Cells expressing anti-cd19 chimeric antigen receptors and methods of use thereof

Abstract

The present disclosure relates to DNA constructs encoding CD19-specific chimeric antigen receptors (CARs) and other recombinant proteins, such as CD70-binding proteins or CD70-specific CARs, as well as engineered immune cells (e.g., CD19-specific CAR T cells or CD19 / CD70 dual CAR T cells) comprising the DNA constructs, methods of making the same, and uses thereof in therapeutic applications.

Description

Cross-references to related applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 659,501, filed June 13, 2024, and U.S. Provisional Application No. 63 / 594,261, filed October 30, 2023, the contents of which are hereby incorporated by reference in their entirety.

[0002] sequence list This application contains a sequence list submitted electronically in XML file format and hereby incorporated in its entirety by reference. The XML copy was created on October 18, 2024, named AT-058_03WO_SL.xml, and has a size of 112,901 bytes. Technical Field

[0003] This disclosure generally relates to engineered immune cells (e.g., CAR T cells) and their use in therapeutic applications. Background Technology

[0004] Adoptive transfer of engineered immune cells, such as chimeric antigen receptor (CAR) T cells, shows great promise in cancer treatment, particularly for hematologic malignancies and other autoimmune diseases. CD19 is commonly expressed in hematologic cancer cells, such as non-Hodgkin's lymphoma. Currently, several autologous CAR T-cell therapies targeting CD19 have been approved for the treatment of hematologic malignancies. Autologous CD19 CAR T-cell therapy also shows promise in treating CD19-related autoimmune diseases. Some pathogenic B cells have been shown to also express CD70, and both B cells and T cells are associated with autoimmune diseases (see, for example, Ulutekin et al., Cell Reports Medicine, 2024 Jan 16;5(1):101351.doi: 10.1016 / j.xcrm.2023.101351. Epub Dec 21, 2023; Han et al., Seminars in Arthritis and Rheumatism, 2016, 45:496-501).

[0005] Allogeneic CAR T cells (where CAR T cells are generated from healthy donor cells rather than the patient's own cells) can be prepared and used as off-the-shelf products. This promises to reduce manufacturing costs, improve drug consistency, shorten patient wait times, and allow more patients to access CAR T therapy. However, allogeneic CAR T cells may be recognized as foreign by the host and rejected by the patient's immune system, thus limiting the expansion and persistence of the infused allogeneic CAR T cells. Furthermore, although CD19 is an effective target for, for example, non-Hodgkin's lymphoma, preclinical and clinical studies have observed a tendency for pathogenic target cells (such as tumor cells) to downregulate CD19 after CAR T cell therapy (see Spiegel et al.). people , 2021, NatureMedicine 27:1419-1431).

[0006] Therefore, there is a need to improve anti-CD19 CAR T cells to give them higher potency, increased persistence and / or enhanced function. Summary of the Invention

[0007] This document provides engineered immune cells, such as engineered T cells, that contain and / or express anti-CD19 or CD19-specific chimeric antigen receptor (CAR) and CD70-binding protein and / or chimeric cytokine receptor (CCR). This document also provides engineered immune cells containing one or more polynucleotides encoding one or more recombinant proteins. In some embodiments, the one or more polynucleotides contain one or more, at least two, or two or more coding sequences that encode CD19-specific CAR, CD70-binding protein, CCR, and / or other recombinant proteins.

[0008] In some embodiments, the engineered immune cells contain or express a CD19-specific CAR and a CD70-binding protein, or a CD70-specific CAR. In some embodiments, the CD19-specific CAR contains the amino acid sequence of SEQ ID NO: 2, 3, 4, or 5, with or without a signal peptide, and the CD70-specific CAR contains the amino acid sequence of SEQ ID NO: 16, 17, 18, or 19, with or without a signal peptide.

[0009] In some embodiments, the engineered immune cells comprise a polynucleotide containing a coding sequence encoding a CD19-specific CAR and a coding sequence encoding a CD70-specific CAR. In some embodiments, the coding sequence encoding the CD19-specific CAR is linked to the coding sequence encoding the CD70-specific CAR via a nucleotide sequence encoding a P2A peptide.

