9,402,919. The receptor may be selected from members of the ecdysone-based receptor family, including:

[0095] As referred to herein, a "proliferative disorder" refers to a disorder characterized by excessive proliferation of cells and intracellular matrix Excessive turnover of proteins significantly contributes to the pathogenesis of several diseases, including cancer. This means that a unified concept is presented.

[0096] As used herein, a "patient" refers to a person suffering from or suffering from a proliferative disorder, such as cancer. The term "mammalian" refers to a mammalian subject who has been diagnosed as having or is suspected of having a pulmonary embolism. In this context, the term "patient" refers to a person who has a higher than average chance of developing a proliferative disorder, such as cancer. Refers to a mammalian subject. Exemplary patients include humans, apes, dogs, pigs, cows, cats, horses, Goats, sheep, rodents, and other mammals that may benefit from the therapies disclosed herein. The patient may be an animal. Exemplary human patients may be male and / or female.

[0097] As used herein, "administering" refers to administering one or more of the combinations described herein. By way of example and not limitation, administering a composition to a patient or subject refers to administering a composition to a patient or subject. Administration of the substance, e.g., injection, can be by intravenous (iv) injection, subcutaneous (sc) injection, intradermal (i d.) injection, intraperitoneal (ip) injection, or intramuscular (im) injection. One or more of such routes can be utilized. Parenteral administration can be, for example, For example, this can be by bolus injection or by gradual perfusion over time. Alternatively, or contemporaneously, administration may be by the oral route. It may also be by bolus or cell pellet depot surgery, and the placement of medical devices. This may also depend on the placement.

[0098] As used herein, a "patient in need thereof" or a "subject in need thereof" refers to a cancer Refers to patients diagnosed with or suspected of having a proliferative disorder such as In one embodiment, the patient is diagnosed with acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), ML), chronic lymphocytic leukemia (CLL), and chronic myelogenous leukemia (CML). The individual may have or develop leukemia, including, but not limited to, leukemia.

[0099] In some embodiments, the compositions described herein comprise a nucleic acid sequence as described herein. A modified or host cell expressing at least one nucleic acid as described herein. The vector containing the nucleic acid sequence may be included in an amount effective to treat or prevent a proliferative disorder. As used herein, the terms "treatment," "treating," and the like refer to the process of achieving a desired pharmacological effect. and / or physiological effect. In some embodiments, the effect is therapeutic. That is, the effect is to partially or completely alleviate the disease and / or adverse symptoms attributable to the disease. For this purpose, the method of the invention involves the use of a nucleic acid sequence of the invention or and administering a "therapeutically effective amount" of a composition comprising a host cell expressing a vector comprising a nucleic acid sequence of The method includes the step of:

[0100] A "therapeutically effective amount" refers to a compound administered at a dosage required for a desired therapeutic result, for a required period of time. A therapeutically effective amount refers to an amount that is effective over a period of time depending on the disease state, age, sex, and weight of the individual. and compositions described herein that induce a desired response in one or more subjects. It may vary depending on factors such as the capabilities of the object.

[0101] Alternatively, the pharmacological and / or physiological effects may be "prophylactic." That is, the effect is to completely or partially prevent the disease or its symptoms.

[0102] A "prophylactically effective amount" refers to the amount needed to achieve a desired prophylactic outcome (e.g., prevention of disease onset). It refers to an amount effective at the required dosage and for the required period of time.

[0103] Chimeric Antigen Receptor In several embodiments described herein, the CAR uses antibody-derived mAbs for target recognition. The extracellular single chain variable domain (scFv) may be included, and the scFv may include a flexible phosphorylation domain. Carrier transmembrane domains and / or CD3ζ for T cell activation, e.g. Typically, in vivo, If T cells can be activated, they transmit the antigen-induced primary TCR signal to the cytokine CD28-derived IL-1 and IL-21 induce the production of cytokines (i.e., IL-2 and IL-21). They then receive the secondary costimulatory signals and then mediate these in an autocrine / paracrine manner. With this in mind, CAR is a a CD28 cytoplasmic signaling domain, or a 4-1BB signaling domain, The chimeric CD may contain signal transduction domains from other costimulatory molecules, such as transduction domains. Costimulation with 28 induces upregulation of antiapoptotic molecules and IL-2 production, as well as peripheral By expanding T cells derived from blood mononuclear cells (PBMC), T cell survival was improved. Improve.

[0104] In one embodiment, a CAR comprises, for example, a transmembrane domain and an endodomain of CD3 zeta. Derived from a monoclonal antibody specific for the tumor-associated antigen CD33 fused to a lysine Such molecules are fusions of single chain variable fragments (scFv) that are This results in the transmission of the zeta signal in response to recognition of its target.

[0105] In one embodiment, a CAR comprises an ectodomain (extracellular), a transmembrane domain, and an end domain. In one embodiment, the CAR ectodomain may have an end domain (intracellular). Signal peptides direct the nascent protein to the endoplasmic reticulum. When the protein is glycosylated and anchored in the cell membrane, any eukaryotic signal The peptide sequence is assumed to be functional. Generally, the amino acid sequence is Using a signal peptide joined to a terminal component (e.g., light chain-linker - In scFvs with heavy chain orientation, the natural signals of the light chain are used). The signal peptide is GM-CSFRa (SEQ ID NO: 16) or IgK (SEQ ID NO: 3 4) Other signal peptides that may be used are those for CD8 alpha and CD28. It contains the signal peptide from which it is derived.

[0106] The antigen recognition domain may be an scFv, but alternatives may exist. The antigen recognition domains derived from the single chains of the transcription factor receptor (TCR) alpha and beta are simple In addition to ectodomains (e.g., the CD4 ectodomain, which recognizes HIV-infected cells), Linked, e.g., cytokines (which result in recognition of cells bearing cytokine receptors) It is envisioned that the IgG antibody may have other recognition components, such as a recognition component that recognizes a given antigen, e.g., a tumor-associated antigen. Almost anything that binds with high affinity to a given target can be used as an antigen recognition region. It is possible.

[0107] Generally, CARs exist in a dimerized form and are composed of an extracellular scFv (VH linked to VL). ) region, stalk domain, transmembrane domain, and intracellular signaling motifs The endodomain of the first generation CAR is expressed as a fusion protein that activates T cells. Second generation CARs induce activation solely through signaling via CD3ζ. via other endodomains, such as 3ζ and CD28, or 4-1BB or OX40 Third generation CARs target CD28, 4-1BB, and through a combination of three signaling motifs, including OX40, CD3ζ, Activates T cells.

[0108] In some embodiments, the invention provides a method for the production of a polypeptide comprising an extracellular domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, a chimeric antigen receptor (CAR) is provided that comprises a null transduction domain. The extracellular domain is a target specific fragment, otherwise referred to as the antigen binding portion or scFv. In some embodiments, the intracellular signaling molecule comprises a heterologous binding element and a stalk domain. The transduction domain, or otherwise the cytoplasmic signaling domain, is a co-stimulatory signaling domain. It contains a null transfer region and a zeta chain portion.

[0109] The costimulatory signaling region comprises the intracellular signaling domain of a costimulatory molecule, such as a CAR Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands. It is a surface molecule required for an efficient response of lymphocytes to antigens.

[0110] In some embodiments, a stalk is provided between the extracellular domain and the transmembrane domain of the CAR. As used herein, the term "stalk domain" refers to a Typically, the transmembrane domain is linked to the cytoplasmic domain within the scFv or polypeptide chain. "Storo" refers to any oligonucleotide or polypeptide that functions to The domain may include a flexible hinge, such as an Fc hinge, and optionally one or two Fc Optionally, the stalk region may comprise a constant domain of a HinG1 derived from IgG1. In an alternative embodiment, the stalk region is a CH2CH3 region of an immunoglobulin. and optionally a portion of CD3. Optionally, the stalk region is CD8α hinge region, Ig The 12 amino acid hinge region of G4-Fc (ESKYGPPCPPCP) (SEQ ID NO:62) , or an IgG4 hinge region.

[0111] The transmembrane domain may be derived from a natural or recombinant source. When the source is natural, the domain may be any membrane-bound or transmembrane protein. Suitable transmembrane domains may be derived from the alpha, beta, and alpha-proteins of the T cell receptor. In some cases, the transmembrane domains of the CD28, CD3 epsilon, and CD4 epsilon and zeta chains are involved. 3zeta, CD45, CD4, CD5, CD8alpha, CD9, CD16, CD22, CD 33, CD37, CD64, CD80, CD86, CD134, CD137, or CD Alternatively, the transmembrane domain may be derived from a synthetic In some embodiments, the amino acid sequence may include hydrophobic residues such as leucine and valine. At one or both ends of the transmembrane domain, phenylalanine, tryptophan, and valine triplets are found. Optionally, in some embodiments, 2 to 10 Short oligonucleotide or polypeptide linkers that are between amino acids in length This may form a link between the transmembrane domain and the cytoplasmic signaling domain of CAR. In some embodiments, the linker is a glycine-serine linker. In this embodiment, the transmembrane domain comprises the CD8α transmembrane domain or the CD3ζ transmembrane domain. In some embodiments, the transmembrane domain comprises a CD8α transmembrane domain. In the embodiment, the transmembrane domain comprises a CD3 zeta transmembrane domain.

[0112] The intracellular domain may include one or more costimulatory domains. The markers are CD8, CD27, CD28, 4-1BB (CD137), ICOS, and DAP10. , DAP12, OX40 (CD134), CD3 zeta, or fragments or combinations thereof. In some cases, the CARs described herein include, but are not limited to, D8, CD27, CD28, 4-1BB(CD137), ICOS, DAP10, DAP 12, OX40 (CD134), or a fragment or combination thereof. Contains one or more of the following domains: Therefore, the CARs described herein bind to CD27, CD28, 4-1BB (CD13 7), ICOS, OX40 (CD134), or a fragment or combination thereof. One or more of the co-stimulatory domains, or two or more of the co-stimulatory domains Optionally, the CAR described herein is a CAR that is capable of targeting CD8, CD28, 4-1BB ( CD137), DAP10, DAP12, or a fragment or combination thereof. The co-stimulatory domains may include one or more, or two or more, of In some cases, the CARs described herein can be used to target CD28, 4-1BB (CD137 ), or a fragment or combination thereof. In some cases, the term "a" includes "a plurality," or "two or more." The CARs used are those that bind the costimulatory domains CD28 and 4-1BB (CD137), or Optionally, the CARs described herein include co-stimulatory The domains CD28 and OX40 (CD134), or their respective fragments In some cases, the CAR described herein comprises a co-stimulatory domain, CD8 and and CD28, or fragments of each of these. The CAR comprises a costimulatory domain, CD28, or a fragment thereof. The CARs described herein contain a co-stimulatory domain, 4-1BB (CD137), and In some cases, the CAR described herein comprises a co-stimulatory domain. OX40 (CD134), or a fragment thereof. The CAR comprises the costimulatory domain CD8, or a fragment thereof. The CARs described herein comprise at least one co-stimulatory domain, DAP10, or In some cases, the CAR described herein comprises at least one co-stimulatory fragment. It comprises the cytoplasmic domain of DAP12, or a fragment thereof.

[0113] The intracellular signaling domain, also known as the cytoplasmic domain, of the CAR of the present disclosure. The study found that CAR-infused immune cells were able to activate at least one of their normal effector functions. The term "effector function" refers to a specialized cellular function. The effector function of a cell can be, for example, cytolytic activity, or the secretion of cytokines. In some cases, the domain contains a helper activity. The term effector function refers to a set of proteins that transmit signals and direct cells to perform specialized functions. The term refers to the portion of a protein that is attached to the cytoplasm. Typically, the entire intracellular signaling domain is available. However, in many cases it is not necessary to use the entire chain. Truncation of the intracellular signaling domain To the extent that the moiety is used to transmit an effector function signal, such The truncated portion can be used in place of the complete chain, thus allowing intracellular signaling The term domain refers to an intracellular signaling domain sufficient to transmit an effector function signal. In some embodiments, the intracellular The domain further comprises a signaling domain for T cell activation. The signaling domains for T cell activation are TCR zeta, FcR gamma, and FcR vector. , CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a , CD79b, or CD66d. The signaling domain for activation includes a domain derived from CD3ζ.

[0114] In some embodiments herein, an isolated nucleic acid encoding a chimeric antigen receptor (CAR) is provided. (a) a CD33 antigen-binding domain; (b) a stalk domain; (c ) a transmembrane domain; (d) a co-stimulatory protein comprising 4-1BB or CD28, or both; (e) a CD3 zeta signaling domain; optionally, (f An isolated nucleic acid is provided that contains a truncated epidermal growth factor receptor (HER1t or HER1t-1). As shown.

[0115] Within the scope of the present invention are nucleic acid sequences encoding functional portions of the CARs described herein. Functional moieties include, for example, those that recognize target cells or detect, treat, or otherwise inhibit disease. or preventive ability to a similar extent, comparable to, or greater than the parent CAR. The portion of the CAR that is retained in the

[0116] In some embodiments, the CAR is provided at the amino or carboxy terminus of the moiety, or both. additional amino acids at either end that are not found in the amino acid sequence of the parent CAR The additional amino acids are the biological function of the functional moiety. For example, the present invention can be used to recognize target cells, detect cancer, or treat or prevent cancer. It is desirable that the additional amino acids do not interfere with the biological function of CAR. It is more desirable to enhance the therapeutic activity of the CAR as compared to the biological activity of the parent CAR.

[0117] As used herein, the term "functional variant" refers to a variant encoded by a nucleic acid sequence of the present invention. A CAR that has substantial or significant sequence identity or similarity to the CAR being tested. A functional variant of a polypeptide or protein refers to a variant of that polypeptide or protein. Functional variants are, for example, variants described herein that retain the biological activity of CAR. A mutant of a CAR (parent CAR) that inhibits target cells to the same extent as the parent CAR The parent CAR includes mutants that retain the ability to recognize the CAR to a low or higher degree. In view of the nucleic acid sequence to be loaded, the nucleic acid sequence encoding the functional variant of CAR is For example, about 10% identical, about 25% identical, about 30% identical, about 50% identical, % identical, about 65% identical, about 80% identical, about 90% identical, about 95% identical, or about 99% identical It can be one.

[0118] The CARs described herein (including functional portions and functional variants thereof) are For example, glycosylation, amidation, carboxylation, phosphate modification, etc., via disulfide bridges. C, which has been modified, esterified, N-acylated, cyclized, or converted to an acid addition salt. AR, and / or, optionally, dimerized or polymerized CAR, or Contains jugate CAR.

[0119] antigen binding portion In some embodiments, the CARs described herein are otherwise capable of binding to an antigen-binding moiety. In some embodiments, the antibody comprises a target-specific binding element, referred to as a binding domain, that binds to an antigen on a tumor cell. The tumor antigen of interest can be targeted by engineering a desired antigen-binding moiety that specifically binds to the tumor antigen. In the context of the present invention, the CAR described herein is modified to target "Tumor antigen" or "hyperproliferative disorder antigen" or "antigen associated with a hyperproliferative disorder" refers to antigens common to specific hyperproliferative disorders, such as cancer.

[0120] In several embodiments, the antigen-binding portion of the CAR described herein is specific for CD33. CD33-specific CARs, when expressed on the cell surface, Redirects T cell specificity to human CD33. In some embodiments, antigen binding The variable domain is a light chain variable domain of an anti-CD33 monoclonal antibody specific for the target antigen. (VL) and a variable domain heavy chain (VH) having a glycine-serine linker or A single chain comprising a VL and a VH, connected by a flexible linker such as an itro linker. In some embodiments, the scFv includes M195, m2H12, , DRB2, and / or My9-6. In some embodiments, the scFv is human In some embodiments, the antigen-binding portion is a modified scFv, e.g., hM195. For example, VH-linker-VL or VL-linker-VH can be arranged from the N-terminus to the C-terminus. The VH and VL may be tethered together.

[0121] In some embodiments, the CAR described herein has the amino acid sequence of SEQ ID NO:1 (h M195 VL) with at least 90%, 91%, 92%, 93%, 94%, 95%, VL polypeptides having 96%, 97%, 98%, 99% or 100% identity. The antigen-binding portion comprises

[0122] In some embodiments, the CAR described herein has the amino acid sequence of SEQ ID NO:3 (h M195 VH) at least 90%, 91%, 92%, 93%, 94%, 95%, VH polypeptides having 96%, 97%, 98%, 99% or 100% identity The antigen-binding portion comprises

[0123] In some embodiments, the CAR described herein comprises a Gly-Ser linker (sequence No. 6) or a humanized anti-CD33 mAb clone with a functional variant of the linker The antibody comprises the VL (SEQ ID NO: 1) and VH (SEQ ID NO: 3) of the antigen-binding portion of hM195, nothing.

[0124] In some embodiments, the CAR described herein has the amino acid sequence of SEQ ID NO:8 (h M195 VH, VL, and linker) 3%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity The antigen-binding portion comprises a polypeptide that binds to the antigen.

[0125] In some embodiments, the CAR described herein has the amino acid sequence of SEQ ID NO:9 (M 2H12 VH) at least 90%, 91%, 92%, 93%, 94%, 95%, VH polypeptides having 96%, 97%, 98%, 99% or 100% identity The antigen-binding portion comprises

[0126] In some embodiments, the CAR described herein has the amino acid sequence of SEQ ID NO: 10 ( M2H12 VL) at least 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, 99% or 100% identity to The antigen-binding portion comprises

[0127] In some embodiments, the CAR described herein has the amino acid sequence of SEQ ID NO: 11 ( DRB2 VH) with at least 90%, 91%, 92%, 93%, 94%, 95%, VH polypeptides having 96%, 97%, 98%, 99% or 100% identity The antigen-binding portion comprises

[0128] In some embodiments, the CAR described herein has the amino acid sequence of SEQ ID NO: 12 ( DRB2 VL) at least 90%, 91%, 92%, 93%, 94%, 95%, VL polypeptides having 96%, 97%, 98%, 99% or 100% identity. The antigen-binding portion comprises

[0129] In some embodiments, the CAR described herein has the amino acid sequence of SEQ ID NO: 13 ( My9-6 VH) at least 90%, 91%, 92%, 93%, 94%, and 95% , 96%, 97%, 98%, 99% or 100% identity to The antigen-binding portion comprises

[0130] In some embodiments, the CAR described herein has the amino acid sequence of SEQ ID NO: 14 ( My9-6 VL) at least 90%, 91%, 92%, 93%, 94%, and 95% , 96%, 97%, 98%, 99% or 100% identity to The antigen-binding portion comprises

[0131] In some embodiments, the antigen-binding portion has at least one amino acid sequence identical to that of SEQ ID NO:16. Also 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or has a GM-CSFRa signal peptide with 100% identity.

[0132] Stork Domain In some embodiments, the CD33 CAR of the present invention comprises an antigen-binding portion and a T cell membrane. In some embodiments, the stalk domain comprises an endogenous stalk domain. Establish the optimal transmembrane distance between the effector and the target. In some embodiments, the stalk domain is The polypeptide provides the antigen-binding domain with flexibility to reach its target. In this case, the stalk domain is the CD8 alpha hinge domain.

[0133] In some embodiments, the CD8 alpha hinge domain has the amino acid sequence of SEQ ID NO:22. With at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, This includes polypeptides having 98%, 99% or 100% identity.

[0134] Transmembrane domain In some embodiments, the CAR is fused to the extracellular domain of the CAR stalk domain. In one embodiment, the naturally occurring domains in a CAR include a transmembrane domain that is In some embodiments, the transmembrane domain is associated with one of: It is a membrane-spanning, hydrophobic alpha helix.

[0135] The transmembrane domain may be derived from natural or synthetic sources. When the source is natural, the domain may be any membrane-associated or transmembrane protein. The transmembrane domain of particular use in the present invention may be derived from the alpha domain of the T cell receptor. chain, beta chain, or zeta chain, CD28, CD3 epsilon, CD3 zeta, CD45, C D4, CD5, CD8 alpha, CD9, CD16, CD22, CD33, CD37, C It can be derived from CD64, CD80, CD86, CD134, CD137, and CD154 (i.e. Alternatively, the transmembrane domain may be a synthetic In some cases, the transmembrane domain contains hydrophobic residues such as leucine and valine. In some embodiments, the synthetic transmembrane domain may include a phenylalanine at each end. , tryptophan, and valine triplets will be found. In some embodiments, short oligonucleotide linkers between 2 and 10 amino acids in length are used. The polypeptide linker connects the transmembrane domain of the CAR with the cytoplasmic signaling domain. In some embodiments, the linker can be a glycine-serine linker. He is a breaker.

[0136] In some embodiments, the transmembrane domain in the CAR described herein is a CD8A receptor. In some embodiments, the CD8 alpha transmembrane domain is At least 90%, 91%, 92%, 93%, 94%, or more of the amino acid sequence of sequence number 18; Polypeptides with 95%, 96%, 97%, 98%, 99% or 100% identity Includes do.

[0137] In embodiments, the transmembrane domain in the CAR described herein is a CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain is a transmembrane domain of SEQ ID NO: 20. At least 90%, 91%, 92%, 93%, 94%, 95%, 96% %, 97%, 98%, 99%, or 100% identity to the polypeptide.

[0138] Cytoplasmic domain (costimulatory and signaling domains) The intracellular signaling domain, also known as the cellular signaling domain, of the CAR described herein The cytoplasmic domain mediates at least some of the normal effector functions of CAR-loaded immune cells. The term "effector function" refers to a specialized cellular function that The effector function of T cells can be, for example, cytolytic activity or cytokine production. In some cases, the secretion of cytokines may be involved in the function of the helper pathway. The term "in" refers to a molecule that transmits effector signals and performs a specialized function. The term refers to the part of a protein that directs a cell to a specific target. Usually refers to the entire intracellular signaling domain. Although the entire chain can be used, in many cases it is not necessary to use the entire chain. The main cleavage portion is used, so long as it transmits an effector function signal. Such truncated portions can be used in place of the complete chain. The term signaling domain refers to a domain that is sufficient to transmit an effector function signal. It is intended to include any truncated portion of the intracellular signaling domain.

[0139] Examples of intracellular signaling domains for use in the CARs described herein include , which act cooperatively to induce signal transduction following antigen receptor binding; Cytoplasmic sequences of the T cell receptor (TCR) and co-receptors, as well as any derivatives of these sequences The invention may include derivatives or variants, and any synthetic sequences that have the same functional capability.