[0010] This document also provides a polynucleotide comprising a first coding sequence encoding a CD19-specific CAR and a second coding sequence encoding a CD170-specific CAR. In some embodiments, the coding sequence encoding the CD19-specific CAR is linked to the coding sequence encoding the CD70-specific CAR via a nucleotide sequence encoding a P2A peptide. In some embodiments, the CD19-specific CAR comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, or 5, with or without a signal peptide, and the CD70-specific CAR comprises the amino acid sequence of SEQ ID NO: 16, 17, 18, or 19, with or without a signal peptide.

[0011] In some embodiments, the polynucleotide comprises a truncated PGK promoter, a first coding sequence, and a second coding sequence, wherein the truncated PGK promoter comprises a deletion of at least 50 nucleotides from the 5' end of a PGK promoter, or a wild-type or long PGK promoter having the nucleotide sequence of SEQ ID NO: 34, wherein the truncated PGK promoter is operatively linked to the first coding sequence and the second coding sequence, and wherein the first coding sequence or the second coding sequence encodes an anti-CD19 chimeric antigen receptor (CAR). In some embodiments, the polynucleotide comprises a truncated PGK promoter, a first coding sequence, and a second coding sequence from 5' to 3', wherein the truncated PGK promoter comprises a deletion of at least 50 nucleotides from the 5' end of a long PGK promoter having the nucleotide sequence of SEQ ID NO: 34, wherein the truncated PGK promoter is operatively linked to the first coding sequence and the second coding sequence, and wherein the first coding sequence or the second coding sequence encodes an anti-CD19 chimeric antigen receptor (CAR). In some embodiments, the first coding sequence and the second coding sequence are linked by a P2A peptide.

[0012] In some embodiments, the anti-CD19 CAR comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the anti-CD19 CAR further comprises a CD8 hinge domain and a transmembrane domain. In some embodiments, the CD8 hinge domain comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 10 or 99. In some embodiments, the anti-CD19 CAR further comprises a CD3z signaling domain and / or a 4-1BB co-stimulatory domain. In some embodiments, the CD3z signaling domain comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, the 4-1BB co-stimulatory domain comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the anti-CD19 CAR comprises the amino acid sequence of SEQ ID NO: 5 or 7, with or without a signal peptide. In some embodiments, the anti-CD19 CAR comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 5 or 7, with or without a signal peptide. In some embodiments, the coding sequence encoding the anti-CD19 CAR comprises a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 6 or 8.

[0013] In some embodiments, the truncated PGK promoter comprises a deletion of at least 100 nucleotides, at least 150 nucleotides, at least 200 nucleotides, at least 250 nucleotides, at least 300 nucleotides, or at least 350 nucleotides from the 5' end of a long PGK promoter having the nucleotide sequence of SEQ ID NO: 34. In some embodiments, the truncated PGK promoter comprises a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequences of SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38. In some embodiments, the truncated PGK promoter comprises a nucleotide sequence comprising (a) a deletion of about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, or about 390 nucleotides from the 5' end of SEQ ID NO: 34, and having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 35; (b) a deletion of about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, or about 290 nucleotides from the 5' end of SEQ ID NO: 34, and having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 36; (c) a deletion of about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 370, about 380, or about 390 nucleotides from the 5' end of SEQ ID NO: 35. SEQ ID NO: 34 has about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, or about 190 nucleotides deleted from its 5' end, and has at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 37; or (d) has about 50, about 60, about 70, about 80, about 90, or about 100 nucleotides deleted from the 5' end of SEQ ID NO: 34, and has at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 38. In some embodiments, the truncated PGK promoter is substantially composed of or consists of the nucleotide sequences of SEQ ID NO: 35, 36, 37, or 38. In some implementations, the truncated PGK promoter is substantially composed of or consists of a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 35, 36, 37, or 38.

[0014] In some embodiments, the first coding sequence encodes an anti-CD19 CAR, and the second coding sequence encodes a CD70-binding protein. In some embodiments, the first coding sequence encodes a CD70-binding protein, and the second coding sequence encodes an anti-CD19 CAR. In some embodiments, the CD70-binding protein is or comprises an anti-CD70 CAR.