[0140] Signals generated through the TCR alone are generally insufficient for full T cell activation. Thus, a secondary or costimulatory signal is also required. Activation occurs via two distinct classes of cytoplasmic signaling sequences: antigen-dependent primary activation via the TCR; The cytoplasmic signaling sequence (Primary cytoplasmic signaling sequence) that induces activation and the antigen non-activation cytoplasmic synthases that act in a synthase-dependent manner to provide secondary or costimulatory signals It can be said that the signal transduction pathway is mediated by the secondary cytoplasmic signaling sequences (secondary cytoplasmic signaling sequences). .

[0141] Primary cytoplasmic signaling sequences mediate primary activation of the TCR complex, either stimulatory or inhibitory. The primary cytoplasmic signaling sequences that act in a stimulatory manner are the immunoreceptor tyrosine-based The proteins may contain signaling motifs known as ITAMs or activation motifs.

[0142] An example of an ITAM-containing primary cytoplasmic signaling sequence of particular use in the present invention is T CD3 gamma, CD3 delta, CD3 ipsilateral The primary cells derived from the IL-1 receptor agonist, CD5, CD22, CD79a, CD79b, and CD66d In some embodiments, the CAR comprises a cytoplasmic signaling sequence. The cytoplasmic signaling molecule contains a cytoplasmic signaling sequence derived from CD3 zeta.

[0143] In several embodiments, the cytoplasmic domain of the CAR can be used by itself or as described herein. and any other desired cytoplasmic domains useful in the context of the CAR. and can be designed to include the CD3 zeta signaling domain. The cytoplasmic domain of the CAR contains a CD3 zeta chain portion and a costimulatory signaling region. The costimulatory signaling region may comprise a portion of the CAR that contains the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands. These are cell surface molecules required for efficient lymphocyte responses to antigens. Examples of such molecules are CD27, CD28, 4-1BB (CD137), OX40, and CD30. , CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, C Ligands that specifically bind to D7, LIGHT, NKG2C, B7-H3, and CD83 In some embodiments, the costimulatory molecules include, for example, CD28 and 4-1BB. They can also be used in combination with CD28 and OX40. Thus, the present invention relates to a method for the treatment of inflammatory bowel disorders using 4-1BB and CD28 as costimulatory signaling elements. Although exemplified together, other costimulatory elements are within the scope of the invention.

[0144] Cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR described herein Columns can be concatenated with each other in a random order or in a specified order. Optionally, in some embodiments, a short nucleotide sequence is provided, which is between 2 and 10 amino acids in length. A oligonucleotide or polypeptide linker may form the linkage. The -serine doublet provides a particularly suitable linker.

[0145] In one embodiment, the cytoplasmic domain comprises the signaling domain of CD3 zeta and In another embodiment, the cytoplasmic domain comprises 28 signaling domains. The signaling domain of 4-1BB and the signaling domain of 4-1K. In embodiments, the cytoplasmic domain comprises the signaling domain of CD3 zeta, as well as the signaling domain of CD 28 and 4-1BB signaling domains.

[0146] In one embodiment, the cytoplasmic domain in the CAR described herein is a cytoplasmic domain of 4-1BB. The signal transduction domain of CD3 zeta is included in the 4- The signaling domain of 1BB has at least 9 copies with the polypeptide sequence of SEQ ID NO:24. 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or or a polypeptide sequence having 100% identity thereto, The main sequence is at least 90%, 91%, 92%, 93%, or Polypeptides with 94%, 95%, 96%, 97%, 98%, 99% or 100% identity Contains the polypeptide sequence.

[0147] In one embodiment, the cytoplasmic domain in a CAR described herein is linked to the CD28 signaling domain. The nucleic acid sequence of the nucleotide ... However, in this case, the signaling domain of CD28 has the polypeptide sequence of SEQ ID NO:28. of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9 8%, 99%, or 100% identity to a polypeptide sequence of CD3 zeta The signaling domain of the present invention has at least 90% identity with the polypeptide sequence of SEQ ID NO: 26, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 1 100% identity to the polypeptide sequence.

[0148] In one embodiment, the cytoplasmic domain in the CAR described herein is a cytoplasmic domain of 4-1BB. It contains the signal transduction domain and the signal transduction domain of CD3 zeta, and is involved in the signal transduction of 4-1BB. The signal transduction domain comprises the amino acid sequence set forth in SEQ ID NO:24 and is identical to that of CD3 zeta. The signaling domain comprises the amino acid sequence set forth in SEQ ID NO:26.

[0149] Further genetic elements Cell therapy holds great promise for the treatment of human diseases, but the cells themselves or their Significant toxicity from these transgene products has hampered clinical exploration. In some embodiments described herein, immune effector cells comprising a CAR described herein The immune effector cells infused into a mammalian subject, e.g., a human, If a damaging effect results from their use, the action of such immune effector cells may be modulated. Therefore, in certain embodiments, the In addition to the CD33-specific chimeric antigen receptor, a second gene is also The second gene can be introduced into the modified immune effector cells described herein. The gene is, in effect, a "kill switch" that allows for the depletion of CD33 CAR-containing cells. In certain embodiments, the "kill switch" refers to at least one of HER1 and CD20 or a functional fragment thereof, and optionally a signal polypeptide. The present invention relates to a method for the preparation of a HER1 polypeptide or a CD20 polypeptide comprising the peptide sequence or a fragment thereof. The HER1 tag or CD20 tag are

[0150] In certain embodiments, the second gene is epidermal growth factor receptor (HER1) or In some embodiments, the second gene is a HER1 tag, which is a fragment or variant of these. The gene is truncated human epidermal growth factor receptor 1 (e.g., HER1t or HER1t-1) Optionally, the second gene is a truncated human epidermal growth factor In some embodiments, HER1, HER1t, and HER1 At least one of the t-1s is FDA-approved cetuximab or HER2 1. Administering any antibody that recognizes HER1t and / or HER1t-1. This provides a safety mechanism by allowing depletion of infused CAR-T cells via In some embodiments, the HER1t gene is a nucleic acid sequence having at least one homologue of the HER1t gene with the nucleic acid sequence of SEQ ID NO:32. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% In some embodiments, the nucleotide sequence includes a nucleotide sequence having 100% or 100% identity to the H The ER1t-1 gene has at least 90%, 91%, 92%, or 100% identity with the nucleic acid sequence of SEQ ID NO:54. %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical Truncated HER1 sequences, such as HER1t and H ER1t-1 inhibits the EGF ligand while keeping cetuximab binding to the receptor intact. Binding to EGFR, homodimerization and heterodimerization of EGFR, and EGFR-mediated signaling (Ferguson, K., 2008. A structure-based view of f Epidermal Growth Factor Receptor regulation. Annu Rev Biophys, Volume 37, pp. 353-373).

[0151] In a further embodiment of the invention, in addition to a therapeutic CD33-specific chimeric antigen receptor In some cases, the second gene introduced is a CD20 tag. a full length CD20 polypeptide, or a truncated CD20 polypeptide (CD20t-1) In some cases, the CD20 tag, e.g., CD20 or CD20t-1, may also be used as a FDA Infused CAR-T cells via administration of rituximab therapy approved in It also provides a safety mechanism by allowing depletion of CD 20 tags have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, Polypeptides with 4%, 95%, 96%, 97%, 98%, 99%, or 100% identity In certain embodiments, the CD20 tag has a peptide sequence. and having at least 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98%, 99% or 100% identity In some embodiments, the CD20 tag has a nucleic acid sequence of SEQ ID NO: 35. , at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% %, 99%, or 100% identity to the CD20 gene In some embodiments, the CD20 tag is encoded by the nucleic acid sequence of SEQ ID NO:57. , at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% %, 99%, or 100% identity to the CD20t-1 gene It is encoded by a gene.

[0152] In embodiments, the CAR vector comprising the CAR described herein is represented by SEQ ID NO: 36 nucleic acid sequences with at least 90%, 91%, 92%, 93%, 94%, 95%, 9 A full-length C containing a nucleic acid sequence with 6%, 97%, 98%, 99%, or 100% identity Further comprising a D20 tag.

[0153] In some embodiments, a kill tag, e.g., a HER1t tag, a HER1t-1 tag, a CD The gene encoding the CD20 tag or CD20t-1 tag was then inserted into the CD33 CAR. ' At the end, in frame, e.g., Thosea asigna virus The transgenic mice can be genetically modified to express the T2A peptide, but not limited to the T2A peptide, via a self-cleaving peptide. In some embodiments, the T2A peptide is fused to the amino acid sequence of SEQ ID NO:30. of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9 The amino acid sequence has 8%, 99%, or 100% identity.

[0154] In some embodiments, the kill tag gene is incorporated into the pFUGW lentiviral plasmid backbone. and is cloned in frame with the CD33 CAR gene. The gene was cloned into the Sleeping Beauty transposon vector. In another embodiment, the kill tag is cloned into a separate lentiviral vector. In yet other embodiments, HER1t, HER1t-1, CD20, or CD 20t-1, etc., into a separate Sleeping Beauty transposon vector. In certain embodiments, the kill tag is a signal peptide. For example, the GM-CSFRa signal peptide is The null peptide has at least 90%, 91%, 92% identity with the amino acid sequence of SEQ ID NO:16. , 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity In certain embodiments, the signal peptide has the sequence of SEQ ID NO: 34. , at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% %, 99%, or 100% identity to the IgK. The signal peptide is selected from IgE and CD8α, variants and fragments thereof. It is possible.

[0155] An exemplary vector encoding the CAR and kill tag described herein is shown in FIG. As shown in.

[0156] Exemplary CAR open reading frames Exemplary CARs and Human Carcinomas Encompassed by the Methods and Compositions Described Herein The CD33 receptor open reading frame is in Table 1.

[0157] [Table 1]

[0158] In some embodiments herein, an isolated nucleic acid encoding a CAR is provided, the CAR being Sequence No. SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO: 49, at least 90%, 9 1%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 10 Isolated nucleic acids, including polypeptides having 0.0% identity to the nucleic acid sequence of the present invention, are presented.

[0159] In each of the embodiments listed in Table 1, which involve "hM195scFv" The antigen-binding portion of the CAR has at least 90%, 90%, or more affinity to the amino acid sequence of SEQ ID NO:8. 1%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 10 In some embodiments, the polypeptide has a sequence identity to hM195scFV. hM195scFv has at least 90%, 91%, or more identical amino acid sequence to the amino acid sequence of SEQ ID NO:16. %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identity to the GM-CSFRa signal peptide.

[0160] In each of the embodiments in Table 1 with "CD8a", the membrane of the CAR The spanning region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 3%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity and the stalk domain comprises a CD8 alpha transmembrane domain comprising a polypeptide having a sequence At least 90%, 91%, 92%, 93%, 94%, or more of the amino acid sequence of sequence number 22; Polypeptides with 95%, 96%, 97%, 98%, 99% or 100% identity The CD8a gene is CD8a-containing.

[0161] In each of the embodiments in Table 1 with "CD28m", the CAR The intracellular domain has at least 90%, 91%, 92%, or 100% identity with the amino acid sequence of SEQ ID NO:28. %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical The CD28 includes CD28 having an amino acid sequence having the above-mentioned properties.

[0162] In each of the embodiments in Table 1 with "T2A", the OR of CAR F is a self-cleaving tosea nucleotide that allows the production of multiple gene products from a single vector. In some embodiments, the peptide comprises a Thoseaasigna virus (T2A) peptide. The T2A peptide has at least 90%, 91%, 92%, or 100% identity with the amino acid sequence of SEQ ID NO:30. %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical It has an amino acid sequence having the same properties.

[0163] In the embodiments in Table 1 with "HER1t", the ORF of CAR The authors report that the efficacy and safety of infused CA1970-related inflammatory bowel disease was improved by administering FDA-approved rituximab therapy. Truncated human epidermal growth factor provides a safety mechanism by allowing depletion of RT cells The HER1t gene described herein includes the HER1t gene set forth in SEQ ID NO: At least 90%, 91%, 92%, 93%, 94%, 95% with 31 amino acid sequences , 96%, 97%, 98%, 99% or 100% identity to In Table 1, unless otherwise stated, HER1t The tag is the GM-CSFRa signal peptide ("GM-CSFRsp") (SEQ ID NO: 16). In certain embodiments, HER1t has another tag, e.g., HER1t-1 or CD20t-1. In an embodiment involving "Ksp", the signal peptide has the amino acid sequence of SEQ ID NO: 34, At least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99%, or 100% identical amino acid sequence to IgK.

[0164] In some embodiments in Table 1, CAR ORF has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 2%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% The costimulatory molecule includes a costimulatory molecule having a polypeptide sequence having the same identity as the costimulatory molecule.

[0165] Some embodiments in Table 1 with "FL CD20" In the present invention, the CAR ORF has at least 90%, 91% or more identical amino acid sequence to the amino acid sequence of SEQ ID NO: 36. , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% The CD20 tag comprises a full length CD20 tag comprising a polypeptide sequence having the identity of Infused CAR-T cells via administration of rituximab therapy approved in This provides a safety mechanism by allowing the depletion of In this form, the CAR ORF can be under the control of an inducible promoter for gene transcription. In one aspect, the inducible promoter is an inducible promoter of a gene switch ligand. Optionally, the inducible promoter can be RHEOSWITCH®. A gene switch, induced by small molecule ligands, and two polypeptides, ecdysone It may be a receptor-based gene switch.

[0166] Cytokines In some embodiments, a CAR described herein is administered to a subject, comprising a cytokine. Optionally, the therapeutic agent is administered in combination with one or more additional therapeutic agents, including, but not limited to, , cytokines include at least one chemokine, interferon, interleukin , lymphokines, tumor necrosis factors, or variants or combinations thereof. In some cases, the cytokine is an interleukin. IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15 , IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL- 22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, I L-29, IL-30, IL-31, IL-32, IL-33, and their functional In some embodiments, the cytokine is at least one of a variant and a fragment. In some embodiments, the cytokine is Soluble IL-15, soluble IL-15 / IL-15Rα complex (e.g., ALT-803 In certain cases, the interleukin is membrane-bound IL-15 (mbIL- 15), or a fusion of IL-15 with IL-15Rα. In one embodiment, mbIL-15 is co-expressed with the engineered immune effector cells described herein. In some embodiments, the chimeric IL-15 is a membrane-bound form of the mbIL-15. 15 is inflated with full-length IL-15Rα or functional fragments or mutants thereof. full-length IL-15 (e.g., a native IL-15 polypeptide), or In some cases, the IL-15 is mediated by IL-15Rα. In some cases, mbIL-15 is indirectly linked via a linker. “Tethered IL-15 augments antitumor activity and promotes a stem-cell memory su bset in tumor-specific T cells,” PNAS 2016. By combining these two, the cytokine is expressed in the same immune effector cells as the CAR. In this embodiment, the cytokine described above acts as an inducible promoter for gene transcription. In one aspect, the inducible promoter can be under the control of a gene switch ligand. Optionally, the inducible promoter is RHEOSWI. Two polypeptides, such as the TCH® gene switch, that are inducible by small molecule ligands It may be an ecdysone receptor-based gene switch.

[0167] In a further embodiment, an immune effector cell expressing a CAR described herein expresses membrane-bound IL-15 ("mIL-15 or mbIL-15"). In some embodiments, mbIL-15 is a fusion protein of IL-15 and IL-15Rα. In a further embodiment, mbIL-15 comprises the amino acid sequence of SEQ ID NO: 37: At least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99%, or 100% identity.

[0168] In some embodiments, mbIL-15 is administered in combination with an HER1t, HER2, or HER3 antibody, as described herein. mbI is expressed with a cell tag such as CD20t-1, CD20t-1, or CD20. L-15 binds HER1t, HER-1t-1, CD20t-1, or CD20 and inflammasomes. It can be expressed in Reme.

[0169] In some embodiments, mbIL-15 inhibits the regulation of an inducible promoter for gene transcription. In one aspect, the inducible promoter is under the control of an inducible gene switch ligand. Optionally, the inducible promoter can be a RHEOSWITCH promoter. (R) Gene Switch, a Small Molecule Ligand-Induced, Two-Polypeptide , may be an ecdysone receptor-based gene switch.

[0170] Viral-Based Delivery Systems The present invention also provides delivery systems, such as viral-based systems, which incorporate a nucleic acid of the invention. Representative viral expression vectors include adenovirus-based vectors (e.g., Cruc Adhesive tape is commercially available from Adhesive Tape, Inc. (Leiden, Netherlands). vectors based on endoviruses (e.g., the Per.C6 system), lentivirus-based vectors (e.g., the Li Lentils manufactured by FE Technologies (Carlsbad, Calif.) vectors (e.g., pCFB-EGSH pFB-ERV) and herpes viruses. In embodiments, the viral vector is a lentiviral vector. Vectors derived from retroviruses such as these allow the long-term delivery of transgenes to daughter cells. This allows stable integration and propagation, achieving long-term gene transfer. Lentiviral vectors are a suitable tool for transfecting non-proliferating cells such as hepatocytes. Derived from oncoretroviruses, such as murine leukemia viruses, in that they are capable of transducing Lentiviral vectors also have the added advantage of being less immunogenic. Generally, and in some embodiments, a suitable vector comprises at least A replication origin, promoter sequence, convenient restriction endonucleases, and a sequence that is functional in another organism. ase site, and one or more selectable markers (e.g., those described in WO 01 / 9658 No. 4, International Publication No. WO 01 / 29058, and U.S. Pat. 326,193).

[0171] In some embodiments, the scaffold comprises: (a) a CD33 antigen binding domain; and (b) a stalked (c) the transmembrane domain; (d) 4-1BB or CD28, or both. (e) a costimulatory signaling domain containing a CD3 zeta signaling domain; A lentiviral vector is provided that contains a nucleic acid sequence encoding a chimeric antigen receptor (CAR). Optionally, the vector is a truncated epidermal growth factor receptor (HER1t or HE R1t-1), CD20t-1, or full-length CD20.

[0172] Optionally, a scaffold and (1) a truncated epidermal growth factor receptor, e.g., HER1t or (2)(a) CD33 antigen binding, and (b) the stalk domain; (c) the transmembrane domain; (d) 4-1BB or (e) a costimulatory signaling domain that includes a co-stimulatory domain of CD28 or both; and A nucleic acid sequence encoding a chimeric antigen receptor (CAR) containing a D3 zeta signaling domain. A vector containing the sequence is presented.

[0173] Optionally, a scaffold and (1) a full-length CD20, a truncated CD20, or a variant thereof. , and (2) (a) a CD33 antigen-binding domain, (b) a stalk domain, (c) (d) costimulatory antibodies that contain a transmembrane domain, such as 4-1BB or CD28, or both (e) a chimeric antibody comprising a CD3 zeta signaling domain. A vector comprising a nucleic acid sequence encoding a progenitor receptor (CAR) is provided.

[0174] In some embodiments, a nucleic acid encoding a CD33-specific CAR is delivered to a lentivirus. The vector containing the backbone components is cloned. An exemplary backbone component is pFUGW and pSMPUW. pFUGW lentiviral vectors. The backbone is a self-inactivating (SIN) lentiviral vector backbone that ingests unwanted HIV-1 viruses. Removal of viral sequences results in the development of neoplasms, deleterious mutations, and the proliferation of infectious particles. In some embodiments, the CD33 CAR encoding The vector also encodes mbIL-15 in a single construct. CD33 CAR and mbIL-15 are delivered on two separate lentiviral vectors In some embodiments, mbIL-15 is encoded by a truncated epidermal growth factor receptor tyrosine kinase inhibitor. In embodiments, the CD33 CAR is expressed in conjunction with mbIL-15 and Together with the cell tag, they can be co-expressed from a single lentiviral vector. In one embodiment, the CD33 CAR can be under the control of an inducible promoter. In embodiments, the mbIL-15 may be under the control of an inducible promoter. The inducible promoter can be an inducible promoter of a gene switch ligand. Inducible promoters can be used in a variety of applications, including gene switches such as RHEOSWITCH® gene switches. Small molecule ligand-induced, two-polypeptide ecdysone receptor-based gene expression It may be a gene switch.

[0175] In one embodiment, the CD33 CAR described herein comprises an anti-CD33 scFv, Human CD8 hinge and transmembrane domains, and human 4-1BB signaling domain In another embodiment, the C-terminal domain of the present invention comprises a human CD3 zeta signaling domain. The D33 CAR contains an anti-CD33 scFv, a human CD8 hinge, and a transmembrane domain. a human 4-1BB signaling domain and a human CD3 zeta signaling domain; Optionally, the antibody may include a truncated epidermal growth factor receptor (HER1t or HER1t-1) tag. Other suitable vectors may integrate randomly into the DNA of a host cell. In the case where the expression vector contains a recombination site that allows specific recombination with the host cell chromosome, Such an expression vector for integration may be used in a host cell. The endogenous expression control sequences of the chromosome of the desired protein can be used to effect expression of the desired protein. Examples of specific integrating vectors are available from, for example, Invitrogen (Carlsbad, Calif. (e.g., pcDNA™5 / FRT), or pExchange-6 from Stratagene (LaJolla, Calif.) cre-lox systems such as those found in Core Vectors Examples of vectors that randomly integrate into a host cell chromosome include For example, pcDNA3.1 ( (when introduced in the absence of T antigen), and Promega (Madison, Wis. (ACT)FLEXI™ from . Motor elements, such as enhancers, regulate the frequency of transcription initiation. Typically, The promoter elements are located within the region 30–110 bp upstream of the initiation site, but A number of promoters have recently been shown to contain functional elements downstream of the initiation site. If the elements are inverted or displaced relative to each other, To preserve promoter function, the spacing between promoter elements should be In the case of the thymidine kinase (tk) promoter, activity begins to decline If the spacing between promoter elements is increased to a distance of 50 bp before Individual elements may co-activate transcription depending on the promoter. It is believed that they may function in an operative manner or independently.

[0176] One example of a suitable promoter is the cytomegalovirus (CMV) immediate early promoter. The promoter sequence is a promoter sequence that can be used to express any polynucleotide sequence operably linked to it. A strong constitutive promoter capable of driving high level expression of the nucleic acid sequence. - Array.

[0177] Another example of a suitable promoter is human elongation growth factor 1 alpha 1 (hEF1a1). In some embodiments, the vector construct comprising the CAR described herein is a hEF Includes functional variants of 1a1.