[0015] In some embodiments, the first coding sequence encodes an anti-CD19 CAR, and the second coding sequence encodes a chimeric cytokine receptor (CCR).

[0016] In some embodiments, the CD70-binding protein is a recombinant CD70-binding protein. In some embodiments, the CD70-binding protein comprises an anti-CD70 antibody or an antigen-binding fragment thereof, and a transmembrane domain. In some embodiments, the anti-CD70 antibody comprises the amino acid sequence of SEQ ID NO: 16 and / or 17. In some embodiments, the anti-CD70 antibody comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the CD70-binding protein further comprises a hinge domain. In some embodiments, the hinge domain and transmembrane domain comprise a CD8 hinge domain and a CD8 transmembrane domain. In some embodiments, the CD8 hinge domain comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 10 or 99. In other embodiments, the hinge domain and transmembrane domain comprise a CD28 hinge domain and a CD28 transmembrane domain. In some embodiments, the CD70-binding protein further comprises a CD3 signaling domain. In some embodiments, the CD70-binding protein does not comprise a co-stimulatory domain. In some embodiments, the CD70-binding protein does not comprise a 4-1BB co-stimulatory domain. In some embodiments, the CD70-binding protein comprises a CD3 signaling domain comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the CD70-binding protein comprises the amino acid sequence of SEQ ID NO: 19 or 21, or comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity with SEQ ID NO: 19 or 21, with or without a signal peptide. In some embodiments, the coding sequence encoding the CD70-binding protein comprises a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 20 or 22.

[0017] In some embodiments, the CCR includes a thrombopoietin receptor / myeloproliferative leukemia protein receptor (TPOR / MPLR) transmembrane and JAK-binding domains and an intracellular recruitment domain. In some embodiments, the TPOR / MPLR transmembrane domain and JAK-binding domain comprise the amino acid sequence of SEQ ID NO: 25 or 51. In some embodiments, the intracellular recruitment domain comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, the CCR comprises the amino acid sequence of SEQ ID NO: 27 or 29, or an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity with SEQ ID NO: 27 or 29, with or without a signal peptide. In some embodiments, the coding sequence encoding the CCR comprises a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 28 or 30.

[0018] In some embodiments, the polynucleotide further comprises a 5' homologous arm and a 3' homologous arm. In some embodiments, the length of the 5' homologous arm and the 3' homologous arm is each at least about 100, at least about 150, at least about 200, at least about 250, or at least about 300 nucleotides or more. In some embodiments, the 5' homologous arm and the 3' homologous arm each comprise a nucleotide sequence homologous to the nucleotide sequence of the T cell receptor α constant region (TRAC) gene.

[0019] In this regard, this article provides vectors containing polynucleotides, and engineered immune cells (e.g., engineered T cells or CAR T cells) containing the vector or polynucleotides. In some embodiments, the vector is an adeno-associated virus (AAV) vector or a lentiviral vector.

[0020] In some embodiments, immune cells contain polynucleotides integrated into the TRAC gene. In some embodiments, the integration of polynucleotides into the human TRAC gene is mediated by a nuclease. In some embodiments, the nuclease is a CRISPR nuclease. In some embodiments, the nuclease is not TALEN. In some embodiments, the integration of polynucleotides into the human TRAC gene is mediated by a CRISPR nuclease, and said integration prevents or reduces the expression or activity of the TRAC gene. In some embodiments, the CRISPR nuclease is Cas9 or Cas12, such as Cas12i. In some embodiments, the CRISPR nuclease causes a double-strand break, thereby creating an integration site in a target sequence within the TRAC gene, and the polynucleotide integrates into the integration site via AAV-mediated site-specific integration, optionally wherein said integration site is located in the first, second, or third exon of the TRAC gene, and further optionally wherein said target sequence comprises the nucleic acid sequence GACCCTGCC or SEQ ID NO: 92.

[0021] In some embodiments, the immune cell further comprises an additional polynucleotide containing a promoter operatively linked to one or more coding sequences, wherein the one or more coding sequences express a CCR or CD70 binding protein, as described herein. In some embodiments, the one or more coding sequences of the additional polynucleotide further express a PD-1 dominant-negative receptor. In some embodiments, the additional polynucleotide further comprises a 5' homologous arm and a 3' homologous arm. In some embodiments, the 5' homologous arm and the 3' homologous arm are each at least 100 nucleotides in length. In some embodiments, the 5' homologous arm and the 3' homologous arm each contain a nucleotide sequence homologous to the nucleotide sequence of the CD52 gene. In some embodiments, the promoter comprises the EFS promoter as described herein or a truncated PGK promoter. In some embodiments, the additional polynucleotide is integrated into the CD52 gene. In some embodiments, the integration of the additional polynucleotide into the human CD52 gene is mediated by a CRISPR endonuclease. In some embodiments, the CRISPR endonuclease is Cas9 or Cas12, such as Cas12i, and integration inhibits or reduces the expression or activity of the CD52 gene. In some embodiments, the CRISPR endonuclease causes a double-strand break, thereby creating an integration site in a target sequence within the CD52 gene, and the polynucleotide integrates into the integration site via AAV-mediated site-specific integration.

[0022] In some aspects, this disclosure provides an immune cell or immune cell population comprising a polynucleotide integrated into a human TRAC gene, wherein the polynucleotide comprises a nucleic acid sequence comprising the 5' region of the human TRAC gene, a nucleic acid sequence encoding a CD19-specific CAR and a CD70-binding protein or a CD70-specific CAR, and the 3' region of the human TRAC gene, wherein the integrated polynucleotide inhibits or reduces the expression or activity of the human TRAC gene. In some embodiments, the anti-CD19 CAR comprises amino acids of SEQ ID NO: 5 or 7, and the CD70-binding protein (or CD70-specific CAR) comprises amino acids of SEQ ID NO: 19 or 21. In some embodiments, the polynucleotide encodes a CD19-specific CAR and a CD70-specific CAR linked by a P2A sequence. In some embodiments, the polynucleotide integration into the human TRAC gene is mediated by a CRISPR endonuclease, such as Cas9, Cas12, or Cas12i. In some embodiments, a CRISPR endonuclease causes a double-strand break, thereby creating an integration site in a target sequence within the TRAC gene, and the polynucleotide integrates into the integration site via AAV-mediated site-specific integration. In some embodiments, the integration site is located in exon 1, exon 2, or exon 3 of the TRAC gene. In some embodiments, the integration site is located in exon 1 of the TRAC gene. In some embodiments, the target sequence comprises the nucleic acid sequence GACCCTGCC or SEQ ID NO: 92.

[0023] In another aspect, this disclosure provides an immune cell comprising a polynucleotide encoding an anti-CD19 CAR and a CD70 binding protein, wherein the anti-CD19 CAR comprises the amino acid sequence of SEQ ID NO: 5 or 7, and the CD70 binding protein comprises the amino acid sequence of SEQ ID NO: 19 or 21. In some embodiments, the polynucleotide encodes a polypeptide of a CD19-specific CAR and a CD70-specific CAR linked by a P2A peptide, the polypeptide comprising an amino acid sequence that is at least 85%, 88%, 90%, 95%, 98%, 99%, or 100% identical to the polypeptide sequence of SEQ ID NO: 97. In some embodiments, the polynucleotide comprises a nucleic acid sequence that is at least 85%, 88%, 90%, 95%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 96. In some embodiments, the polynucleotide is integrated into the human TRAC gene by a nuclease. In some embodiments, the nuclease is a CRISPR nuclease. In some embodiments, the nuclease is not a TALEN nuclease. In some embodiments, the polynucleotide is integrated into the human TRAC gene via a CRISPR endonuclease, wherein the CRISPR endonuclease causes a double-strand break, thereby creating an integration site in a target sequence within the TRAC gene, and the polynucleotide integrates into the integration site via AAV-mediated site-specific integration. In some embodiments, the integrated polynucleotide inhibits or reduces the expression or activity of the human TRAC gene. In some embodiments, the integration site is located in the first, second, or third exon of the TRAC gene. In some embodiments, the integration site is located in the first exon of the TRAC gene. In some embodiments, the target sequence includes the nucleic acid sequence GACCCTGCC or SEQ ID NO: 92.

[0024] In some implementations, a polynucleotide encoding a CD19 CAR and CD70 binding protein or an anti-CD70 CAR is operatively linked to the truncated PGK promoter disclosed herein.