[0178] However, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus ( MMTV), human immunodeficiency virus (HIV) LTR (long terminal repeat epeat) promoter, MoMuLV promoter, avian leukosis virus promoter -, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter In addition, actin promoter, myosin promoter, hemoglobin promoter, and and creatine kinase promoter, Other constitutive promoter sequences may also be used, including, but not limited to, the Furthermore, it is not intended that the present invention be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use may be effected, when such expression is desired, by the use of a polynucleotide to which it is operably linked. To turn on expression of a gene sequence or to turn off expression when expression is not desired. An example of an inducible promoter is the metallothionein promoter. promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter. These include, but are not limited to, cyclin promoters.

[0179] To assess the expression of the CAR or portions thereof described herein, The expression vectors used can also be transfected or infected via viral vectors. A selection marker that facilitates the identification and selection of expressing cells from a cell population that is desired to express the It may also contain a car gene or a reporter gene or both. The selectable marker is carried on a separate piece of DNA and is used in the co-transfection procedure. Both selectable markers and reporter genes are used to regulate expression in a host cell. To make this possible, the appropriate regulatory sequences can be used to flank the marker. For example, antibiotic resistance genes such as the neomycin resistance gene (neo) and the ampicillin resistance gene In some embodiments, the truncated epidermal growth factor receptor (HER1t or HE The R1t-1) tag can be used as a selectable marker gene.

[0180] The reporter gene is used to identify potentially transfected cells and to identify the regulatory sequences. In general, reporter genes can be used to assess the functionality of a gene. Not present in or expressed by the organism or tissue of the scipient, Expression of a polypeptide that is manifested by some easily detectable property, e.g., enzymatic activity. The gene that encodes the appropriate peptide after DNA is introduced into the recipient cell. At each time point, the reporter gene expression is assayed. The enzymes used in the study were luciferase, beta-galactosidase, and chloramphenicol acetyltransferase. phosphatase, a gene encoding secreted alkaline phosphatase, or a gene encoding green fluorescent protein Protein genes (e.g., Ui-Tei et al., FEBS Letters 479: 79-82 (2000)). Suitable expression systems are well known and can be prepared using known techniques or purchased commercially. Generally, the smallest 5' fragment that gives the highest level of reporter gene expression is chosen. The construct with the linking region is identified as a promoter. The region can be linked to a reporter gene and the drug can be used to induce promoter-driven transcription. The present invention can be used to assess the ability of a therapeutic agent to modulate

[0181] In some embodiments, the vectors described herein drive expression of a transgene. hEF1a1 promoter, bovine growth hormone polyA sequence, and wood thymus In addition to the Jack hepatitis virus posttranscriptional regulatory element (WPRE), the pFUGW plasmid contains It may contain the LTR sequences from which it is derived.

[0182] Methods for introducing and expressing genes into cells are well known. In the context of expression vectors The vector is then transfected into a host cell, e.g., a mammalian cell, by any method in the art. , bacterial cells, yeast cells, or insect cells. The expression vector can be introduced into the host cell by physical means or by chemical means. The gene may be introduced via intracellular or biological means.

[0183] For introducing polynucleotides into host cells, e.g., immune effector cells. The chemical methods used are calcium phosphate precipitation, lipofection, gene gun, and microinjection. In the art, vectors and / or exogenous factors include, but are not limited to, vector injection, electroporation, and the like. Methods for generating cells containing recombinant nucleic acids are well known. See, for example, Sambrook et al. ular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (2001 In some embodiments, the polynucleotide is introduced into a host cell using a method such as The methods for transfection are calcium phosphate transfection or polyethylenimine (PEI) ) transfection.

[0184] To introduce a polynucleotide of interest into a host cell, e.g., an immune effector cell, Biological methods for this purpose include the use of DNA and RNA vectors. Vectors, and especially retroviral vectors, are used to deliver genes to mammalian cells, e.g. It has become the most widely used method for inserting the vector into mouse cells. - lentivirus, poxvirus, herpes simplex virus I, adenovirus, and adeno-associated viruses, etc. See, e.g., U.S. Patent No. 5,350,674. See specification and US Pat. No. 5,585,362.

[0185] Chemical means for introducing polynucleotides into host cells include polymer complexes, nanoparticles, and Capsules, microspheres, beads, as well as oil-in-water emulsions, micelles, mixed micelles In vivo, the present invention provides a method for the preparation of a liquid suspension comprising administering to a subject a liquid suspension comprising administering to said subject a liquid suspension, ... and administering to said subject a liquid suspension, including to said subject a liquid suspension, and Exemplary colloids for use as delivery vehicles in vitro and in vivo The liquid vesicle is a liposome (eg, an artificial membrane vesicle).

[0186] When a viral delivery system is used, an exemplary delivery vehicle is a liposome. Introduction of nucleic acids into a host cell (in vitro, ex vivo, or in vivo) In another embodiment, the nucleic acid can be associated with a lipid. The lipid-associated nucleic acid is distributed in aqueous lipids that are interspersed within the lipid bilayer of the liposome. It can also be encapsulated inside liposomes, and is compatible with both liposomes and oligonucleotides. They can also be attached to liposomes via associated linking molecules and incorporated into the liposomes. They can be incorporated into liposomes, or can be dispersed in solutions containing lipids. It can be mixed with lipids, it can be combined with lipids, it can be in lipids It may also be included as a suspension of the compound, or may be included or complexed with micelles. or may be otherwise associated with lipids. The lipid / expression vector related composition is not limited to any particular structure in solution. For example, the composition may be present in a bilayer structure as a micelle, which may be referred to as a "collapsing" The composition may also be simply dispersed in a solution. The lipids may also be dispersed in water and possibly form aggregates that are not uniform in size or shape. A lipid is a fatty substance, which may be natural or synthetic. For example, lipids In nature, lipid droplets occur in the cytoplasm, as well as long-chain aliphatic hydrocarbons, Fatty acids, alcohols, amines, aminoalcohols, and aldehydes, and their derivatives The present invention includes a class of compounds containing

[0187] Lipids suitable for use can be purchased from commercial sources. Dimethyl phosphate ("DMPC") was purchased from Sigma, St. Louis, Mo. Dicetyl phosphate ("DCP") is available from K & K Laboratories. Cholesterol ("Ch") can be purchased from Sigma-Aldrich (Plainview, NY). oi) is available from Calbiochem-Behring; Dimethylphosphatidylglycerol ("DMPG") and other lipids are available from Avanti Pharmaceuticals. It can be purchased from Olar Lipids, Inc. (Birmingham, Ala.). Stock lipid solutions in chloroform or chloroform / methanol can be stored at approximately -20°C. Chloroform is the only solvent that evaporates more readily than methanol. "Liposome" means an encapsulated lipid bilayer or lipid aggregate. In general, the present invention includes a variety of single lipid vehicles and multi-layer lipid vehicles formed by the creation of A liposome is a vesicular structure with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes can be characterized as having a structure in which the multilamellar liposomes are separated by an aqueous medium. Multilamellar liposomes have multiple lipid layers bounded by a phospholipid. The lipid components undergo autotransition before forming the closed structure. , which traps water and dissolved solutes between the lipid bilayer (Ghosh et al., Glycobiology 5: 505-10 (1991)). However, there are also compositions in solution that have structures different from the normal vesicle structure. For example, lipids may have a micelle structure, and the lipid molecules may form heterogeneous structures. Lipofectamine-nucleic acid complexes are also envisioned. do.

[0188] Non-viral-based delivery systems Nucleic acids encoding CARs described herein can also be used to deliver CARs to immune effector cells. " refers to a synthetic DNA transposon system for introducing NA sequences into vertebrate chromosomes. Non-viral based transposon-based systems such as the Sleeping Beauty (SB) transposon system An exemplary SB transposon system can also be used for the introduction of the vector. For example, U.S. Pat. Nos. 6,489,458 and 8,227,432 disclose This is described in Sleeping Beauty Translator and illustrated in FIG. The splicon system is a system that expresses Sleeping Beauty (SB) transposase and SB transposase. It is composed of transposons.

[0189] DNA transposons are simple cuts from one DNA site to another. Translocation is a process that involves the transfer of a defined DNA segment to a single DNA fragment. They can be excised from a molecule and placed within the same DNA molecule or genome, or within different DNA molecules or This is a precise process that often transfers the Tc1 / marine gene to another site in the genome. Like r-type transposases, SB transposase also transfers transposons to recipient The insertion site is located at a TA dinucleotide pair in the target DNA sequence. This may be at another site within the offspring, or it may be in another DNA molecule (or chromosome). There are approximately 200 million TA sites in the mammalian genome, including the genome. During the transposon integration process, the TA sequence is duplicated. This duplication is responsible for the prominent role of transposition. Transposases are a characteristic of transcription and are used in some experiments to confirm mechanisms. In some cases, the transposase may be encoded within a transposon, and the transposase may be provided by another source. In some cases, the transposon may be a non-autonomous element. Transposons cannot excise and reinsert independently after insertion, so gene trees The SB transposon is most useful as a vector for the introduction of genes into the vertebrate genome. It is also envisaged that they will be used as non-viral vectors for gene therapy. Specifically, the Sleeping Beauty (SB) system is used to genetically modify T cells. (Hackett et al., Mol Ther 18:674-83, (2010)) was adapted from (Cooper et al., Blood 105:1622-31, (2005)). This involves two steps: (i) redirecting T cell specificity; SB transposon [i.e., chimeric antigen receptor (CAR)] and SB Electrotransfer of DNA plasmid expressing transposase (Jin et al. , Gene Ther 18:849-56, (2011), Kebriaei et al., Hum Gene Ther 23:444-50, (2012) ), and (ii) designer artificial antigen-presenting cells (AaPCs) derived from the K562 cell line. )(AaPC(Activating and Propagating Cell) The propagation and propagation of T cells stably expressing the integrated sequences in contact with the In one embodiment, the SB transposon system is Such coding sequences include those encoding L-21, and / or chimeric antigen receptors. The Sing system is described, for example, in h et al., Cancer Res (8):68 (2008). April 15, 2008 and Maiti et al., J Immunoth er. 36(2): 112-123 (2013).

[0190] In certain embodiments, the CD33 CAR and mbIL-1 5 is encoded within a transposon DNA plasmid vector and encodes the SB transposase In certain embodiments, the vectors described herein are encoded in separate vectors. The CD33 CAR is encoded within a transposon-based DNA plasmid vector and expressed in mb- IL15 is encoded within a second transposon-based DNA plasmid vector and expressed in the SB transposon. The endonuclease is encoded in a third DNA plasmid vector. mbIL-15 binds to kill tags, such as HER1t, HRt-1, CD20, or Encoded by CD20t-1.

[0191] Exogenous nucleic acid may be introduced into a host cell or the cell may be otherwise transfected with an inhibitor of the invention. Regardless of the method used to expose the host cell to the recombinant DNA sequence, To identify the gene(s) that are involved in the synthesis of the protein(s), various assays can be performed. Such assays include, for example, , Southern and Northern blotting, RT-PCR and PCR, etc. "Molecular biology" assays, well known in the art; for example, immunological means (ELISA and Western blot) blot), or an assay as described herein for identifying an agent comprising: Assays falling within the scope of the present invention include assays to detect the presence or absence of a particular peptide. This includes "biochemical" assays.

[0192] In some embodiments, the SB11 transposon system, the SB100X transposon system, the S B110 transposon system, piggyBac transposon system (see, e.g., Wilson et al., "PiggyBac Transposon-mediated ed Gene Transfer in Human Cells,” Molecular Therapy 15:139-145 (2007). ), and / or the piggyBat transposon system (e.g., Mitra et al., “ Functional characterization of piggyBat from the bat Myotis lucifugus unveils an active mammalian DNA transposon,” Proc. Natl. Acad. Sci USA 110:234-239 (2013) (see, e.g., J. Med. Soc. 2014; 2013; 201 ... For further transposase and transposon systems, see their respective No. 7,148,233, which is incorporated herein by reference in its entirety. No. 03, No. 8,227,432, U.S. Patent Publication No. 2011 / 01170 No. 72, Mates et al., Nat Genet, 41(6):753-61 (2009). doi: 10.1038 / ng.343. Epub 2009 May 3, Gene Ther., 18(9):849-56 (2011). doi: 10.1038 / gt.2011.40. Epub 2011 Mar 31 and Ivics et al., Cell. 91(4):501-10, (1997) do.

[0193] In another embodiment, CD33 is expressed via integration of a recombinase and an expression vector. CAR and cytokines, mbIL-15, and / or HER1t / HER1t Other genetic elements, such as the -1 / CD20 / CD20t-1 tag, can be used to express immune effectors. Such vectors can be integrated into the DNA of a host cell. In some cases, the expression vector is integrated randomly, whereas in other cases, the expression vector is integrated specifically into the host cell chromosome. Such integrative expression vectors may contain recombination sites that allow for recombination of the host. Endogenous expression control sequences of the host cell's chromosomes can be used to effect expression of the desired protein. In some embodiments, the sequences on the donor polynucleotide that flank the integration site are The presence of sequences homologous to the sequence facilitates targeting of integration. Targeting of integration using the donor polynucleotides described herein is conventional. knockout or deletion of genes by homologous recombination, This can be accomplished according to the technique used to create the look-in. the presence of sequences on the donor polynucleotide that are homologous to sequences flanking the integration site; and contacting the cell with a donor polynucleotide in the presence of a site-specific recombinase. This promotes integration targeting by both activating the site-specific reconstitution. A recombinase, or simply a recombinase, is a conserved intercalation enzyme that mediates the synthesis of nucleotides between compatible recombination sites. As used herein, site-specific recombination refers to a polypeptide that catalyzes site-specific recombination. In addition to naturally occurring polypeptides, target recombinases include derivatives, mutants, and and / or fragments, as well as native polynucleotides, that encode recombinases that retain activity. This includes derivatives, variants, and / or fragments that

[0194] The recombinase is introduced into the target cell prior to the introduction of the targeting vector. It can be introduced at the same time as this, or it can be introduced later. The enzyme can be, for example, incorporated into liposomes, particles, or by microinjection. Alternatively, the protein can be introduced directly into cells using a suitable Expression vectors are used to express DNA encoding the recombinase or messenger RNA. A polynucleotide, which is A, can also be introduced into a cell. The components of the recombination vector are expression vectors that contain sequences encoding the recombinase of interest. However, expression of the recombinase can be achieved in other ways, e.g. For example, expression of the recombinase can be controlled by a regulatable promoter (i.e., the expression can be selectively controlled). It can also be regulated by placing it under the control of a promoter that can be induced or repressed by .

[0195] Recombinases come from the integrase or resolvase families. The integrase family of recombinases has over 100 members, Examples include FLP, Cre, and lambda integrase. - also called the tyrosine family or lambda integrase family, DNA The catalytic hydroxyl group of tyrosine is used for nucleophilic attack on the phosphodiester bond of Typically, members of the tyrosine family first nick the DNA and then This then forms a double-strand break. Examples of tyrosine family integrases are: Cre integrase, FLP integrase, SSV1 integrase, and lambda (lambda) integrase. In the solvase family, a conserved serine residue provides covalent attachment to the DNA target site. (Grindley, et al., (2006) Ann Rev Biochem 16:16).

[0196] In one embodiment, the recombinase is SPβc2 recombinase, SF370.1 recombinase. recombinase, Bxb1 recombinase, A118 recombinase, and ΦRv1 recombinase The present invention relates to an isolated polypeptide comprising a nucleic acid sequence encoding a recombinase selected from the group consisting of Examples of serine recombinases are provided herein by reference in their entirety. No. 9,034,652, the disclosure of which is incorporated herein by reference in its entirety. It is described in.

[0197] Recombinases for use in the practice of the present invention can also be produced recombinantly. The desired recombinase activity can be obtained by the synthesis of a polypeptide having the desired recombinase activity and purified as described above. The polypeptides can be prepared by ammonium sulfate precipitation of proteins, a method known in the art. Methods, size fractionation, affinity chromatography, HPLC, ion exchange chromatography affinity chromatography (e.g., Thorpe & Smith, Proc. Nat. Acad. Sci. 95:5505-5510, 1998). The protein can be purified to the desired degree of purity by protein purification methods.

[0198] In one embodiment, the recombinase is administered to the recombinase where recombination is desired by any suitable method. The conjugation site can be introduced into a eukaryotic cell containing the conjugation site. Introducing functional proteins into cells by microinjection or other methods Methods for this purpose are well known. Introduction of purified recombinase protein results in the transient presence of the protein. , and its functionality, which is often the preferred embodiment. The gene encoding the recombinase is contained in an expression vector used to transform the cell. The method comprises: incorporating a polynucleotide encoding a recombinase into a polynucleotide sequence in a eukaryotic cell; The gene is contained within an expression vector operably linked to a promoter that mediates expression of the octide. The recombinase polypeptide also includes a message encoding the recombinase polypeptide. Recombinases can also be introduced into eukaryotic cells by means of sender RNA. It is preferred that the nucleic acid fragment be present only as necessary for insertion into the genome to be modified. Therefore, the lack of persistence associated with most expression vectors is not detrimental. The recombinase gene is expected to be inserted into the target vector prior to introduction of the exogenous polynucleotide of interest. It can be installed at the same time as this, or it can be installed later. In one embodiment, the recombinase gene is located in the vector carrying the polynucleotide to be inserted. The recombinase gene is still present in the vector, and the recombinase gene is still present in the polynucleotide to be integrated. It is possible.

[0199] In one embodiment, the method for site-specific recombination comprises a first recombination site and a second recombination site. providing a recombination site; and introducing the first recombination site and the second recombination site into a prokaryotic cell. contacting the nucleic acid with a recombinase polypeptide of and a step of introducing a recombinase polypeptide into the first recombination site and the second recombination site. The first recombination site is capable of mediating recombination with an attP or The first recombination site is attB, the second recombination site is attB or attP, and the second recombination site is attB or attP. If the junction site is attB, the second recombination junction site is attP, and the first recombination junction site is attP. If the first recombination junction is attP, the second recombination junction is attB. In this case, the recombinase is a Phage recombinase, phage recombinase of Streptococcus pyogenes enzymes, phage recombinases from Bacillus subtilis, Mycobacterium tuberculosis U. tuberculosis phage recombinase and Mycobacterium smegmatis atis) phage recombinase.

[0200] Further embodiments include the introduction of a site-specific recombinase into the cell whose genome is to be modified. One embodiment provides a method for obtaining site-specific recombination in a eukaryotic cell, comprising: providing a eukaryotic cell containing a first recombination junction site and a second recombination junction site; The first recombination junction site and the second recombination junction site are bound to a prokaryotic recombinase polynucleotide. and contacting the peptide with the recombination site resulting in recombination between the recombination sites. a recombinase polypeptide that binds the first recombination junction site and the second recombination junction site; The first recombination junction site is capable of mediating recombination of the phage genome. The junction site (attP) or the recombination junction site (attB) of the bacterial genome, and the second The recombination junction is attB or attP, and the first recombination junction is attB , then the second recombination junction is attP and the first recombination junction is at If the second recombination junction site is attB, the recombinase The phage recombinase of Listeria monocytogenes Streptococcus pyogenes phage recombinase, Bacillus subtilis phage recombinase Phage recombinase of Mycobacterium tuberculosis and the phage recombinase of Mycobacterium smegmatis In one embodiment, the recombinase is selected from the group consisting of The enzymes used were A118 recombinase, SF370.1 recombinase, and SPβc2 recombinase. recombinase, ΦRv1 recombinase, and Bxb1 recombinase. In one embodiment, recombination results in integration.

[0201] Cells containing CD33 CAR and vector Provided herein are modified cells that express the CARs described herein. In certain embodiments, the modified cells described herein are immune effector cells. In some embodiments of the present disclosure, the scaffold and the endothelial cell line comprise a medicament for use in treating a chronic inflammatory disease. or HER1t-1), and (2) (a) the CD33 antigen-binding domain, (b) the streptavidin-binding domain, (c) a transmembrane domain; (d) 4-1BB or CD28, or any of the foregoing. (e) a costimulatory signaling domain that contains both the CD3 zeta signaling domain and the CD3 zeta signaling domain. and a nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising the domain. Immune effector cells are presented.

[0202] In certain embodiments, the antibody comprises: (a) a CD33 antigen-binding domain; (b) a stalk domain; (c) a transmembrane domain; and (d) 4-1BB or CD28, or both. (e) a CD3 zeta signaling domain, and f) A chimeric antigen receptor containing a truncated epidermal growth factor receptor (HER1t or HER1t-1) Immune effector cells containing receptors (CARs) are presented.

[0203] In some embodiments of the present invention, (1) a kill switch, a selection marker, a biomarker or a combination thereof, and (2)(a) CD33 (b) antigen-binding domain, (b) stalk domain, (c) transmembrane domain, (d) 4-1B B or CD28, or both, and e) Immunoglobulins containing a chimeric antigen receptor (CAR) containing a CD3 zeta signaling domain In some embodiments, the cell tag is HER1t, HER1 t-1, CD20t-1, or CD20.

[0204] In some embodiments, the immune effector cells are T cells, natural killer (NK) cells, or In some embodiments, the immune cells include T cells, cytotoxic T lymphocytes (CTLs), and regulatory T cells. The cells exhibit anti-tumor immunity when the CD33 antigen-binding domain binds to CD33. .

[0205] Engineered immune effector cells Expressing one or more heterologous genes or heterologous polypeptides as described herein The immune effector cells modified as described herein are presented. CAR, as well as HER1t tag, HER1t-1 tag, CD20 tag, and CD20 Immune effector cells modified to express at least one of the t-1 tags are Optionally, the CD33 CAR, mbIL-15, and HER1t tag, HER1t-1 tag, CD20 tag, and CD20t-1 tag. The immune effector cells are modified to express at least one of the antigens. do.

[0206] As used herein, "T cell" or "T lymphocyte" refers to a T cell that is involved in cell-mediated immunity. A "T cell" or "T lymphocyte" is a type of lymphocyte that plays a central role in The presence of T cell receptors (TCR) on the cell surface allows B cells and natural killer cells They can be distinguished from other lymphocytes, such as NK cells.

[0207] In some embodiments, the engineered immune effector cells are T cells and / or natural T cells or T lymphocytes are modified immune cells that contain killer cells. Exemplary T cells include helper T cells, cytotoxic T cells, T H17 cells, stem cell memory T cells (TSCM), naive T cells, memory T cells , effector T cells, regulatory T cells, or natural killer T cells.

[0208] Helper T cells (TH cells) mediate the maturation of B cells into plasma cells and memory B cells. and in immune processes, including activation of cytotoxic T cells and macrophages, among others. TH cells support white blood cells. In some cases, TH cells are activated by expression of the CD4 glycoprotein on the cell surface. Helper T cells are known as CD4+ T cells because of their ability to bind antigens. C) When a peptide antigen is presented by MHC class II molecules expressed on the surface of the Upon activation, helper T cells divide rapidly and mediate active immune responses. secrete small proteins called cytokines that help These cells include TH1, TH2, TH3, TH17, Th9, or TNF-α. There are several subtypes that secrete different cytokines to facilitate different types of immune responses. Signaling from APCs induces T Commits cells to a particular subtype.

[0209] Cytotoxic T cells (TC cells or CTLs) destroy virus-infected cells and tumor cells. These cells also express endothelial cells on their surface, which are involved in the destruction of the endothelial cells and in the rejection of transplants. These T cells express the CD8 glycoprotein and are therefore known as CD8+ T cells. These cells bind to antigens associated with MHC class I molecules, which are present on the surface of all nucleated cells. Regulatory T cells recognize their targets by combining with IL-1. Through 0, adenosine, and other molecules, CD8+ cells are inactivated into an anergic state. This may prevent autoimmune diseases.

[0210] Memory T cells are antigen-specific T cells that persist for a long time after an infection has cleared. Memory T cells are a subset of the immune system that can reactivate upon reexposure to their cognate antigen. Rapidly expand into large numbers of effector T cells, providing the immune system with the ability to fight off previous infections. Memory T cells are a subtype of stem cell memory T cells (T SCM), central memory T cells (TCM cells) and two types of effector memory -T cells (TEM cells and TEMRA cells). Memory cells are CD4+ cells. Memory T cells may be CD8+ cells or CD8+ cells. , CD45RO, CD45RA, and / or CCR7.

[0211] Regulatory T cells (Treg cells), formerly known as suppressor T cells, Regulatory T cells play a role in maintaining immune tolerance. The primary role of regulatory T cells is to regulate T cell-mediated immunity. Blocking the immune response and evading the negative selection process in the thymus The aim is to suppress this.

[0212] Natural killer T cells (NKT cells) act as a bridge between the adaptive and innate immune systems. Conventional T cells recognize peptide antigens presented by major histocompatibility complex (MHC) molecules. Unlike normal cells, NKT cells recognize glycolipid antigens presented by a molecule called CD1d. Upon activation, these cells undergo functions ascribed to both Th and Tc cells. These may play a role in the production of cytokines and release of cytolytic / cell killing molecules. NKT cells also recognize some tumor cells and cells infected with herpes viruses. , it is also possible to eliminate these.

[0213] Natural killer (NK) cells are a type of cytotoxic lymphocyte of the innate immune system. In some cases, NK cells act as a frontline defense against viral infections and / or tumor formation. NK cells detect MHC presented on infected or cancerous cells. It is possible to induce cytokine release and subsequent lysis and apoptosis. NK cells further induce the activation of stress cells in the absence of antibody and / or MHC. It is possible to detect the presence of IgG4, thereby allowing for a rapid immune response.

[0214] Dosage of Engineered Immune Effector Cells In some embodiments, an amount of engineered immune effector cells is administered to a subject in need thereof. The dose is determined based on efficacy and potential to induce cytokine-associated damaging effects. In some cases, the amount of engineered immune effector cells is determined based on the potential for development of a therapeutic agent. Approximately 10 engineered immune effector cells 5 ~about 10 9 Optionally, modified immune efflux The amount of engineered immune effector cells is approximately 10 5 ~about 10 8 Includes In some cases, the amount of engineered immune effector cells is 100 mg / kg of engineered immune effector cells. -about 10 cells 5 ~about 10 7 In some cases, the amount of engineered immune effector cells comprises 1 Approximately 10 engineered immune effector cells per kg 6 ~about 10 9 Including pieces. In some cases, modifications The amount of immune effector cells is approximately 10 engineered immune effector cells per kg. 6 ~about 1 0 8 In some cases, the amount of engineered immune effector cells is 100 mg / kg of engineered immune effector cells. 10 effector cells 7 ~about 109 Optionally, modified immune effector cells The amount of cells is approximately 10 engineered immune effector cells per kg. 5 ~about 10 6 Includes pieces. Therefore, the amount of engineered immune effector cells is approximately 1 kg of engineered immune effector cells. 10 6 ~about 10 7 In some cases, the amount of engineered immune effector cells may include Approximately 10 engineered immune effector cells 7 ~about 10 8 Optionally, modified immuno-F The amount of engineered immune effector cells is approximately 10 8 ~about 10 9 Including In some cases, the amount of engineered immune effector cells may be 100 mg / kg of engineered immune effector cells. Approximately 10 9 In some cases, the amount of engineered immune effector cells may include Approximately 10 engineered immune effector cells per 8 Optionally, the modified immune effector - The amount of cells is approximately 10 engineered immune effector cells per kg. 7 Including pieces. The amount of engineered immune effector cells is approximately 10 engineered immune effector cells per kg. 6 In some cases, the amount of engineered immune effector cells may include 100 mg of engineered immune effector cells per kg. Effector cells approx. 10 5 Includes pieces.

[0215] In some embodiments, the engineered immune effector cells are CD33-specific CAR-T cells. In some cases, the amount of CD33-specific CAR-T cells is 1k Approximately 10 CAR-T cells per gram 5 ~about 10 9Optionally, CD33-specific C The amount of CAR-T cells was approximately 10 CAR-T cells per kg. 5 ~about 10 8 Includes pieces. Therefore, the amount of CD33-specific CAR-T cells was approximately 10 CAR-T cells per kg. 5 ~about 10 7 In some cases, the amount of CD33-specific CAR-T cells may include Approximately 10 CAR-T cells 6 ~about 10 9 Optionally, a CD33-specific CAR- The amount of T cells is approximately 10 CAR-T cells per kg. 6 ~about 10 8 Including pieces. The amount of CD33-specific CAR-T cells was approximately 10 CAR-T cells per kg. 7 ~about 1 0 9 In some cases, the amount of CD33-specific CAR-T cells may include C About 10 AR-T cells 5 ~about 10 6 Optionally, CD33-specific CAR-T cells The amount of CAR-T cells per kilogram is approximately 10 6 ~about 10 7 In some cases, CD The amount of 33-specific CAR-T cells was approximately 10 CAR-T cells per kg. 7 ~about 10 8 Pieces In some cases, the amount of CD33-specific CAR-T cells is 100 mg / kg of CAR-T cells. about 10 cells 8 ~about 10 9 In some cases, the amount of CD33-specific CAR-T cells includes Approximately 10 CAR-T cells per kg 9 Optionally, a CD33-specific CAR - The amount of T cells is approximately 10 CAR-T cells per kg. 8In some cases, CD The amount of 33-specific CAR-T cells was approximately 10 CAR-T cells per kg. 7 Including pieces. By combining the two, the amount of CD33-specific CAR-T cells was approximately 1 CAR-T cell per kg. 0 6 In some cases, the amount of CD33-specific CAR-T cells may include C About 10 AR-T cells 5 Includes pieces.

[0216] Source of immune effector cells In certain aspects, the embodiments described herein provide redirected, antigen-specific Generate allogeneic immune effector cells (e.g., T cells, NK cells, or NK T cells) A method of infecting and / or expanding a cell comprising injecting a DNA encoding a CAR into a cell. A (or RNA) construct containing an expression vector, and then Optionally, the cells are stimulated with feeder cells, recombinant antigens, or antibodies against the receptor. In certain embodiments, the method comprises the step of growing the cells by inducing the expression of a CAR. The cells (or cell populations) engineered to produce these products include stem cells, iPS cells, immune effector cells, and cells, or precursor cells of these cells.

[0217] Sources of immune effector cells can include both allogeneic and autologous sources. By combining these cells, immune effector cells can be generated from stem cells or induced pluripotent stem cells (iPSCs). Thus, cells for modification according to the embodiment may be derived from umbilical cord blood, They can be isolated from peripheral blood, human embryonic stem cells, or iPSCs. For example, allogeneic T cells. The cells are engineered to contain a chimeric antigen receptor (and, optionally, to lack a functional TCR). In some embodiments, the immune effector cells can be modified to human peripheral blood mononuclear cells. Primary human T cells, such as T cells derived from peripheral blood MCs (PBMCs). They can also be harvested from the bone marrow after stimulation with G-CSF (granulocyte colony-stimulating factor). Alternatively, they can be harvested from umbilical cord blood. After infusion (depending on the expression construct for CAR), the cells are either immediately infused or cryopreserved. In certain embodiments, after transfection, the cells are Within about 1, 2, 3, 4, 5 or more days after transfer, ex vivo In this way, they can be bred as bulk populations for days, weeks, or months. In a further embodiment, after transfection, the transfected cells can be cloned. A single expression cassette or vectors that are cloned and integrated or maintained episomally Clones confirming the presence of the plasmid and expression of the chimeric antigen receptor were identified by ex vivo analysis. The clones selected for expansion are then expanded in ivo. The recombinant T cells are capable of specifically recognizing and lysing target cells that are or other cytokines that bind to the common gamma chain (e.g., IL-7, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL They can be expanded by stimulation with IL-15, IL-21, and other cytokines. Recombinant T cells can be expanded by stimulation with artificial antigen-presenting cells. The recombinant T cells can also be expanded in contact with artificial antigen-presenting cells, They can also be expanded with antibodies such as OKT3, which crosslinks CD3 on the surface. Recombinant T cell subsets were isolated using magnetic bead-based isolation and / or fluorescence-activated cell sorting. The use of preparative techniques allows for further selection and further culture with AaPCs. In a further aspect, the genetically modified cells can be cryopreserved.

[0218] T cells also reside in bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from the site of infection, and peritoneal It can also be obtained from a number of sources, including water, pleural effusion, splenic tissue, and tumors. In certain embodiments, any number of T cell lines available in the art are used. In certain embodiments of the invention, the T cells are Ficoll (registered trademark)- Blood is collected from the subject using any number of techniques known to those of skill in the art, such as (i) ELISA (registered trademark) separation. In some embodiments, cells derived from the circulating blood of an individual can be obtained from an The apheresis product typically contains T cells, monocytes, granulocytes, Contains lymphocytes, including B cells, other nucleated white blood cells, red blood cells, and platelets. In this study, cells collected by apheresis are washed to remove the plasma fraction, and the cells are then In the present invention, the cells are placed in an appropriate buffer or medium for the processing steps. In an embodiment, the cells are washed with phosphate buffered saline (PBS). So, the cleaning solution is calcium deficient, and sometimes magnesium deficient, but not all, Many divalent cations may be absent. Initial activation steps in the absence of calcium As one of ordinary skill in the art will readily appreciate, the washing step may be used to enhance the activity of the preparation. Following the original instructions, centrifuge in a semi-automated "flow-through" centrifuge (e.g., Cobe 299 1 Cell Processor, Baxter CytoMate, or Haemonetics This can be achieved by methods known to those skilled in the art, such as using Cell Saver 5). After washing, the cells can be incubated in, for example, Ca2+-free PBS, Mg2+-free PBS, or the like. , Plasmalyte A, or other saline solutions with or without buffers The cells can be resuspended in a variety of biocompatible buffers, such as PBS. The undesired components of the lysis sample are removed and the cells are resuspended directly in culture medium. It can also be done.

[0219] In another embodiment, the centrifugation through a PERCOLL® gradient, By using counter-flow centrifugal elutriation to lyse red blood cells and deplete monocytes, T cells can be isolated. Isolate from peripheral blood lymphocytes. CD3 + , CD28 + , CD4 + , CD8 + , CD45RA + , and CD45RO + Specific subpopulations of T cells, such as T cells, can also be identified using positive or negative selection methods. For example, in one embodiment, T cells can be isolated by DYN Anti-CD3 / anti-CD28 antibodies, such as ABEADS® M-450 CD3 / CD28 T. With 28 (i.e., 3 x 28) conjugated beads, sufficient for the desired T cell positive selection In one embodiment, the incubation time is about 30 minutes. In further embodiments, the time period is between 30 minutes and 36 hours, or longer. In a further embodiment, the time range is between 0 and 100 ms, and any integer value therebetween. In yet another embodiment, the time is at least 1, 2, 3, 4, 5, or 6 hours. In one embodiment, the incubation time is 24 hours. For the isolation of T cells from patients with leukemia, long incubation times, such as 24 hours, have been used. The use of a time course of immunosuppression can increase cell yield. T cells are in low numbers compared to other cell types, such as in the situation of isolating TILs In any situation, longer incubation times may be used to isolate T cells. Furthermore, the use of long incubation times can improve capture of CD8+ T cells. Efficiency may also be increased. Thus, simply by shortening or lengthening the time, The T cells are allowed to bind to the CD3 / CD28 beads and / or the beads to T cells (as further described herein). This allows the T cell subpopulation to be preferentially isolated at the initiation of culture or at other points during the process. In addition, the antibodies can be positively or negatively selected in advance on beads or other surfaces. By increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies, Subpopulations of cells may be preferentially identified as positive or negative at the initiation of culture, or at any other desired time point. Those skilled in the art will appreciate that, in the context of the present invention, multiple rounds of selection can be used to negatively select. It will be appreciated that a selection procedure may also be used. and using the "unselected" cells in the activation and expansion steps. It may also be desirable to have "unselected" cells that can be subjected to further rounds of selection. You can also call.

[0220] Enrichment of T cell populations by negative selection directed against surface markers unique to negatively selected cells This can be achieved by a combination of antibodies that are present on negatively selected cells. A cocktail of monoclonal antibodies directed against specific cell surface markers was used to identify cell types that are highly susceptible to inflammatory cytokines. and / or selection via negative magnetic immunoadhesion or flow cytometry. For example, CD4 + The cells were enriched by negative selection using monoclonal antibody citrate. The IL-1 receptors typically express CD14, CD20, CD11b, CD16, HLA-DR, and C In certain embodiments, the antibody is typically directed against CD4. + , CD25 + , C D62L hi , G.I.T.R. + , and FoxP3 + Regulatory T cells expressing Alternatively, in certain embodiments, it may be desirable to select regulatory T cells: Can be depleted by anti-CD25 conjugated beads or other similar selection methods .

[0221] To isolate the desired cell population by positive or negative selection, the concentration and The surface (e.g., particles such as beads) can be varied. The volume in which the beads and cells were mixed together was significantly reduced (i.e., the concentration of cells was reduced). It may be desirable to increase the cell-to-bead contact (e.g., by increasing the cell-to-bead contact). In one embodiment, a concentration of 2 billion cells per ml is used. A concentration of 1 billion cells per liter is used. In a further embodiment, Concentrations of greater than 100 million are used. In further embodiments, concentrations of 10 million cells per ml are used. , 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 450 In yet another embodiment, a cell concentration of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 100, 200, 35, 40, 50, 60, 70, 80, 90, 100, 15 ...200, 35, 40, 75, 80, 85, 90, 95, or 100 million cells In a further embodiment, a cell concentration of 125 million or A concentration of 150 million may be used. The use of higher concentrations may improve cell yield, cell activation, and and can result in increased cell expansion. Furthermore, the use of high cell concentrations CD28-negative T cells, or many tumor cells (i.e., leukemic blood, tumor tissue, etc.) More efficient isolation of cells that may express a low level of the target antigen of interest, such as cells from samples containing Such cell populations may have therapeutic value. For example, the use of high cell concentrations may be desirable in cases where CD28 expression is normally low. Yes, CD8 + Allows for more efficient selection of T cells.

[0222] In a related embodiment, it may be desirable to use low concentrations of cells. (e.g., particles such as beads) is significantly diluted to obtain a mixture of particles and cells. This minimizes the interaction of the cells expressing high amounts of the desired antigen with the particles. For example, CD4 + T cells express high levels of CD28 and dilute concentrations of CD8 + In one embodiment, the cell concentration used is 5 x 10 cells 6 In another embodiment, the concentration used is 100 cells per ml. Approximately 1×10 5 1 x 10 cells per ml 6 and any integer value between these. Possible.

[0223] In another embodiment, the cells are cultured at varying speeds and lengths at 2-10° C. or at room temperature. The mixture can be incubated on a rotator for a period of time to allow the cells to separate.

[0224] The stimulated T cells can also be frozen after a washing step. After a washing step to remove platelets, the cells can be suspended in a freezing solution. Many freezing solutions and freezing parameters are known in the art and in this context One method that may be useful is to use a solution containing 20% ​​DMSO and 8% human serum albumin. PBS with 10% dextran 40 and 5% dextrose, 20% culture medium containing 31% human serum albumin and 7.5% DMSO, or .25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% dextran 40 and 5% dextrose, 20% human serum Albumin and 7.5% DMSO, or, for example, Hespan and Plas The cells were then frozen in a 5% CO2-freezing medium containing malyte A or another suitable cell freezing medium. , frozen to -80°C at a rate of 1°C per minute in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used, as well as storage at -20°C or in liquid nitrogen. Rapid, uncontrolled freezing may also be used.

[0225] In certain embodiments, the cryopreserved cells are thawed as described herein. The cells were incubated at room temperature for 1 hour before being washed and activated using the method of the present invention. do.

[0226] In certain embodiments, the expanded cells described herein may also be required. Collection of a blood sample or apheresis product from the subject during the period prior to the point Therefore, the source of cells to be expanded can be any desired time point. The desired cells, such as T cells, can then be harvested and used in a T cell therapy, such as those described herein. Therapeutic methods for treating T cell-related disorders, including those described herein, for treating any number of diseases or conditions that would benefit from T cell therapy. In one embodiment, the blood sample or Apheresis is generally performed from a healthy subject. In certain embodiments, blood samples are Apheresis or apheresis is generally performed on patients who are at risk of developing a disease but have not yet developed the disease. The cells are collected from healthy subjects without any mutations and the cells of interest are isolated and frozen for subsequent use. In certain embodiments, the T cells are expanded and frozen at a later time point. In certain embodiments, a sample can be obtained from a patient using the methods described herein. The subjects are harvested shortly after diagnosis for the particular disease described, but prior to any treatment. In further embodiments, the cells are treated with natalizumab, efalizumab, antiviral agents, chemotherapy, method, radiation, cyclosporine, azathioprine, methotrexate, mycophenolate and immunosuppressants such as FK506, antibodies, or CAMPATH, anti-CD3 antibodies, Toxan, Fludarabine, Cyclosporine, FK506, Rapamycin, Mycophenolate by drugs such as acids, steroids, FR901228, and other immunosuppressants such as irradiation The subject may be administered any number of appropriate treatment modalities, including, but not limited to, These drugs are isolated from calcium-dependent blood samples or apheresis. Inhibits calcineurin (cyclosporine and FK506), a phosphatase or inhibit p70S6 kinase, which is important in growth factor-induced signaling ( rapamycin) (Liu et al., Cell 66:807-815, (1991); Henderson et al., Immun 73: 316-321, (1991); Bierer et al., Curr. Opin. Immun 5:763-773, (1993). In embodiments, the cells are isolated for a patient and administered bone marrow or stem cell transplant, fludarabine chemotherapy drugs such as cyclophosphamide, external beam radiation therapy (XRT), or OK T cell depletion therapy using antibodies such as T3 or CAMPATH (e.g., The mixture is frozen (either before, at the same time as, or after) for later use. In this study, cells were isolated in advance and treated with a drug that reacts with CD20, such as Rituxan. Can be frozen for subsequent use for treatment following any B cell depletion therapy. Cut.

[0227] In a further embodiment of the invention, the T cells are obtained directly from the patient after treatment. In some cases, after certain cancer treatments, particularly treatments with drugs that impair the immune system, patients The resulting T cells are then harvested, usually immediately following treatment, during the recovery period from the treatment. Cell quality may be optimal or their ability to be expanded ex vivo. It has been observed that the force can be improved using the methods described herein. Following ex vivo modification using , these cells have demonstrated extended engraftment and in vivo This may be a favorable condition for expansion in vivo. Thus, within the context of the present invention, During this recovery phase, blood cells, including T cells, dendritic cells, or other cells of the hematopoietic lineage, are It is envisioned that the mobilization (e.g., GM-C In subjects using a SF mobilization regimen and a conditioning regimen , in particular, repopulation, recirculation of specific cell types in a defined time window following treatment. The present invention can create conditions favorable for cell proliferation, regeneration, and / or proliferation. include T cells, B cells, dendritic cells, and other cells of the immune system.

[0228] T cell activation and expansion Either before or after modifying the T cells to express a CAR as described herein. In addition, T cells can be produced by, for example, the methods described in U.S. Pat. Specification No. 55, Specification No. 6,905,680, Specification No. 6,692,964, Specification No. 6,692,964, Specification No. 5,858,358, Specification No. 6,887,466, Specification No. 6,905,6 Specification No. 81, Specification No. 7,144,575, Specification No. 7,067,318, Specification No. 7,067,318, Specification No. 7,172,869, Specification No. 7,232,566, Specification No. 7,175,8 Specification No. 43, Specification No. 5,883,223, Specification No. 6,905,874, Specification No. Nos. 6,797,514, 6,867,041, and U.S. patent applications. Generally using the method described in Publication No. 20060121005, It can be activated and expanded.

[0229] Generally, the T cells described herein are those that express a CD3 / TCR complex-associated signal. and a surface having a ligand attached thereto that stimulates costimulatory molecules on the T cell surface. In particular, the T cell population is expanded by contact with a surface Anti-CD3 antibody or its antigen-binding fragment, or anti-CD2 antibody immobilized on a substrate Protein kinase C activators (e.g., The T cell surface is stimulated by contact with an antigen (e.g., bryostatin). To costimulate accessory molecules, ligands that bind to accessory molecules are used. For example, a population of T cells may be stimulated with anti-CD3 antibodies and / or CD4+ antibodies under appropriate conditions to stimulate T cell proliferation. and anti-CD28 antibodies. + T cells or CD8 + T cell proliferation To stimulate the cells, they can be contacted with anti-CD3 and anti-CD28 antibodies. Examples of 28 antibodies include 9.3, B-T3, and XR-CD28 (Diaclone, Besancon). on, France), as well as other methods commonly known in the art. (Berg et al., Transplant Proc. 30(8):3975-3977, (1998), Haanen et al., J. Exp. Med. 190(9):13191328, (1999), Garland et al., J. Immunol Meth. 227(1-2): 53-63, (1999).

[0230] In certain embodiments, the primary and costimulatory signals for T cells are Each signal is produced by a different protocol. For example, the agent producing each signal is in solution. In some cases, the surface is coupled to the substrate. In other cases, the surface is coupled to the substrate. In some cases, the agents may be coupled to the same surface (i.e., in a "cis" configuration). ), or may be coupled to a separate surface (i.e., a "trans" configuration). Alternatively, one agent can be coupled to a surface and the other agent can be in solution. In one embodiment, an agent that provides a costimulatory signal is bound to the cell surface and stimulates primary The agent that provides the activation signal is either in solution or coupled to a surface. In certain embodiments, both agents are in solution. In another embodiment, the agent is: It can be in a soluble form and then binds to an Fc receptor or an antibody, or a drug. In this respect, the present invention is , artificial antigen-presenting cells (a) contemplated for use in T cell activation and expansion APC) is described, for example, in U.S. Patent Application Publication No. 20040101519 and Please refer to the specification of the same application, No. 20060034810.

[0231] In one embodiment, the two agents are on the same bead, i.e., "cis," or The nucleotides are immobilized on separate beads, i.e., in trans, on the beads. Specifically, the agent that provides the primary activation signal is an anti-CD3 antibody or its antigen. The binding fragment and the agent that provides the costimulatory signal are anti-CD28 antibodies or antibodies that bind to this antigen. In one embodiment, the antibody is a synthetic fragment and both agents are co-immobilized to the same beads with equal molecular weights. In terms of morphology, CD4 + Bind to beads for T cell expansion and proliferation In certain embodiments of the invention, a 1:1 ratio of each antibody bound to beads is used. The ratio of anti-CD3:CD28 antibodies used was compared to the expansion observed using a 1:1 ratio. In one particular embodiment, a 1:1 ratio is used to observe increased T cell expansion. A 1- to 3-fold increase is observed compared to the expansion observed using the ratio of In an embodiment, the ratio of CD3:CD28 antibodies bound to the beads is between 100:1 and 1:10. 0, and all integer values ​​therebetween. In one embodiment of the invention, the anti-CD3 antibody More anti-CD28 antibody is bound to the particles, i.e., the ratio of CD3:CD28 is In certain embodiments of the invention, the binding of anti-CD28 antibodies to beads In one particular embodiment, the ratio of anti-CD3 to anti-CD3 bound to beads is greater than 2:1. A 1:100 ratio of CD3:CD28 is used. A ratio of 1:75 of combined CD3:CD28 is used. A 1:50 ratio of CD3:CD28 bound to the antibody is used. A 1:30 ratio of CD3:CD28 bound to the IgG antibody is used. In another embodiment, a 1:10 ratio of CD3:CD28 bound to the beads is used. uses a 1:3 ratio of CD3:CD28 bound to beads. The format uses a 3:1 ratio of CD3:CD28 bound to beads.

[0232] Particle to cell ratios ranging from 1:500 to 500:1, and any integer value between these. The ratio of the IgG antibody to the IgG antibody can be used to stimulate T cells or other target cells. As can be readily appreciated, the ratio of particles to cells may depend on the size of the particles relative to the target cells. For example, small sized beads can only bind to a small number of cells, whereas large beads can In certain embodiments, the binding of the cells to the particle may be achieved by the following steps: The ratio may range from 1:100 to 100:1, and any integer value therebetween, and In certain embodiments, the ratio is from 1:9 to 9:1, including any integer value therebetween. These may also be used to stimulate T cells. Anti-CD3 / anti-CD28 coupled particles to T cells which results in stimulation of the T cells is as mentioned above. As mentioned above, the values ​​can vary, but certain values ​​are 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1: 2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10 :1, and 15:1, with one ratio being at least a 1:1 ratio of particles to T cells. In one embodiment, a particle to cell ratio of 1:1 or less is used. In one particular embodiment, the particle:cell ratio is 1:5. In a further embodiment, the particles The ratio of to cells may vary depending on the day of stimulation. For example, in one embodiment, the particles The ratio of particles to cells was 1:1 to 10:1 on day 1, and additional particles were added to the cells. Then, add more every day or every other day for up to 10 days, until the final ratio is 1:1 to 1:1. 0 (based on cell count on the day of addition). In one particular embodiment, the particles The ratio of cells to 1:1 on the first stimulation day and 1:1 on the third and fifth stimulation days. In another embodiment, the particles are adjusted to a final ratio of 1:1 on day 1. ratio, and to 1:5 on the third and fifth stimulation days, followed daily or every other day. In another embodiment, the ratio of particles to cells is 2:1 on the first stimulation day. and adjusted to 1:10 on the third and fifth stimulation days. , particles were mixed at a final ratio of 1:1 on day 1, and on the third and fifth stimulation days. on a daily or alternate day basis to a 1:10 ratio. In particular, the ratio may be determined by the size of the particles, as well as the This will vary depending on the size and type of cell.

[0233] In further embodiments described herein, immune effector cells, such as T cells, The cells are then combined with drug-coated beads, the beads are then separated from the cells, and the cells are then purified by centrifugation. In an alternative embodiment, the drug-coated beads and the cells are incubated with the drug-coated beads prior to incubation. In a further embodiment, the beads and cells are first cultured together using a magnetic force. Ligation of cell surface markers results in enrichment by applying a force such as This leads to an increase in the amount of

[0234] By way of example, anti-CD3 and anti-CD28 conjugated paramagnetic beads (3× 28 beads) are contacted with T cells to ligate cell surface proteins. In one embodiment, cells (e.g., T cells 10 4 ~10 9 pcs) and B (e.g., DYNABEADS® M-450 CD3 / CD28 T paramagnetic beads or MACS (registered trademark) from Miltenyi Biotec The beads are then mixed with PBS (calcium and magnesium) in a buffer solution, e.g. (without divalent cations such as nesium). Again, those skilled in the art will appreciate that It can be readily appreciated that any cell concentration may be used. For example, the target cells may be It is extremely rare in samples, sometimes containing only 0.01% of the sample, and not in the entire sample (all samples). i.e., 100%) may contain the desired target cells. In certain embodiments, the particles and cells are mixed together in a mixing vessel. By significantly decreasing the amount of β-cells (i.e., increasing the cell concentration), the maximum contact between the cells and the particles was obtained. For example, in one embodiment, about 2 cells per ml In another embodiment, a concentration of more than 100 million cells per ml is used. In further embodiments, 10 million, 15 million, 200 million, or 300 million cells per ml are used. 0 million, 25 million, 30 million, 35 million, 40 million, 45 million, or 5000 In yet another embodiment, a cell concentration of 75 million cells per ml is used. Use cell concentrations of 80, 85, 90, 95, or 100 million In further embodiments, 125 million or 150 million cells per ml. Use of high concentrations can improve cell yield, cell activation, and cell expansion. Furthermore, the use of high cell concentrations may result in an increase in the number of CD28-negative T cells. This allows for more efficient capture of cells that may have low expression of the target antigen of interest, such as In certain embodiments, such cell populations may have therapeutic value. For example, the use of high cell concentrations may be desirable in cells that normally express low levels of CD28. CD8 + Allows for more efficient selection of T cells.

[0235] In one embodiment described herein, the mixture is incubated for a period of time ranging from several hours (about 3 hours) to about 14 days. or any integer value between these. The mixture can be cultured for 21 days. In one embodiment of the present invention, the beads and The beads and T cells are cultured together for about 8 days. The cells are cultured together for 2 to 3 days. The T cell culture period is 60 days or Stimulation over several cycles may also be desirable, so as to exceed the The appropriate conditions include factors necessary for growth and survival, such as serum (e.g., fetal bovine serum or human serum), interleukin 2 (IL-2), insulin, IFN-gamma, IL-4 , IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ, and T NF-alpha or any other additives for cell growth known to those skilled in the art. 2. The appropriate medium (e.g., Minimum Essential Medium or RPMI Medium 1640) may contain factors such as , or X-vivo 15 (Lonza). Other additives for cell growth The surfactant, plasmanate, and N-acetylcysteine ​​and 2-mercapto The medium includes, but is not limited to, RPMI 1640, AIM-V, DMEM, MEM, Alpha-MEM, F-12, X-Vivo 15 and and X-Vivo 20, Optimizer, Amino Acid Addition, Sodium Pyruvate, and vitamins, serum-free or adequate serum (or plasma) supplementation, or prescribed A set of hormones and / or sites sufficient for T cell proliferation and expansion Antibiotics, such as penicillin and streptomycin, are used in laboratory cultures. The target cells are only incorporated into the cell culture that is infused into the subject. The conditions necessary to support the growth of the cells, e.g., appropriate temperature (e.g., 37°C) and atmosphere (e.g., For example, keep the animals under a constant air / 5% CO2 atmosphere.

[0236] T cells exposed to varying stimulation times can exhibit different characteristics. For example, typical Blood products or apheresis peripheral blood mononuclear cell products are used to generate cytotoxic T cell populations. T cell population or suppressor T cell population (T C , CD8 + ) a larger population of helper T cells (T H , CD4 + ) by stimulating the CD3 and CD28 receptors. T cell expansion in vivo begins around day 8–9, primarily in T H Consists of cells But after about 8-9 days, more and more T C resulting in a population of T cells containing a population of Therefore, depending on the purpose of the treatment, the subject will be mainly H Infusing a T cell population containing Similarly, it may be advantageous to C When antigen-specific subsets of cells are isolated, It may be beneficial to expand this subset to a greater extent.

[0237] Furthermore, in addition to the CD4 and CD8 markers, other phenotypic markers were also significantly fluctuates widely, but for the most part, fluctuates reproducibly during the course of the cell expansion process. Such reproducibility would therefore be useful for tailoring the activation of T cell products for specific purposes. This enables the ability to

[0238] In some cases, the immune effector cells (e.g., T cells) of the embodiments are expressed as AaPCs ( activating and propagating cells) AaPCs are also called artificial antigen-presenting cells (aAPCs). For example, antigen presenting cells (APCs) can be used in the therapeutic compositions and cells of the embodiments. In one embodiment, the AaPCs are useful in the preparation of therapeutic products. For general guidance on the preparation and use of antigen-presenting systems, see See, for example, U.S. Pat. No. 6,225,042, each of which is incorporated by reference. The specification, the specification of the same patent No. 6,355,479, the specification of the same patent No. 6,362,001, and the specification of the same patent No. No. 6,790,662, U.S. Patent Application Publication No. 2009 / 0017000 and WO 2009 / 0004142, and WO 2007 / 1030 In yet further aspects of the embodiment, the trans Culture of transgenic CAR cells allows for the expansion and proliferation of immune effector cells expressing CARs. Stimulating, dendritic cells or AaPCs (activating and propaga The method includes culturing the transgenic CAR cells in the presence of a ting cell. In yet further embodiments, the AaPCs comprise a CAR-binding antibody or or a fragment thereof. The AaPC ​​optionally activates or costimulates T cells. The additional molecule may optionally include a membrane-bound Cγ cytokine. In yet a further embodiment, the AaPCs may be inactivated or It has been irradiated or tested for and found to be free of infectious agents. In yet a further embodiment, transgenic CAR cells in the presence of AaPC Cell cultures were treated with soluble cytokines such as IL-15, IL-21, and / or IL-2. The method comprises culturing the transgenic CAR cells in a medium containing an agonist. 0:1 to about 1:10, about 3:1 to about 1:5, about 1:1 to about 1:3 (immune effector cells To AaPC), or any range of ratios derivable therein. For example, the co-culture of T cells and AaPCs can be at a ratio of about 1:1, about 1:2, or about 1:3. do.

[0239] In one embodiment, the AaPCs may express CD137L. In certain embodiments, the cells may further express D19, CD64, CD86, or mIL15. AaPCs express at least one antibody, such as OKT3 and / or UCHT1. In one embodiment, the AaPCs are inactivated (e.g., irradiated In one embodiment, the AaPCs are examined for infectious agents and In some cases, it has been confirmed that such AaPCs are not included. Methods for culturing a CAR-modified T cell population with AaPCs are known. The cells were cultured at about 10:1 to about 1:10, about 3:1 to about 1:5, about 1:1 to about 1:3 (T cells) The method may include culturing at a ratio of 100 to 150 AaPCs (AaPCs to AaPCs), or any range derivable therein. For example, the co-culture of T cells and AaPCs may be at a ratio of about 1:1, about 1:2, or about 1:3. In one aspect, the culturing step comprises administering to the subject an aminobisphosphonate (e.g., zoledro) The method may further comprise culturing the bacteria with a phosphate buffer (phosphate phosphate).

[0240] In a further embodiment, the population of transgenic CAR cells is cultured using a method according to any one of claims 7, 14, 21, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 Culture and incubation for not more than 35, 42, 49, 56, 63, or 70 days In one embodiment, the stimulation is of the transgenic CAR T cell: Another embodiment includes co-culture with AaPCs that promotes the proliferation of CAR-positive T cells. In this study, a population of transgenic CAR cells was cultured for one, two, three, or four cycles of stimulation. 4, 5, 5, 6, 7, 8, 9, or 10 Optionally, the transgenic cells are stimulated no more than once. In some special cases, the embodiment is not cultured ex vivo in the presence of C. The method further comprises the steps of: after the transfection step and / or the culturing step, and enriching the resulting culture for immune effector cells (e.g., T cells) that express the CAR. The enrichment step further includes a step of enriching the cells by fluorescence activated cell sorting (FACS) and a step of enriching the cells by eluting the cells with CAR. In a further embodiment, the method can include isolating cells expressing a CAR. Sorting for cells includes the use of a CAR-binding antibody. The enrichment step also includes: In yet a further aspect of the embodiment, the method comprises the steps of: The method further includes a cryopreserved sample of the population of transgenic CAR cells.

[0241] In some cases, the AaPCs can be subjected to peptide, MHC fragmentation without further processing. The antibody is incubated with a peptide of optimal length that allows direct binding to the molecule. Alternatively, the cells may express the antigen of interest (i.e., in the case of MHC-independent antigen recognition). Additionally, in some cases, the APCs may be capable of expressing a specific CAR polypeptide or a general CAR polypeptide. R polypeptide (e.g., uAaPC (universal activating a These cells can express antibodies that bind to endothelial propagating cells. The method is described in WO 2014 / 190273, which is incorporated herein by reference. In addition to the peptide-MHC molecule or antigen of interest, Thus, the AaPC ​​system may also include at least one exogenous support molecule. Supporting molecules can be used in combination with other molecules such as costimulatory molecules and adhesion molecules. Exemplary costimulatory molecules include CD70 and B7 B7.1 (formerly known as B7 and also as CD80) These include, inter alia, the CD28 molecule on the surface of T cells and / or the CT It binds to the LA-4 molecule, which mediates, for example, the expansion and proliferation of T cells, the differentiation of Th1, and short-term It affects the survival of T cells and the secretion of cytokines such as interleukin (IL) 2. Adhesion molecules include carbohydrate-binding glycoproteins such as selectins, integrins, etc. Transmembrane-associated glycoproteins, calcium-dependent proteins such as cadherins, and e.g. For example, intercellular adhesion molecules (ICAMs), which promote cell-cell or cell-matrix contacts ) may include single-pass transmembrane immunoglobulin (Ig) superfamily proteins such as Exemplary adhesion molecules include LFA-3 and ICAMs, such as ICAM-1. Selection, cloning, preparation, and expression of exemplary support molecules, including adhesivity molecules For techniques, methods, and reagents useful in Nos. 6,225,042, 6,355,479, and No. 6,362,001.

[0242] The cells selected to become AaPCs preferably have intracellular antigen processing, Intracellular peptide trafficking and / or MHC class I or class II molecules Either they have defective intracellular peptide loading in the offspring or they are poikilothermic (i.e., are less sensitive to temperature stress than mammalian cell lines) or are defective and mutated Preferably, the cells selected to become AaPCs also have both endothelial and endothelial properties. Exogenous MHC class I or class II molecules and supporting molecules introduced into the At least one endogenous counterpart (e.g., an endogenous MHC class I component) to the construct and / or the endogenous support molecules described above). Furthermore, AaPCs lack the ability to differentiate into cellular components prior to their modification to generate AaPCs. Preferably, the AaPC ​​retains the defective and poikilothermic properties that the cells possessed. Exemplary AaPCs include: Insect cell lines, etc., TAP (transporter associated with Constitute or are derived from cell lines deficient in antigen processing Exemplary poikilothermic insect cell lines include the Drosophila Drosophila cell lines (see, e.g., Schneider 1972). Exemplary methods for the preparation, growth, and culture of eider 2 cells are described in U.S. Pat. 225,042, 6,355,479, and 6,362,0 This is presented in Specification No. 01.

[0243] In one embodiment, the AaPCs are also subjected to freeze-thaw cycles. In the ice cream, a suitable receptacle containing AaPCs is placed in the ice cream container so that freezing occurs quickly. , a suitable amount of liquid nitrogen, solid carbon dioxide (i.e., dry ice), or similar cryogenic material. The AaPCs can be frozen by contacting them with C by excision of AaPC ​​from cryogenic material and exposure to ambient room temperature conditions, or Accelerated melting process using a lukewarm water bath or warm hands to help reduce melting time In addition, AaPCs can be frozen and stored for extended periods of time prior to thawing. Frozen AaPCs can also be thawed and then frozen prior to further use. It can also be dried. Preferably, it is dissolved in dimethyl sulfoxide (DMSO), polyethylene Preservatives that may have adverse effects on the freeze-thaw procedure, such as PEG, PEG-1, and other preservatives. Preservatives that are used in the manufacture of AaPCs should be absent from media containing AaPCs that undergo freeze-thaw cycles or should be Such preservatives can be essentially removed, for example, by introducing AaPC ​​into a medium essentially lacking such preservatives. do.

[0244] In a further embodiment, after inactivation, cell growth, replication, or nucleic acid expression is essentially Cross-linking inactivates xenogenic and endogenous nucleic acids in AaPCs to prevent In one embodiment, AaPCs can be cultured to express exogenous MHC and supporting molecules, Such molecules are presented on the surface of AaPCs, and the selected molecules are delivered to the presented MHC molecules. , and is inactivated at a time point subsequent to loading of one or more peptides. Such inactivated AaPCs loaded with selected peptides are capable of proliferation or replication. Although this is considered qualitatively impossible, the presentation function of the selected peptide is retained. Crosslinking also prevents bacterial and viral infection without substantially compromising the antigen-presenting cell function of AaPCs. Crosslinking also results in AaPC ​​that is essentially free of contaminating microorganisms such as bacteria. Help alleviate safety concerns about cell therapy products developed using aPCs While maintaining important AaPC ​​functions. See, for example, U.S. Patent Application Publication No. 20090017000, which is incorporated herein by reference. Please refer to the specification.

[0245] In certain embodiments, modified antigen presenting cells (APCs) are further provided. To propagate immune effector cells in ivo, such cells can be used, e.g., In a further embodiment, the modified APCs themselves can be used as described above. This induces the expansion and proliferation of immune effector cells in vivo. The modified APC of the embodiment itself can be used as a therapeutic agent. In other embodiments, the modified APCs are expressed as endogenous immune effector cells specific for a target antigen. and / or stimulate activation of adoptively transferred immune effectors specific for the target antigen. They can be used as therapeutic agents that can increase the activity or prolong the survival of cells. do.

[0246] As used herein, the term "modified APC" refers to a modified APC that contains at least a first transgene The modified AP refers to a cell comprising the first transgene encoding an HLA. C is for the expression of antigens so that the antigens are presented on the surface of APCs in complex with HLA. In some embodiments, the modified APC can further comprise a second transgene. In a further embodiment, the modified APC can be a T cell. or cell types that do not normally present antigens, such as T cell precursors (referred to as "T-APCs" ). Thus, in some aspects, the modified APC of the embodiment can be made from: The target antigen is expressed on the surface of the modified APC in complex with an epitope of the target antigen. The first transgene encodes an antigen and the second encodes a human leukocyte antigen (HLA). In certain specific embodiments, the HL transgene expressed in the modified APC. A is HLA-A2.

[0247] In some aspects, the modified APC of the embodiments comprises at least a third gene encoding a costimulatory molecule. The co-stimulatory molecule may be a membrane-bound Cγ cytokine. In certain aspects, the costimulatory cytokine can be a membrane-bound In some further embodiments, the modified APC is an IL-15, such as an IL-15 recombinant protein. (or deleted) genes. For example, PD-1, LIM-3, CTLA-4, or Inhibitory genes such as TCR are edited to reduce or eliminate gene expression. The modified APC of the embodiment can be a transfected APC that encodes any target antigen of interest. For example, the target antigen may be an infectious disease antigen or a tumor-associated antigen ( TAA).

[0248] In one embodiment of the present disclosure, the immune effector cells described herein are administered at point-of-care. In some cases, the point-of-care facility may be a hospital in the vicinity of the subject. In some cases, the immune effector cells are administered to a patient at a hospital or institution (e.g., a medical institution). By modifying / introducing melanoma receptors and / or cytokines into immune effector cells, and then rapidly infused into the subject. Immune effector cells are modified with the vectors described herein via electroporation. In other embodiments, the vector may be a non-viral vector, such as a viral vector. In one case, the non-viral vector is Sleeping Bea uty transposon system. In some cases, the modified immune effector cells are In some cases, the engineered immune effector cells do not undergo an induction and activation step. No incubation or culturing step is performed. In other cases, the chimeric receptor and cytokines into said immune effector cells, The effector cells are modified and then rapidly infused into the subject. By modifying / introducing receptors and cytokines into the cells, immune effectors - modifying the cells and then administering at least 0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 2 Infuse immediately within 1, 22, 23, or 24 hours. In other cases, the chimeric recipient and cytokines into cells to modify / introduce them to modify NK cells or T cells. Then, to the control, 0 days, <1 day, <2 days, <3 days, <4 days, <5 days , <6 days, or <7 days. The source of cells can include both allogeneic and autologous sources. The effector cell can be a T cell or a NK cell. In one case, the chimeric receptor In another embodiment, the cytokine is mbIL-15. In other cases, immune effector cells expressing CD33 CAR may be involved in the regulation of HER1 In another embodiment, the HER1t tag can include a t tag or a HER1t-1 tag. No. 32 or 54, or a variant or fragment thereof. The mbIL-15 is represented by SEQ ID NO: 37, or a variant or fragment thereof. In other cases, mbIl-15 is also a HER1t tag or a HER1t-1 tag. In yet another embodiment, the CD33 CAR and / or mbIL-15 Expression is modulated by the gene switch expression system described herein.

[0249] Therapeutic applications In some embodiments herein, the lentiviral vector (LV) transduced For example, LVs are described as CD4+ immune effector cells (e.g., T cells). The antigen recognition domain of 33 was identified as CD8α hinge stalk domain and its variants. , the intracellular domain of CD3 zeta, CD28, 4-1BB, or any combination thereof. and the intracellular domain of CD3 zeta. By this, the transduced T cells can induce a CAR-mediated T cell response.

[0250] In some embodiments described herein, the specificity of the primary T cells is determined based on the CD33 tumor antigen. The present invention therefore also provides the use of CAR to redirect the expression of a mammalian for stimulating a T cell-mediated immune response against a target cell population or target tissue in 1. A method comprising administering to a mammal a T cell expressing a CAR, R comprises the stalk domain of CD33, e.g., the intracellular domain of human CD3 zeta The antibody comprises a zeta chain portion and a binding portion that specifically interacts with a costimulatory signaling domain. Well, we also provide a method.

[0251] In one embodiment, the present disclosure provides a method for the preparation of a method for the preparation of a T cell expressing a CD33-specific CAR of the present invention. The CAR T cells are genetically modified and then infused into a recipient in need. The infused cells are capable of killing tumor cells in the recipient. Unlike antibody therapy, CAR T cells are not replicable in vivo. This results in long-term survival that may lead to sustained tumor control. It is possible to do so.

[0252] In one embodiment, the CAR T cells described herein are capable of in vivo They can undergo robust T cell expansion and persist for long periods of time. In embodiments, the CAR T cells of the invention are designed to inhibit further tumor formation or growth. These cells then evolve into specific memory T cells that can be reactivated in the same way.

[0253] The CAR modified T cells described herein can also be used in ex vivo immunization and and / or as a type of vaccine for in vivo treatment of mammals. In some embodiments, the mammal is a human. In terms of immunity, i) cell expansion and proliferation, ii) delivery of the nucleic acid encoding the CAR to the cell, and / or iii) cryopreservation of the cells. Once in tro, the immune effector cells are administered to a mammal.

[0254] Ex vivo procedures are well known and are discussed more fully below. Upon receipt, cells are isolated from the mammal (e.g., human) and express the CAR disclosed herein. Genetically modify (i.e., transduce in vitro) a vector that expresses The CAR-modified cells are administered to a mammalian recipient. The mammalian recipient can be a human. Alternatively, the cells can be autologous to the recipient. may be allogeneic, syngeneic, or xenogeneic to the recipient.

[0255] A procedure for ex vivo expansion of hematopoietic stem and progenitor cells is described. See U.S. Pat. No. 5,199,942, which is incorporated herein by reference. and can be applied to the cells of the present invention. Suitable methods are also known, and thus the present invention relates to the ex vivo expansion of cells. The present invention is not limited to any particular method of propagation. Briefly, ex vivo propagation of T cells is performed. The culture and expansion of hematopoietic stem cells and hematopoietic progenitor cells from mammalian (2) recovering such cells from peripheral blood collection or bone marrow explants of subjects; and and expanding the cells in vivo. In addition to the cellular growth factors described in the document, flt3-L, IL-1, IL-3 Other factors, such as , and c-kit ligand, are also required for cell culture and expansion. It can be used.

[0256] In addition to using cell-based vaccines for ex vivo immunization, The present invention also provides a method for eliciting an immune response directed against an antigen in a patient. Compositions and methods for in vivo immunization are also provided.

[0257] Generally, cells activated and expanded as described herein are useful in treating immune-compromised individuals. In particular, the present invention can be used in the treatment and prevention of diseases occurring in the body. CAR modified T cells can be used to treat myeloid malignancies, such as acute myeloid leukemia (AML). In certain embodiments of the invention, the cells are selected from the group consisting of cells at risk of developing AML. The present invention therefore provides a method for treating or preventing AML. The method comprises administering a therapeutically effective amount of a CAR modified antibody of the present invention to a subject in need thereof. In some embodiments, the method includes administering the mutant T cells to a patient as described herein. Cells activated and expanded as described are useful for the treatment of relapsed or refractory AML. It can be used in the following cases.

[0258] Briefly, the pharmaceutical compositions of the present invention comprise a method for treating a target cell population as described herein, comprising administering to a subject a therapeutic agent, such as a therapeutic agent comprising administering to a subject ... In combination with one or more pharma- ceutical or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffered saline, such as neutral buffered saline, phosphate buffered saline, etc. Solution; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol compounds; proteins; polypeptides or amino acids such as glycine; antioxidants; EDTA or or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. In some embodiments, the compositions of the present invention are formulated for intravenous administration. Formulate into a drug.

[0259] The pharmaceutical compositions described herein may be administered in a form appropriate for the disease to be treated (or prevented). The amount and frequency of administration will depend on the condition of the patient and the severity of the patient's disease. The appropriate dosage will be determined by clinical trials, depending on factors such as the type and severity of the disease. It is possible.

[0260] Refers to an "immunoeffective amount," an "antitumor effective amount," an "antitumor effective amount," or a "therapeutic amount" In this case, the exact amount of the composition of the present invention to be administered will depend on the age, weight, size of the tumor, and other factors of the patient (subject). The procedure may be determined by a physician, taking into account the size, degree of infection or metastasis, and individual differences in the condition. In general, pharmaceutical compositions comprising the T cells described herein can be used in any of the above ranges. Includes numbers, 10 cells per kg of body weight 4 ~10 9 10 cells per kg of body weight 5 ~ 10 6 It may be stated that the T cell compositions may be administered in individual doses. The cells can be administered in multiple doses in the same amount. The drug can be administered by injection (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676 (1988). Optimal dosage and treatment for a particular patient The regimen would be used by one of ordinary skill in the medical arts to monitor the patient for signs of disease. This can be readily determined by adjusting treatment accordingly.

[0261] In certain embodiments, activated T cells are administered to a subject and then subsequently rebleed. (or perform apheresis) and activate T cells derived therefrom in accordance with the present invention. It may be desirable to reinfuse the patient with these activated and expanded T cells. This process can be performed multiple times, every few weeks. In one embodiment, T cells can be activated from a blood sample of 10cc to 400cc. In certain embodiments, T cells are administered at 20 cc, 30 cc, 40 cc, 50 cc, 60 cc Can be activated from 70cc, 80cc, 90cc, or 100cc of blood. Without being bound by theory, this multiple blood draw / multiple reinfusion process The use of the protocol can be used to select for certain T cell populations. In this embodiment, the subject composition is an active ingredient in a lymphocyte-derived leukocyte antigen (LLA) that is administered to a patient after lymphodepletion via radiation or chemotherapy. It may be desirable to administer engineered T cells.

[0262] The compositions described herein can be administered by aerosol inhalation, injection, ingestion, implantation, or the like. The compositions described herein can be administered in any convenient manner, including by transplantation or by injection. The composition is administered to the patient subcutaneously, intradermally, intratumorally, intralymph node, intrabone marrow, or intramuscularly. In one embodiment, the drug may be administered intravenously, by intravenous (iv) injection, or intraperitoneally. In another embodiment, the T cell compositions of the present invention are administered to the patient by intradermal or subcutaneous injection. In an embodiment, the T cell compositions of the present invention are administered by iv injection. It can be injected directly into a tumor, lymph node, or site of infection.

[0263] The dosage of the above treatments administered to a patient will depend on the condition being treated and the recipient of the treatment. The dosages will vary with the exact nature of the compound. Scaling of dosages for human administration will be determined at the This can be done according to accepted practices in the art. For example, the treatment doses listed above are , 1 × 10 CAR+ cells per kg 4 5 x 10 CAR+ cells per kg 6 pcs Exemplary doses are 1 x 10 CAR+ cells per kg. 4 piece, 1kg 1×10 CAR+ cells per 5 3 x 10 CAR+ cells per kg 5 Pieces per kg 1×10 CAR+ cells 6 or 5 x 10 CAR+ cells per kg 6 It can be individual Therefore, the appropriate dose can be adjusted for adult patients and for pediatric patients. It can also be adjusted to.

[0264] Alternatively, a typical set of immune effector cells administered to a mammal (e.g., a human) may be The amount can be, for example, in the range of 1 million to 100 billion cells, although amounts below this exemplary range are not limiting. Amounts that are equal to or greater than the dose of the host cells of the present invention are also within the scope of the present invention. For example, the dose of the host cells of the present invention is , about 1 million to about 50 billion cells (for example, about 5 million cells, about 25 million cells, Approximately 500 million cells, approximately 1 billion cells, approximately 5 billion cells, approximately 20 billion cells, approximately 30 billion cells, Approximately 40 billion cells, or a range defined by any two of the preceding values), cells Approximately 10 million to 100 billion cells (for example, approximately 20 million cells, approximately 30 million cells, approximately 10 ... Approximately 40 million cells, approximately 60 million cells, approximately 70 million cells, approximately 80 million cells, Approximately 90 million cells, approximately 10 billion cells, approximately 25 billion cells, approximately 50 billion cells, approximately 7 cells 5 billion cells, approximately 90 billion cells, or a range defined by any two of the preceding values. 100 million cells to 50 billion cells (for example, 120 million cells, 200 million cells, 50 million cells, approximately 350 million cells, approximately 450 million cells, approximately 650 million cells 10,000 cells, approximately 800 million cells, approximately 900 million cells, approximately 3 billion cells, approximately 30 billion cells, approximately 450 cells The value may be any number between 0 and 100 million, or a range defined by any two of the preceding values.

[0265] The therapeutic or prophylactic efficacy is monitored by periodic evaluation of treated patients. For repeated administration over several days or more, depending on the condition, Treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be used. In some cases, this may be useful and is within the scope of the present invention. The desired dosage may be administered as a single bolus injection of the composition. The composition may be delivered by multiple doses or by multiple boluses of the composition. The composition may also be delivered by continuous infusion.

[0266] Immune effectors expressing the disclosed nucleic acid sequences, or vectors containing these nucleic acid sequences. The composition comprising the target cells may be co-administered to a mammal with one or more additional therapeutic agents. "Co-administration of" refers to administration of one or more additional The therapeutic agent and a composition comprising the host cell of the present invention or the vector of the present invention are administered in one or more administering the two or more additional therapeutic agents close enough together to enhance the effect of the two or more additional therapeutic agents; In this respect, the immune effector cells described herein, or A composition comprising a vector as described herein may be administered concomitantly with one or more additional therapeutic agents. Alternatively, the first therapeutic agent may be administered first, and one or more additional therapeutic agents may be administered second. Alternatively, the disclosed immune effectors can be administered to Cells or compositions comprising the vectors described herein and one or more additional therapeutic agents. The therapeutic agents can also be administered simultaneously.

[0267] In a composition comprising the host cell of the invention and / or the vector of the invention (or Examples of therapeutic agents that may be co-administered with the above-mentioned compositions (or included in the kit) include: Interleukins, cytokines, interferons, adjuvants, and chemotherapeutic agents In some embodiments, the additional therapeutic agent is IFN-alpha, IFN-beta, IFN-gamma, GM-CSF, G-CSF, M-CSF, LT-beta, TNF-alanine pha, growth factors, and hGH, human Toll-like receptors, TLR1, TLR2, and T TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, and TLR 10 ligands.

[0268] A Bcl-2 inhibitor, a STAT3 inhibitor, or a STAT5 inhibitor may also be included in the composition. or co-administered with (or in a kit for) cancer Cells may also overexpress anti-apoptotic proteins such as Bcl-2, which may lead to the formation of naturally occurring This results in an abnormally high level of resistance to apoptotic mediators or pharmacological agents. In cases where activation of the pathways that mediate STAT5 and STAT3 is prolonged, In some cases, drug therapy and immunotherapy approaches can be combined to achieve more than one apoptosis. Utilizing the cis pathway may increase tumor cell death and reduce resistance to therapy. The use of small molecule inhibitors is expected to mediate the expression of pro-apoptotic and anti-apoptotic molecules in tumor cells. The balance between inflammatory cytokines and T cell proliferation sensitizes tumor cells to death or inhibits their ability to encounter tumor-specific T cells. The shift in Bcl-2 expression can be sufficient to reduce resistance to killing during exposure. The increase is seen in blood cancers such as acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL). It has been observed in malignant and solid tumors (i.e., melanoma, breast cancer, and ovarian cancer). Prolonged activation of STAT3 or STAT5 has been shown to be a key factor in the pathogenesis of AML, CLL, and chronic osteoporosis. It has been observed in myeloid leukemia, large granular lymphocytic leukemia, and leukemia cell lines. Small molecule inhibitors targeting Bcl-2, STAT3, or STAT5 are In one embodiment, the composition is co-administered with and / or co-cultured with the One bcl-2 inhibitor is ABT-737. targets these Bcl-2 family proteins, but not A1 or Mc l-1 is part of a group of non-targeting, BH3 mimetic small molecule inhibitors (SMIs). BT-737 has affinity for Bcl-2, Bcl-xL, and Bcl-w. However, it is significantly larger than conventional BCL-2 inhibitors. ABT-199, a novel therapeutic agent for the treatment of chronic lymphocytic leukemia, inhibits Bcl-2 tandem repeat inhibition in patients with chronic lymphocytic leukemia. It is designed to block the function of proteins.

[0269] STAT3 inhibitors and STAT5 inhibitors are described in Fagard et al., STAT3 inhibitors for cancer. ancer therapy, JAK-STAT, 2:1 (2013), e22882, DOI: 10.4161 / jkst.22882 and Furqan , et al., Journal of Hematology & Oncology 2013, 6:90 doi:10.1186 / 1756-8722-6-90 In one embodiment, the STAT3 inhibitors and the STAT5 inhibitors are described in In addition to the JAK / STAT pathway effectors, three major negative regulators Class:SOCS (suppressors of cytokine signaling ng), PIAS (protein inhibitors of activated stats), and PTPs. Tyrosine phosphatases inhibit the activity of JAKs. The best characterized of these regulators is the mouse The sweetin gene product, SHP-1, contains two SH2 domains. They contain kinases that bind to phosphorylated JAKs or phosphorylated receptors to mediate these activating signals. Other tyrosine phosphatases, such as CD45, may facilitate the dephosphorylation of signaling molecules. Plays a role in regulating JAK / STAT signaling through a subset of receptors Thus, the JAK / STAT pathway is a potential drug target for the compositions of the present disclosure. It is expected that this will be the case.

[0270] "Antifoaming agents" are used to prevent foaming during processing, coagulation of aqueous dispersions, and air bubbles in the finished thin film. Advantages of the Invention: Anti-foaming Agents: The foaming agent is a silicone emulsion or sesquioleate. Contains sorbitan sesquoleate.

[0271] "Antioxidants" include, for example, butylated hydroxytoluene (BHT), sodium ascorbate, etc. Contains sodium thorium, ascorbic acid, sodium metabisulfite, and tocopherol. In certain embodiments, antioxidants enhance chemical stability when needed.

[0272] The formulations described herein contain antioxidants, metal chelators, thiol-containing compounds, and These may benefit from the use of stabilizing agents such as stabilizing agents, ... and other common stabilizing agents. Examples of such stabilizing agents are (a) about 0.5% to about 2% w / v glycerol; (b) about 0.1% to about 1% w / v methionine, (c) about 0.1% to about 2% w / v monothioglycerol, (d) about 1 mM (e) about 0.01% to about 2% w / v ascorbic acid; (f) 0.003% to about 0.02% w / v polysorbate 80, (g) 0.001% to about 0. 0.5% w / v polysorbate 20, (h) arginine, (i) heparin, (j) desulfate (k) cyclodextrin, (l) pentosan polysulfate, and other heparins. iodides, (m) divalent cations such as magnesium and zinc, or (n) combinations thereof This includes, but is not limited to:

[0273] "Binders" impart cohesive properties and are, for example, alginic acid and its salts; carboxylates; Methylcellulose, methylcellulose (e.g., Methocel®), hydrocellulose, Hydroxypropyl methylcellulose, Hydroxyethyl cellulose, Hydroxypropyl cellulose cellulose (e.g., Klucel®), ethyl cellulose (e.g., Ethyl cellulose cel®), and microcrystalline cellulose (e.g., Avicel®). Any cellulose derivative; microcrystalline dextrose; amylose; magnesium aluminum silicate nium;polysaccharide acid;bentonite;gelatin;polyvinylpyrrolidone / vinyl acetate copolymer -; Crospovidone; Povidone; Starch; Pregelatinized starch; Tragacanth, dextrin sucrose (e.g., Dipac®), glucose, dextrose, sugar Honey, mannitol, sorbitol, xylitol (e.g., Xylitab® ), and sugars such as lactose; acacia, tragacanth, ghatti gum, isapol lotus mucus of the genus K, polyvinylpyrrolidone (e.g., Polyvidone® CL, K Ollidon® CL, Polyplasdone® XL-10) , arabinogalactan from larch (Veegum brand), polyether These include natural or synthetic gums such as ethylene glycol, waxes, and sodium alginate.

[0274] "Carrier" or "carrier material" includes any excipient commonly used in medicines, including, but not limited to, Compatibility with compounds disclosed herein, such as lutinib and anti-cancer compounds, and The preferred carrier materials are selected based on the release profile characteristics of the desired dosage form. Examples of suitable agents include binders, suspending agents, disintegrating agents, fillers, surfactants, solubilizers, stabilizers, lubricants, etc. "Pharmaceutically compatible carrier materials" include acacia, gelatin, etc. , colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin Trithrin, glycerin, magnesium silicate, polyvinylpyrrolidone (PVP), cholesterol Cholesterol, cholesterol esters, sodium caseinate, soy lecithin, taurocholesterol Phosphatidylcholine, Sodium Chloride, Tricalcium Phosphate, Dipotassium Phosphate , Cellulose and Cellulose Conjugates, Sugar, Sodium Stearoyl Lactylate, Caramel These may include ginan, monoglycerides, diglycerides, pregelatinized starch, etc. For example, but not limited to, Remington: The Science and Practice of Pharmacy, Nineteenth h Ed (Easton, Pa.: Mack Publishing Company, 1995), Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975, Liberm. an, HA and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980, and Pharmaceutical Dosage Forms and Drug Delivery Systems, S See eventh Ed. (Lippincott Williams & Wilkins 1999).

[0275] "Dispersing agents" and / or "viscosity modulating agents" are used in the liquid medium or granulation process. It also includes materials that control the diffusion and homogeneity of the drug through blending. In some embodiments, these agents may also be incorporated into the coating or eroding matrix. Exemplary diffusion enhancers / dispersing agents include, for example, hydrophilic polymers, Solubilizers, Tween 60 or Tween 80, PEG, polyvinyl chloride PVP (commercially known as Plasdone®) ), and for example, hydroxypropyl cellulose (e.g., HPC, HPC-SL, and and HPC-L), hydroxypropyl methylcellulose (e.g., HPMC K100 , HPMC K4M, HPMC K15M, and HPMC K100M), carboxy Sodium methylcellulose (carboxymethylcellulose Sodium ), methylcellulose, Hydroxyethyl cellulose, Hydroxypropyl cellulose, Hydroxypropyl phthalate Pyrmethylcellulose, Hydroxypropylmethylcellulose stearate acetate (HPM CAS), carbohydrate-based dispersants such as amorphous cellulose, magnesium aluminum silicate nium, triethanolamine, polyvinyl alcohol (PVA), vinylpyrrolidone / Vinyl acetate copolymer (S630), with ethylene oxide and formaldehyde, 4- (1,1,3,3-Tetramethylbutyl)-phenol polymer (as tyloxapol) are also known), poloxamers (e.g., bromines of ethylene oxide and propylene oxide), Pluronics F68®, a tungsten copolymer from Pluronics F88®, and Pluronics F108®); and roxamine (e.g., the successive synthesis of propylene oxide and ethylene oxide to ethylenediamine) Poloxamine 90, a tetrafunctional block copolymer derived from the selective addition of 8® (BASF Corporation, Parsippany, NJ ), also known as Tetronic 908®, polyvinylpyrrolidone K12, Polyvinylpyrrolidone K17, Polyvinylpyrrolidone K25, or Polyvinyl Pyrrolidone K30, Polyvinylpyrrolidone / vinyl acetate copolymer (S-630), Poly Ethylene glycol (for example, polyethylene glycol is about 300 to about 6000, may have a molecular weight of about 3350 to about 4000, or about 7000 to about 5400), Sodium carboxymethylcellulose, methylcellulose, polysorbate 80, algin sodium phosphate, e.g. gums such as tragacanth and acacia, guar gum, xanthan, including santhang gum; sugars, such as sodium carboxymethylcellulose; Cellulose derivatives such as methylcellulose and sodium carboxymethylcellulose, poly Resorbate 80, Sodium Alginate, Polyethoxylated Sorbitan Monolaurate , polyethoxylated sorbitan monolaurate, povidone, carbomer, polyvinyl alcohol Cellulose includes cellulose (PVA), alginate, chitosan, and combinations thereof. Alternatively, a plasticizer such as triethyl cellulose can also be used as a dispersant. In particular, dispersing agents useful in liposomal and self-emulsifying dispersions are dimyristoyl Phosphatidylcholine, natural from chicken eggs Phosphatidylcholine, natural from chicken eggs Phosphatidylglycerol, cholesterol, and isopropyl myristate .

[0276] Combinations of one or more erosion promoters with one or more diffusion promoters may also be used. can be used in the present compositions.

[0277] The term "diluent" refers to a compound used to dilute the compound of interest prior to delivery. Diluents can also provide a more stable environment, thus stabilizing the compound. In the art, salts dissolved in buffers (which can also be used for p H) in a variety of solutions, including but not limited to phosphate buffered saline solutions. In certain embodiments, the diluent is used as an unspecified diluent to provide the bulk of the composition. To facilitate compression or to provide a homogeneous blend for capsule filling, Such compounds are, for example, lactose, starch, mannitol, etc. Microcrystalline cellulose such as cellulose acetate, sorbitol, dextrose, Avicel®; Dibasic calcium phosphate, dicalcium phosphate dihydrate; tricalcium phosphate, phosphate Calcium; Anhydrous Lactose, Spray-Dried Lactose; Pregelatinized Starch, Di-Pac( Compressible sugars such as Amstar®; mannitol, hydroxypropyl methylcellulose, cellulose, hydroxypropyl methylcellulose acetate stearate, sucrose-based Diluent, Powdered Sugar; Monobasic Calcium Sulfate Monohydrate; Calcium Sulfate Dihydrate; Calcium Lactate Sodium trihydrate, dextrate; hydrolyzed cereal solids, amylose; flour Powdered cellulose, calcium carbonate, glycine, kaolin, mannitol, sodium chloride, Contains inositol, bentonite, etc.

[0278] "Fillers" are lactose, calcium carbonate, calcium phosphate, dibasic calcium phosphate Calcium sulfate, crystalline cellulose, cellulose powder, dextrose, dextran Dextrate, dextran, starch, alpha starch, sucrose, xylose Lithol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene Glycol and other compounds.

[0279] "Lubricants" and "glidants" mean additives that prevent, reduce, or inhibit adhesion or friction of materials. Exemplary lubricants are, for example, stearic acid, calcium hydroxide, lubricating Do not use any hydrocarbons such as mineral oil, sodium stearyl fumarate, or mineral oil, or hydrogenated soybean oil (S Hydrogenated vegetable oils such as terotex®, high fatty acids, and aluminum, Alkali metal salts and alkaline earth metals such as calcium, magnesium, and zinc Salt, Stearic Acid, Sodium Stearate, Glycerol, Talc, Wax, Stearow et(R), boric acid, sodium benzoate, sodium acetate, sodium chloride, isin, polyethylene glycol (e.g., PEG-4000), or Carbowa x (trademark) and other methoxypolyethylene glycols, sodium oleate, sodium benzoate Sodium, Glyceryl Behenate, Polyethylene Glycol, Magnesium Lauryl Sulfate or sodium lauryl sulfate, Syloid™, Cab-O-Sil® Colloidal silica such as corn starch, starch such as corn starch, silicone oil, surfactants Includes sexual agents, etc.

[0280] "Plasticizer" means a compound used to soften the microencapsulated material; is a film coating that reduces the brittleness of the microencapsulated material. Examples of the copolymerization agent include PEG 300, PEG 400, PEG 600, and PEG 1450. , PEG 3350, and PEG 800; stearin Acid, propylene glycol, oleic acid, triethylcellulose, and triacetin In some embodiments, the plasticizer may also function as a dispersing agent or a humectant.

[0281] "Solubilizers" are triacetin, triethyl citrate, ethyl oleate, and caprylic acid. Ethyl, sodium lauryl sulfate, sodium docusate, vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone Polyvinylpyrrolidone, Hydroxypropyl methylcellulose, Hydroxypropyl Cyclodextrin, ethanol, n-butanol, isopropyl alcohol, cholesterol Terol, bile salts, polyethylene glycol 200-600, glycofurol, trans Compounds such as methacrylate, propylene glycol, and dimethyl isosorbide.

[0282] "Stabilizers" include compounds such as any antioxidant, buffer, acid, preservative, etc.

[0283] "Suspension agents" include polyvinylpyrrolidones, e.g., polyvinylpyrrolidone K12, polyvinylpyrrolidone K22, polyvinylpyrrolidone K32, polyvinylpyrrolidone K42, polyvinylpyrrolidone K52, polyvinylpyrrolidone K62, polyvinylpyrrolidone K72, polyvinylpyrrolidone K82, polyvinylpyrrolidone K9 Nylpyrrolidone K17, Polyvinylpyrrolidone K25, or Polyvinylpyrrolidone K3 0, vinylpyrrolidone / vinyl acetate copolymer (S630), polyethylene glycol ( For example, polyethylene glycol has a molecular weight of about 300 to about 6000, or about 3350 to about 40 00, or from about 7000 to about 5400), carboxymethyl cellulose Sodium stearate, methylcellulose, hydroxypropyl methylcellulose, stearic acid Hydroxymethylcellulose acetate, Polysorbate 80, Hydroxyethylcellulose, Sodium alginates, such as gum tragacanth and gum acacia, guar gum, etc. Gums, xanthans including xanthan gum; sugars, e.g., carboxymethylcellulose sodium sodium, methylcellulose, sodium carboxymethylcellulose, hydroxypropyl cellulose derivatives such as dimethylcellulose and hydroxyethylcellulose, polysorbate 80, sodium alginate, polyethoxylated sorbitan monolaurate, monolaurate Compounds such as polyethoxylated sorbitan urate and povidone.

[0284] "Surfactants" include sodium lauryl sulfate, sodium docusate, T Tween 60 or Tween 80, Triacetin, Vitamin E TPGS, Monoacetate Sorbitan oleate, polyoxyethylene sorbitan monooleate, polysorbate ( polysorbate), poloxamer, bile salts, glyceryl monostearate, oxide Copolymers of ethylene and propylene oxide, such as Pluronic® (B Some other surfactants include compounds such as polyoxyethylene glycerin Fatty acid esters and vegetable oils, such as polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylene alkyl ethers and alkyl phenyl ethers, such as Octoxynol 10, Octoxynol 40. In some embodiments, the surfactant can be incorporated to enhance physical stability or for other purposes.

[0285] The "viscosity enhancer" is, for example, methylcellulose, xanthan gum, carboxymethylcellulose, cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, stearyl Hydroxypropyl methylcellulose acetate, Hydroxypropyl methyl phthalate Cellulose, carbomer, polyvinyl alcohol, alginate, acacia, chitosan, and combinations thereof.

[0286] "Moisturizing agents" are oleic acid, glyceryl monostearate, and sorbitan monooleate. , Sorbitan monolaurate, Triethanolamine oleate, Polyisobutene monooleate Polyoxyethylene sorbitan monolaurate, sodium dodecyl sulfate Docusate, sodium oleate, sodium lauryl sulfate, sodium dodecyl sulfate Docusate, triacetin, Tween 80, Vitamin E TPGS, This includes compounds such as ammonium salts.

[0287] Kits and Compositions One aspect of the present disclosure is a method for producing a CAR comprising the steps of: Alternatively, genes that may be incorporated for safety reasons, e.g., HER1t or HER1t-1 , and functional variants thereof, or CD20 or CD20t-1, and functional variants thereof The present invention also relates to kits and compositions comprising a first vector comprising a functional mutant. The kits and compositions each encode a different protein or subset of proteins. The vectors may be viral vectors, They can be non-viral, episomal or integrative vectors. In some embodiments, the vector may be a transposon, e.g., sleepi ng beauty transposon.

[0288] In some embodiments, the kits and compositions include not only vectors, but also cells, and and interleukins, cytokines, interleukins, and chemotherapeutic agents, adjuvants In one embodiment, the cell is a T cell. In one embodiment, the kit and composition comprises IL-2. In one embodiment, the kit and composition include a Bcl-2 inhibitor. In some embodiments, the kit comprises a STAT3 inhibitor, a STAT5 inhibitor, or a STAT6 inhibitor. comprises IL-15, or mbIL-15.

[0289] In certain embodiments herein, one or more of the methods described herein may be Kits and articles of manufacture for use with the method are disclosed. Such kits include vials, test One or more vessels, such as tubes, each of which may be used in the methods described herein. A container compartmentalized to receive a container containing one of the individual elements used in the Suitable containers include, for example, bottles, vials, and the like. In one embodiment, the containers include glass or plastic. They are made from a variety of materials, including quartz.

[0290] The products presented herein contain packaging materials. Pharmaceutical Packaging Materials Examples of materials include blister packs, bottles, test tubes, bags, containers, bottles, as well as selected Any packaging materials appropriate for the formulation to be packaged and the intended mode of administration and treatment. Including but not limited to:

[0291] For example, the container may contain CAR-T cells (e.g., CD33-specific CAR-T cells described herein). RT cells), and optionally in addition, cytokines and / or and a chemotherapeutic agent. Such kits optionally include a method for the methods described herein. This includes confirmation of the contents of any descriptions, labels or instructions regarding its use in the product.

[0292] The kit typically includes a label listing the contents and / or instructions for use, etc. Typically, a set of instructions also includes a package insert with instructions for use. It is expected that this will be accepted.

[0293] In some embodiments, the indicia is affixed to or associated with the container. In an embodiment, letters, numbers, or other symbols forming the indicia are adhered to the container itself. If the marking is printed, cast or etched on the container, the marking shall be affixed to the container. The indication may also be provided within a receptacle or carrying case that holds the container, e.g. When present as a package insert, the label is associated with the container. Used to indicate that the contents are to be used for a specific therapeutic application. The illustrations may also point to directions for use of the contents, such as in the methods described herein. vinegar.

[0294] array Table 2 lists representative sequences for certain sequences included in the embodiments presented herein. Present a general list.

[0295] [Table 2-1]

[0296] [Table 2-2]

[0297] [Table 2-3]

[0298] [Table 2-4]

[0299] [Table 2-5]

[0300] [Table 2-6]

[0301] [Table 2-7]

[0302] [Table 2-8]

[0303] [Table 2-9]

[0304] [Table 2-10]

[0305] [Table 2-11]

[0306] [Table 2-12]

[0307] [Table 2-13]

[0308] [Table 2-14]

[0309] [Table 2-15]

[0310] [Table 2-16]

[0311] [Table 2-17] EXAMPLES

[0312] These examples are presented for illustrative purposes only and are not intended to be limiting of the scope of the invention as set forth herein. It is not intended to limit the scope of the claims.

[0313] [Example 1] Delivery of CD33 CAR via lentiviral vector: CD33 CAR lentivirus The viral vector was constructed based on the HIV-1 derived vector backbone. The product was then engineered to cleave the CD33 CAR at the 3' end with an in-frame, self-cleaving Tosea Gene fusion via the Thosea asigna virus 2A peptide (T2A) Both genes were expressed in the pFUGW lentiviral plasmid as described below. It was cloned into the character.

[0314] For CD33 CAR lentivirus, VSV-G (vesicular stomatitis virus (VSV) The glycoprotein of HIV-1 (VGA) is used as a substitute for the HIV-1 envelope protein. This results in improved vector stability, target cell targeting, and transduction efficiency. Cronin, J., Zhang, X. & Reiser, J., Altering the tropism of lentiviral vectors through pseudotyping, Curr Gene Ther, 5(4):387-98 (2005)).

[0315] Upon production, CD33 CAR viral particles are assembled and transfected into HEK VSV-G protein was budded from the surface of 293T cells. The vector core and enzyme proteins are carried by the per plasmid, pΔ8.9s o (GagPol) The RNA genome is delivered by the helper plasmid, and the viral particles The Rev protein, which is required for efficient transport and packaging into the offspring, is expressed in pRS Vpu, Vif, Vpr, Nef, and Ta were derived from the V-Rev plasmid. All other HIV-1 accessory proteins, including t, were expressed in the CD33 CAR vector. It was deleted from.

[0316] The pFUGW lentiviral vector backbone is a SIN lentiviral vector backbone .

[0317] Regulatory elements The LV-CD33 CAR vector drives the expression of the transgene, human elongation factor 1 alpha 1 (EF1A1) promoter and bovine growth hormone polyA sequence, and The vector contained a Kozak ribosomal initiation sequence. In addition to the posttranscriptional regulatory element (WPRE), the LTR sequence derived from the pFUGW plasmid was also included. The replication origin was based on the pUC19 plasmid. 1 is a lentiviral vector map.

[0318] [Example 2] How to generate CD33 CAR vectors The LV-CD33 CAR vector was assembled from three sources: (1) The following was obtained from a previously constructed vector, using the restriction enzymes PacI and Blp Digested by I: (a) pFUGW backbone components (b) human elongation factor 1 alpha 1 promoter (EF1A1) promoter, and and an enzyme driving anti-CD33 scFv with GM-CSFRalpha signal peptide. Enhancer (c) CD8α hinge and transmembrane domains (2) The 4-1BB signaling domain module was obtained as a synthetic gene; (3) CD3ζ, T2A transcription linker, GM-CSFRalpha signal peptide, H ER1t depletion marker, bovine growth hormone polyA, and flanking cloning sites The region containing the site was amplified by PCR.

[0319] The three DNA cassettes were synthesized in vitro using homology regions engineered into each cassette. The resulting DNA was annealed together in an in vitro assembly reaction. The lentiviral vector expresses the desired transfection target in the reverse orientation relative to the lentiviral genome. The Stbl4 E. coli strain contained the genes CD33, CAR, and HER1t. After transformation into the ) strain, positive colonies were identified by colony PCR and the three inserts were The fragments were verified by Sanger sequencing. All plasmid sequences were analyzed by Illumina Verified by Next Generation Sequencing.

[0320] Four plasmids, the LV-CD33 CAR vector plasmid, and three helper The parplasmids, pΔ8.9 so (GagPol); pMND-VSVG; and p Calcium phosphate precipitation transfection of RSV-Rev using 10% FBS The vector was produced in HEK-293T cells in DMEM with

[0321] Approximately 18 hours after plasmid transfection, a complete medium change was performed using benzonase ( Approximately 24 hours after medium replacement, the culture supernatant The vector containing the vector was harvested and labeled as harvest 1. Cells were replated with fresh medium The medium was re-fed and returned to the incubator. The supernatant was clarified and stored at between 2-8°C overnight.

[0322] The next day, the cells were observed under a microscope and the vector-containing supernatant was filtered as before. The supernatant from harvest 1 was combined with the filtered supernatant from harvest 2. The combined and pooled supernatants were further clarified by passing through a 0.45 μm filter. The clarified vector harvest was purified using anion exchange membrane chromatography. The purified medium was prepared by filtration, concentrated and diafiltered into the selected medium using a hollow fiber device. The cells were filled with the purified LV-CD33 CAR vector and stored frozen at <-80°C.

[0323] [Example 3] Efficiency of the delivery system Delivery of LV-CD33 CAR lentiviral vector to T cells derived from healthy donors Transduction with CD33 CAR typically results in the expression of CD33 CAR and CD33 CAR-specific markers 12–14 days after transduction. The results showed that 25–50% of cells stably co-expressed HER1t and HER2+. We outlined a typical lentiviral transduction strategy for generating CAR-T cells. FIG. 5 shows the results of lentiviral transduction in healthy donors 12 days after lentiviral transduction. Representative data for CD33 CAR and HER1t expression from a single T cell Dynabeads® Human T-Activator CD 3 / CD28 beads were used to activate purified human CD3+ T cells prior to infection. Transduction with CD33 CAR lentiviral vector was performed at a multiplicity of infection (MOI) of 5. Cells were grown in medium containing IL-2 for 12 days after transduction. Twelve days after transduction, flow cytometry analysis was performed to determine the expression of protein 1, 2, and 3, respectively. Expression of CD33 CAR and HER1t as measured by L and cetuximab mAb The current situation was evaluated.

[0324] Figure 6 shows expression of CD33 CAR from lentivirally transduced cells. , confirmed by Western blot. Culturing T cells or lentiviral-transduced CD33 CAR-T cells and administering proteases Cell lysates were made using RIPA buffer with inhibitors and reducing conditions. To standardize the amount of protein loaded onto the gel, samples were made in donors. For the same purpose, a BCA assay was also performed. After protein transfer, the membrane was blocked and then , 1 μg / ml of anti-CD3ζ antibody (clone 8D3), followed by goat anti-mouse horseradish pepper The bands were detected by chemiluminescence and digitalized. The 16 kDa band was captured by a 3D imaging system. The 60 kDa band represents CD3ζ, whereas the 60 kDa band represents CD3ζ signaling moiety. 33 CAR. Only CD33 CAR-T cells express the endogenous CD3ζ band in addition to the CD3ζ band. The transduced cells and Jurkat T cells showed a 60 kDa band, whereas the non-transduced cells and Jurkat T cells showed a 60 kDa band. , presenting only endogenous CD3ζ.

[0325] FIG. 7 shows the CD33 C cells in cultures after transduction with lentiviral vectors. Ex vivo expansion of AR-T cells. The starting cell population was 2.5×10 5 Data shown are means±SEM from four independent T cell donors. CD33 CAR-T cells were efficiently transduced and demonstrated clinical efficacy ex vivo. The results were expanded to levels that meant a reasonable dose had been achieved.

[0326] [Example 4] Integration of genetic material The delivered genetic material was integrated in the form of a proviral genome. Transgenes introduced into T cells using transduction with Figure 8 shows the expression of CD33-CAR after lentiviral transduction. Copy number assessment for human SUPT1 T cells. Lentivirus-mediated T cell proliferation in mice and healthy donor 270169 (D270169) Determine the number of gene copies integrated into the host genome after viral transduction. Droplet digital PCR (ddPCR) was used to identify the genes involved in transduction. Genomic DNA (gDNA) was obtained after 14 days. Rad's ddPCR system (QX200TM AutoDG ddPCR system) ) with Taqman-based primers and primers for detecting CD33 CAR. All cells contain two copies of the RNaseP gene. Based on the assumption that the couple will have children, RNaseP will be used to evaluate copy number variants (CNVs). SUPT1 / CD33 was used as a standard reference gene for CAR-T cell Expression of the CD33 CAR on D270169 / CD33 CAR-T cells was significantly correlated with the progression of pulmonary circulation. As confirmed by cytometry, the results were approximately 80% and 30%, respectively (data not shown). ) was reported. For D270169 / CD33 CAR-T cells, the assay The samples measured in (referred to as "measurements") are derived from a mixed population, but further calculations to calculate the copy number of the insert (referred to as "back calculation") in T cells expressing the CAR. Data shown are the mean copy number per cell ± S from two experiments. It's D.

[0327] [Example 5] Animal and cell culture models to evaluate the efficacy of gene transfer systems The efficiency of lentivirus-mediated gene transfer, as measured by expression of the transferred gene, was , as assessed by flow cytometry of the transduced cells (Figure 5). Expansion of transduced T cells Growth was performed ex vivo for 12-14 days after transduction to achieve a reasonable dose. vo culture medium (Figure 7).

[0328] In preclinical analyses, IL-1 was observed on the surface of T cells following lentiviral transduction (Figure 8). The levels of CD33 CAR observed in vitro were significantly higher in different CD33-positive In addition to efficiently and specifically eradicating the targeted tumor cell lines (Figure 9), The survival of mice bearing the IL-1 gene was extended to a statistically significant level compared to the control group (Figure 12 ) was sufficient. See also Figures 5 and 6 for further supporting data. stomach.

[0329] The functional capacity of CD33 CAR-T cells was assessed using in vitro and in vivo models. Both were investigated in the ivo model.

[0330] In vitro studies of CD33 CAR-T cells have demonstrated that CD33-expressing A ML cell lines as well as the murine EL4 transduced to express human CD33 on its surface. Cytotoxicity against cell lines and in co-culture with CD33-positive target cells, as determined by IFN-α. These included the secretion of N-γ, IL-2, and TNF-α cytokines. T cells are designed to deplete the infused CAR-T cells by administration of cetuximab. ADCC was observed in the CD33 CAR-T cells, which co-expressed the HER1t protein. The ability of cetuximab to clear tumors was also evaluated in vitro.

[0331] CD33 CAR-T cells are specifically cytotoxic to target cells in a dose-dependent manner (Figure 9). Cytotoxicity at an effector:target (E:T) ratio of 10:1 (B). CD33 CAR-T cells for cytotoxicity and CD3 at an E:T ratio of 1:1 (B) 3 CAR-T cells were measured by a 2-hour europium release assay. Dose-dependent cytotoxicity of CD33 CAR-T cells against 10:1 Between the E:T ratio of 1:1 and 1:2, the target cells expressed CD33 on the surface. Significant cytotoxicity was observed only when transduced T cells expressed the IL-1 agonist. The data presented in the graph show that the cytotoxic effect of the agonist on the target cells was not significant. Mean ± SEM from 4 different donors. ** p-value < 0.01 and *** P-Value <0.001.

[0332] CD33 CAR-T cells out-perform non-transduced T cells when co-cultured with various target cell lines. In comparison, they specifically secreted high levels of a variety of cytokines (Figure 10). After co-culture with various target AML cell lines expressing Evaluation of cytokine production from D33 CAR-T cells. , which were used as a negative control for human CD33 expression. The antigen:target (E:T) ratio was 10:1 and the culture supernatant was purified by QBea For multiplex cytokine analysis using ds(Intellicyt) The expression of human IFNγ, human IL-2, and human TNF secreted into the culture medium. We performed multiplex analysis of CD33 CAR-T cells. After co-culture with target cells expressing β-actin, basal expression of non-transduced T cells with the same target cells was observed. High levels of cytokines, IFNγ, IL-2, and and TNF were detected. The plotted values ​​are the mean ± SD for the test samples, It is expressed in runs.

[0333] Cetuximab targets CD33 CAR-T cells expressing HER1t on their surface at a dose of ADCC was able to eliminate the tumor in a tumor-dependent manner at an E:T ratio of 5:1 (Figure 11). PKH cells were co-incubated with purified effector NK cells for 24 hours. Flow cytometry analysis of 7-AAD uptake using 26-labeled target cells The specific activation of cetuximab against CD33 CAR-T cells was determined by immunohistochemistry. Dose-dependent ADCC was observed. Values ​​plotted are the mean ± SD for the test samples. is expressed in two parts.

[0334] Additionally, fLUC expression was used to monitor tumor burden by bioluminescence imaging. The AML cell line MOLM-13 (MOLM-13 / fLU) was transduced to C) The ability of CD33 CAR-T cells to specifically reduce tumor burden was assessed using immunohistochemistry. In vivo xenograft model of NOD.Cg-Prkdcs in immunodeficient NSG cidIL2rgtm1Wjl / SzJ, NODscid gamma mice Details of the study design are depicted in Table 2. In NSG mice, CD33 CAR-T cells were administered in saline, non-cytosed medium, or in a non-cytosed medium. Transduced T cells and CD33 CAR-T cells were generated from the same healthy T cell donor , when compared to a control treatment group consisting of nonspecific CD19 CAR-T cells. It significantly reduced tumor burden and extended median survival (P<0.001). 33 CAR-T cells expanded in vivo in tumor-bearing mice (Figure 1). 14) Flow cytometry of CD33 CAR-T cells in NSG mice prior to injection. -cytometry analysis and CD33 CAR- Perform T cell detection to assess CAR-T cell persistence and expansion in vivo The CD33 CAR lentivirus induced T cell proliferation in healthy donor T cells (Figure 14A). The CD33 CAR was transduced and expanded ex vivo. - Prior to T cell injection, flow cytometry analysis was performed to assess the expression of CD33 CAR The indicated populations were gated for FSC / SSC / live / CD3+ cells. On day 18 (systemic IV injection into the tail vein), - 10 days after T cell injection), flow cytometry analysis was performed on MOLM-13 tumor-bearing Blood samples, bone marrow samples, and spleen samples collected from CD33 CAR-T-treated mice were A 10-fold increase in FSC was observed in the 100% CI, 0.01 to 0.25, and 0.01 to 0.25. One representative mouse gated based on / SSC / hCD45 / hCD3 expression Data derived from.

[0335] On day 0, immunodeficient NSG mice were transfected with the firefly luciferase (fLUC) gene. MOLM-13 cell line modified to express 5×1 (MOLM-13 / fLUC) 0 5 On day 7, the tumor-bearing mice were imaged using IVIS imaging. The mice were then randomized into various treatment groups. On day 8, mice were treated with either saline alone or non-transduced T cells (mouse 1) via IV injection. Total T cells per animal: 10 7 ), either without cell tag or with HER1t cell tag Accompanying CD33 CAR-T cells (10 CD33 CAR-T cells per mouse) 7 pieces ), or CD19 CAR T cells (1 CD19 CAR-T cell per mouse 0 7 In addition, on day 15, another group of mice was treated with HER1t cell tag CD33 CAR T cells with CD33 CAR+ T cells per mouse 7 Total tumor burden was assessed using bioluminescence imaging with fLU. C expression was assessed over the course of the study. Survival curves according to the various treatment groups are shown in Figure 12. Mice in saline, non-transduced T cells, and CD19 CAR-T cells The median survival time for CD33 CAR T cells without the cell tag was 15 days. Mice treated with CD33 CAR-T cells (dose 1 or All mice treated with 100 mg / kg / day 2 times showed complete survival for up to 29 days.

[0336] The average flux values ​​from the ventral and dorsal views are plotted in Figure 13 for the various treatment groups. Values ​​shown are total flux values ​​expressed as photons / second, averaged for each group. Mean ± SEM is shown. Solid upward arrows indicate the day of CAR-T administration for each group. The dotted upward arrow indicates CD33 CA1 expression in the 2-dose group on day 15. Indicate the second dose of RT.

[0337] [Table 3]

[0338] [Example 6] Cytokine analysis: Plasma samples from mice were analyzed at specific time points within the protocol and Plasma was collected when the mice became moribund and were euthanized for humane reasons. To obtain the ELISA kit, blood was collected into EDTA collection tubes and then centrifuged at 5,000 × g at room temperature. The resulting plasma was then spun for 15 minutes and then subjected to a clean recovery test. The plasma was frozen and stored at -80°C until assayed. aPlex® Human Cytokine / Chemokine / Growth Factor Panel 1 Kit (eB ioscience) is a biomarker for the following analytes: Brain-derived neurotrophic factor (BDNF), epidermal growth factor (EGF), EGFR, eotaxin, fibroblast growth factor 2 (FGF-2), granulocyte / macrophage GM-CSF, GRO alpha (CXCL1), hepatocyte proliferation Growth factor (HGF), interferon (IFN) alpha, IFN gamma (IFNγ), Interleukin (IL)-1 receptor alpha (IL-1RA), IL-1 alpha, I L-1beta, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL- 9, IL-10, IL-12p70, IL-13, IL-15, IL-17A, IL-1 8, IL-21, IL-22, IL-23, IL-27, IL-31, interferon gamma-inducible protein 10 (IP-10; also known as CXCL10), Hematopoietic inhibitory factor (LIF), monocyte chemotactic protein-1 (MCP-1; also known as CCL2 ), macrophage inflammatory protein 1 alpha (MIP-1 alpha; CCL3 (also known as CCL4), MIP-1 beta (also known as CCL4), Nerve growth factor (NGF beta), RANTES (also known as CCL5) gulated upon Activation Normal T cell Ex Pressed and Secreted), Platelet-derived Growth Factor BB (PDGF-B B), placental growth factor 1 (PIGF-1), stem cell factor (SCF), stromal derived factor 1 alpha Tumor necrosis factor alpha (SDF-1 alpha, also known as CXCL12 alpha) TNF-alpha (TNF alpha), TNF-beta (also known as lymphotoxin alpha) ), vascular endothelial growth factor alpha (VEGF-A), and vascular endothelial growth factor D (VEGF A 45-plex magnetic bead-based kit for multiplexed detection of FD The assay was performed according to the manufacturer's specific protocol. Data were provided by Bio- Bio-Ra running Plex Manager software version 6.1 d Bio-Plex® 200 instrument (Bio-Rad, Hercules, The samples were collected on a 1000-well plate (CA).

[0339] On day 11, plasma was collected from the mice to measure human cytokines and human proliferation. Expression of factors was assessed in MOLM-13 and NSG mice. See Figure 22A. Because the T cells introduced into the mice are of human origin, the measurement of human cytokines is Further investigation is needed to determine the mechanism of action of CAR-T cells against the cells. In MOLM-13 tumor-bearing mice treated with saline alone, MCP-1 and MIP1 beta (data not shown), as well as several other growth factors Although some human systemic cytokines such as IFNγ and TNFα were detected, The levels of cytokines were below the detection range (Figure 22A). MOLM-13 tumor-bearing mice also showed similar progression compared to the group treated with saline alone. The T cells continued to show a similar pattern of cytokine and growth factor expression compared to the non-transduced T cells. The levels of cytokines and growth factors observed in the mouse model system were A mixture of human T cells and tumor cells provides a baseline value. Stimulation of T cells (CAR T cells, whether or not they co-expressed the cell tag) Analysis of cytokine expression data revealed that GM-CSF, IL-10, IL-18, and IP-1 In addition to CD1, CD20 produced significant levels of IFNγ and TNF. Administration of 9-CAR-T cells to MOLM-13 tumor-bearing mice was in combination with GM-CSF, IFN-γ, and IFN-γ. For cytokines such as Nγ and IL-18, saline alone or non-transduced T cells The expression levels of these cytokines were slightly increased compared to the control group. Levels were measured using CD33-CAR-T cells (without cell tag) or CD33-CAR- T cells (with cell tag) or CD33-CAR-T cells (with cell tag; 2 doses) ) compared to the levels of the same cytokines detected in mice administered It didn't rise that much.

[0340] Blood samples were analyzed by flow cytometry for the presence of tumor cells as well as adoptively transferred T cells. The samples were collected at the designated time points of the study, as assessed by ELISA. On day 16, the presence of MOLM-13 / fLUC cells was detected in the peripheral blood of mice. The loss of CD33 antigen in MOLM-13 cells may have been underestimated or potentially under-reported. To avoid this possibility, we used CD123 antibody to identify tumor cells. The results were obtained from mice receiving either transduced or non-transduced CD19-CAR-T cells. In peripheral blood samples, MOLM-13 tumor cells were detected. In addition, non-transduced T cells In mice that received CD19-CAR-T cells, the transferred T cells were detected in the blood. The disease was detected as early as day 16 and day 11 after blood cell analysis was started. Blood from mice receiving CD33-CAR-T cells was analyzed using MOLM-13 tumor While most cells are absent or completely absent, T cell populations are clearly present. It was shown that.

[0341] FIG. 22C shows cytotoxicity against additional AML tumor cell lines (THP1 and HL60). The redirected cytotoxic function of CD33-CAR-T cells Further evidence for this is the induction of immune effector cells in tumor cells upon co-culture with CD33 tumor cells. This was achieved by assessing cytokine expression by several CD33-expressing tumors. Tumor cell lines were co-cultured with CD33-CAR-T cells to investigate the effects of IFNγ, TNF-α, and The induction of expression of CD33-C, also known as TNFα, and IL-2 was evaluated. Co-culture of AR-T cells with CD33-expressing tumor cells derived from the same donor It caused a marked increase in expression of IFNγ, TNF, and IL-2 compared to T cells. In addition, the parental EL4 cell line, used as a negative control for tumor targeting, did not show any significant differences in the serologic outcome (Figure 10). Intracellular cytokine staining (ICS) was performed using target-specific This was performed as a further confirmation of the efficacy of CD33-CAR-T cells. The results showed that the IL-16 expression of MOLM-13 tumor cells, THP-1 tumor cells, and HL-60 tumor cells After overnight co-culture, the expression of both IFNγ and IL-2 was measured at the cellular level. whereas co-culture with mouse EL4 cells suppressed the expression of IL-1 at levels seen only by T cells. However, no significant cytokine expression was observed in the control group (Fig. 23E).

[0342] CD33-CAR-T cells were used in an AML patient (donor E261) with relapsed AML disease. Briefly, the cells were generated from cryopreserved PBMCs derived from AML donors. were thawed and T cells were selected and expanded using anti-CD3 / anti-CD28 beads. T cells were transduced with CD33-CAR LV particles and expanded in culture. Characterization of this thawed PBMC sample revealed that from this donor, The T cells present in the spleen were low in frequency and consisted mainly of CD33+ cells. (Figure 23A). For transduction with LV to generate CD33-CAR-T cells, T The cells were isolated, activated and expanded. The remaining cells after T cell isolation were D33+ (Figure EB), and the cytokine granulocyte-macrophage colony-stimulating factor The donor was maintained in culture with the use of GM-CSF. Approximately 41% of the cells were transduced with LV after 12 days of culture. As assessed, CD33-CAR was expressed on the cell surface (Figure 23C). The efficacy of CD33-CAR-T cells derived from this donor was evaluated in patients with MOLM-13 tumor cells or flow cytometry-based co-culture assay with the patient's own CD33-expressing tumor cells The remaining cell populations at the time points assessed (days 0 and 3) were Staining to assess the expression of CD3 (T cells) and CD33 (tumor) on cells According to the study, only co-culture with CD33-CAR-T cells was able to eliminate CD33-expressing tumor cells. In contrast, in a co-culture setting with untransduced (UNT) T cells, No cell killing was observed (Figure 23D).

[0343] In addition, cytokine expression was assessed by flow cytometry using intracellular cytokine staining. Furthermore, CD33-CAR-T cells generated from AML donors also inhibited IFN-γ and IL-1 Intracellular cytokine staining showed that CD33-CAR-T cells (AML Donor E261) after 18 hours of co-culture with the indicated tumor cells. T cells were stained for cell surface staining, then fixed / permeabilized and then stimulated with the cytokine I Stained for FNγ and IL-2. Based on FSC / SSC / Live / CD3 T cells Cells were gated on and analyzed using CD33-CAR-T cells alone or in combination with mouse EL4 tumor cells. Coculture did not show expression of IFNγ or IL-2, whereas MOLM-13 or After co-culture with tumor cells derived from a particular donor, Increased expression of CD33-CAR was also observed (Figure 23E). In addition, increased expression of CD33-CAR was observed in cells expressing CD33-CAR. The expression of cytokines from the CAR-specific T cells was specifically induced, whereas the expression of cytokines from the CAR-specific T cells was not induced by the CAR-specific T cells. No cytokine expression was observed.

[0344] [Example 7] We constructed lentiviral CD33 CARs with diverse signaling domains and regulated their expression. We investigated the CD33 CAR with only the CD3ζ signaling domain, CD28- a CD33 CAR with a CD3z (referred to as CD28z) signaling domain, or CD33 CAR with CD28-CD3z domain with co-expression of HER1t Lentiviral particles with were preactivated according to the schematic outlined in FIG. Human T cells were transduced with the antibody. Flow cytometry analysis was performed 12 to 14 days after transduction. Expression of the CD33 CAR was examined via protein L staining. and HER1t was detected via cetuximab staining (Figures 15A and 15B).

[0345] Specificity of CD33 CARs (with distinct signaling domains) recognizing their targets To support this, co-culture assays were performed on various tumor cell lines. The expression of CD33 on K562 cells was shown (Figure 16A). K562 cells endogenously express CD33. Therefore, we observed expression in K562 / CD123 and in the K562 / CD33 cell line. Note that EL4 was transduced to overexpress CD33. This is a mouse cell line that does not express D33. Cytotoxicity was evaluated using the DELFIA BATDA assay. The drug was used (DELFIA EuTDA cytotoxicity assay; Perkin Elmer). was used to label various tumor targeting cell lines and then co-immunopreserved with CD33 CAR T cells or non- Transduced T cells (UNT) were co-cultured with labeled tumor cells at an effector:target ratio (E:T) The ratio of β-lactams derived from the assay was determined by co-culturing the cells at a ratio of 20:1. The supernatant was collected and developed by adding DELFIA Europium Assay. The results were read on a resolved fluorometer (Figure 16B). ) ratios, and 18 h incubation resulted in robust IFNγ production, and the cultures Supernatants were harvested and assayed for IFNγ production by ELISA according to the manufacturer's instructions. Untransduced T cells (UNT) showed almost no detectable IFNγ in the coculture. Not detected at all (Figure 16C).

[0346] Renal cell transplantation into T cells derived from cryopreserved PBMC samples from patients diagnosed with AML. Transduction with tiviruses is used to transduce T cells derived from T cells of healthy donors. The procedure was carried out similarly to that outlined above. Briefly, anti-CD3 / CD28 beads were Next, we performed lentivirus-mediated CD33 CAR-41BBz-T at varying MOIs. Transduction of 2A-HER1t was used to activate T cells isolated from PBMC samples. The transduced T cell cultures were expanded over a 14 day culture period. CAR expression, as determined by staining for protein L, was examined throughout the culture expansion period. The levels of CD33 CAR were observed on the surface of T cells (data not shown). CD33 CA using donor autologous CD33-expressing tumor cells as well as AML tumor cell lines. The cytotoxic activity of RT cells was confirmed. In one case, a low E:T ratio was used to co-culture At various time points, flow cytometry analysis was performed to assess the presence of T cells. The presence and frequency of CD33-expressing tumor cells present in the co-cultures were determined at various time points. Co-culture of CD33 CAR-T cells with autologous CD33-expressing tumor cells Cytotoxic activity was demonstrated as determined by the reduction or absence of CD33+ tumor cells. (Data not shown). Similar findings were observed in known AML tumor cells tested, such as MOLM-13. was also observed in some strains.

[0347] [Example 8] CD33 CAR by Sleeping Beauty Various CD33 CAR constructs were transduced with the Sleeping Beauty transposase -transposon system (Figure 3). For example, CD33-CD8 a-CD28TM-CD3z constructs were synthesized using various EF1a promoter lengths (short, medium, and We also generated a CD33 CAR construct with a HER1t cell tag. , I looked into it.

[0348] The Sleeping Beauty base was used to mediate integration of the construct into the genome. The CAR construct was introduced into cells via electroporation using the transposon system of On day 0, 20 million PBMCs were incubated with 15 μg of transposon and 5 μg of transposon. 100 μL of Amaxa Hu mixed with sposase (pKan-CMV-SB11) man T cell Nucleofector solution (Model number: VPA- 1002; Lonza, Basel, Switzerland) and The cells were electroporated using Fluorogram U-14. The next day (day 1), cells were counted and CAR expression by protein L staining and HER1t staining using cetuximab Cells were treated with either γ-irradiation (100 Gy) or mitosis at a ratio of 1:1. The AaPC ​​cells used were endogenous CD33 expression. CD64-CD86-41BBL-CD19-mbIL-15 / IL15 with differential expression The CAR T cells were K562-AaPC expressing the Ra-ROR1 antigen. Cultures were stimulated with AaPCs at 1:1. The first round of stimulation included IL-21 (3 0 ng / ml), and then for the remainder of the stimulation, recombinant human IL-2 (50 IU The T cells were supplemented with IL-21 (30 ng / ml) and IL-21 (30 ng / ml) (PeproTech). Cell cultures were typically phenotypically resolved at the end of each stimulation cycle, which lasted for 7 days. Protein L staining or multiparameter flow cytometry Cultures were assayed for CAR expression by recombinant CD33 / Fc protein staining, as detected by The cultures were also subjected to phenotypic analysis for NK cells (CD3- CD56+ population and The proliferation of cytoplasmic endothelial cells (defined as the percentage of the total cell population) was also closely monitored. When the number of cells exceeded 10%, the cells were incubated with magnetic beads for CD56 (Steinberger et al., 2012) according to the manufacturer's instructions. m Cell Technologies and / or Miltenyi Biotech c) were removed from CAR T cell cultures using flow cytometry. Expression of CD33 CAR from multiple donor PBMCs following stimulation with AaPCs The current situation was assessed.

[0349] Specificity of CD33 CAR (with various signaling domains) recognizing its target To support this, co-culture assays were performed with various tumor cell lines. EL4 is a mouse cell line that does not express human CD33 endogenously. Please note that the cytotoxicity was measured using the DELFIA BATDA reagent (DELFI A EuTDA cytotoxicity assay (Perkin Elmer) was used to measure the cytotoxicity of various Tumor-targeting cell lines are labeled and then transduced with CD33 CAR T cells or non-transduced T cells. (UNT) was administered with labeled tumor cells at an effector:target ratio (E:T) of 10:1. T...

Claims

1. (a) a CD33 antigen-binding domain, (b) a transmembrane domain; and (c) Intracellular domain A chimeric antigen receptor (CAR).

2. The CAR of claim 1, wherein the transmembrane domain comprises a polypeptide having at least 90% identity to SEQ ID NO:

18.

3. The CAR of claim 1, wherein the intracellular domain comprises a CD28 or 4-1BB costimulatory signaling domain.

4. The CAR of claim 1, wherein the intracellular domain comprises a polypeptide having at least 90% identity to SEQ ID NO:

28.

5. The CD33 antigen-binding domain, (a) a polypeptide having at least 90% identity to SEQ ID NO:1 and a polypeptide having at least 90% identity to SEQ ID NO:3; (b) a polypeptide having at least 90% identity to SEQ ID NO:9 and a polypeptide having at least 90% identity to SEQ ID NO:10; (c) a polypeptide having at least 90% identity to SEQ ID NO:11 and a polypeptide having at least 90% identity to SEQ ID NO:12; or (d) a polypeptide having at least 90% identity to SEQ ID NO: 13 and a polypeptide having at least 90% identity to SEQ ID NO:

14. The CAR of claim 1 .

6. The CD33 antigen-binding domain, (a) a polypeptide having at least 95% identity to SEQ ID NO:1 and a polypeptide having at least 95% identity to SEQ ID NO:3; (b) a polypeptide having at least 95% identity to SEQ ID NO:9 and a polypeptide having at least 95% identity to SEQ ID NO:10; (c) a polypeptide having at least 95% identity to SEQ ID NO:11 and a polypeptide having at least 95% identity to SEQ ID NO:12; or (d) a polypeptide having at least 95% identity to SEQ ID NO: 13 and a polypeptide having at least 95% identity to SEQ ID NO:

14. The CAR of claim 1 .

7. The CD33 antigen-binding domain, (a) a polypeptide having at least 98% identity to SEQ ID NO:1 and a polypeptide having at least 98% identity to SEQ ID NO:3; (b) a polypeptide having at least 98% identity to SEQ ID NO:9 and a polypeptide having at least 98% identity to SEQ ID NO:10; (c) a polypeptide having at least 98% identity to SEQ ID NO:11 and a polypeptide having at least 98% identity to SEQ ID NO:12; or (d) a polypeptide having at least 98% identity to SEQ ID NO: 13 and a polypeptide having at least 98% identity to SEQ ID NO:

14. The CAR of claim 1 .

8. The CD33 antigen-binding domain, (a) a polypeptide comprising the amino acid sequence of SEQ ID NO:1 and a polypeptide comprising the amino acid sequence of SEQ ID NO:3; (b) a polypeptide comprising the amino acid sequence of SEQ ID NO:9 and a polypeptide comprising the amino acid sequence of SEQ ID NO:10; (c) a polypeptide comprising the amino acid sequence of SEQ ID NO:11 and a polypeptide comprising the amino acid sequence of SEQ ID NO:12; or (d) a polypeptide comprising the amino acid sequence of SEQ ID NO: 13 and a polypeptide comprising the amino acid sequence of SEQ ID NO: 14 The CAR of claim 1 .

9. The CAR described in claim 1, wherein the CD33 antigen-binding domain comprises: (a) a polypeptide having the amino acid sequence of SEQ ID NO:1; and a polypeptide having the amino acid sequence of SEQ ID NO:

3.

10. The CAR described in claim 1, wherein the CD33 antigen-binding domain comprises a polypeptide having the amino acid sequence of SEQ ID NO:

8.

11. The CAR described in claim 1, wherein the intracellular domain comprises a polypeptide having at least 90% identity to sequence number 26.

12. The CAR described in claim 1, further comprising a stalk domain.

13. The CAR described in claim 12, wherein the stalk domain comprises a polypeptide having at least 90% identity to sequence number 22.

14. A CAR described in claim 1, comprising the amino acid sequence of SEQ ID NO:

39.

15. A nucleic acid encoding a CAR described in any one of claims 1 to 14.

16. The nucleic acid described in claim 15, further encoding a cell tag.

17. The nucleic acid of claim 16, wherein the cell tag comprises a truncated epidermal growth factor receptor.

18. The nucleic acid described in claim 17, wherein the cell tag comprises a polypeptide having at least 90% identity to sequence number 54.

19. The nucleic acid of claim 15, further encoding a cytokine.

20. The nucleic acid described in claim 19, wherein the cytokine is membrane-bound IL15.

21. The nucleic acid described in claim 20, encoding a polypeptide having at least 90% identity to sequence number 37.

22. A vector comprising the nucleic acid described in claim 15.

23. The vector of claim 22, wherein the vector is a Sleeping Beauty transposon.

24. An immune effector cell comprising a CAR described in any one of claims 1 to 14.

25. The immune effector cell described in claim 24, further comprising a cell tag.

26. The immune effector cell described in claim 24, further comprising membrane-bound IL15.

27. ​​The immune effector cell described in claim 24, wherein the immune effector cell is a T cell or a natural killer (NK) cell.

28. The immune effector cell described in claim 24, wherein the immune effector cell is a T cell.

29. A composition for use in stimulating a T cell-mediated immune response against a target cell population or target tissue in a human subject, comprising an effective amount of an immune effector cell described in claim 24.

30. The composition of claim 29, wherein the human subject has been diagnosed with cancer.

31. The composition described in claim 29, wherein the human subject has been diagnosed with acute myeloid leukemia (AML).

32. The composition described in claim 29, wherein the human subject has been diagnosed with relapsed or refractory AML.

33. A system for expressing CAR in immune effector cells, comprising the vector described in claim 23 and Sleeping Beauty transposase.

34. A method for producing modified T cells, comprising transfecting a T cell or a precursor cell of a T cell with the vector described in claim 22.

35. A method for stimulating the proliferation or survival of modified T cells, comprising transfecting T cells or T cell precursors in vitro with the vector described in claim 22.

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