[0025] In some implementations, the immune cells are T cells, NK cells, NK-T cells, tumor-infiltrating lymphocytes (TILs), or T cells or NK cells derived from iPSCs.

[0026] In a related aspect, an immune cell is provided comprising a recombinant polynucleotide sequence integrated into the human TRAC gene, wherein the recombinant polynucleotide sequence comprises the 5' region of the human TRAC gene, a polynucleotide as described herein, and the 3' region of the human TRAC gene, wherein the integrated recombinant polynucleotide sequence prevents the expression of the human TRAC gene. In some embodiments, the immune cell further comprises an additional recombinant polynucleotide sequence integrated into the human CD52 gene, wherein the additional recombinant polynucleotide sequence comprises the 5' region of the human CD52 gene, one or more coding sequences, and the 3' region of the human CD52 gene.

[0027] In a related aspect, a population of immune cells and a pharmaceutical composition of immune cells or a population of immune cells are provided. In another aspect, a method of treating a disease condition in a subject is provided, comprising administering immune cells, a population of immune cells, and a pharmaceutical composition to the subject, said pharmaceutical composition comprising immune cells, an effective amount of immune cells, an effective amount of a population of immune cells, or an effective amount of the pharmaceutical composition, as described throughout. In some embodiments, the subject is a human being.

[0028] In some implementations, the disease condition is cancer, including but not limited to non-Hodgkin's lymphoma, large B-cell lymphoma (LBCL), follicular lymphoma (FL), T-cell lymphoma (TCL), B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia (TALL), primary central nervous system lymphoma (PCNSL), mantle cell lymphoma (MCL), chronic lymphocytic leukemia (CLL), and peripheral T-cell lymphoma (PTCL). In some implementations, the immune cells are autologous to the subject. In some implementations, the immune cells are allogeneic to the subject.

[0029] In some implementations, the disease condition is an autoimmune disease or condition, including but not limited to lupus, systemic lupus erythematosus (SLE), lupus nephritis, rheumatoid arthritis, systemic sclerosis, scleroderma, multiple sclerosis (MS), relapsing-remitting multiple sclerosis (RRMS), secondary progressive multiple sclerosis (SPMS), primary progressive multiple sclerosis (PPMS), neuromyelitis optica spectrum disorder (NMOSD), myasthenia gravis, Sjogren's syndrome, stiff-person syndrome, graft-versus-host disease (GvHD), autoimmune hemolytic anemia (AIHA), immune thrombocytopenic purpura (ITP), and myositis. In some implementations, the immune cells are capable of reducing or depleting B cells, pathogenic B cells, autoreactive B cells, pathogenic T cells, autoreactive T cells, CD70+ T cells, or allogeneic reactive T cells. In some implementations, the immune cells are autologous to the subject. In other implementations, the immune cells are allogeneic relative to the subject. Attached Figure Description

[0030] Figures 1A to 1C The results of an in vitro long-term killing assay of anti-CD19 CAR T cells with or without co-expression of chimeric cytokine receptors (CCRs) are shown.

[0031] Figures 2A to 2B The results of in vivo cytotoxicity assays of anti-CD19CAR T cells with or without co-expression of CCR (CD19CAR / CCR) are shown.

[0032] Figure 3A The data depicted showed that, compared with CD19 CAR / CCR T cells generated through site-specific integration, anti-CD19 CAR T cells co-expressing CCR (CD19 CAR / CCR T cells) exhibited reduced in vivo anti-tumor activity. Figure 3B The results showed that the number of CAR+ T cells generated via LVV transduction was higher in vivo compared to CAR T cells generated via site-specific integration. Right now It has higher durability.

[0033] Figures 4A to 4D The results of experiments show the effects of different promoters on the expression of CCR and CAR in T cells generated through site-specific integration. Figure 4A The median fluorescence intensity (MFI) of CD19 CAR / CCR T cells at the end of CAR T cell production is shown. Figure 4B The results of CAR T cell killing of target Raji cells in a continuous restimulation assay based on flow cytometry are shown. Figure 4C The CAR MFI during continuous restimulation assays based on flow cytometry is shown. Figure 4D The CAR+ T cell count during this period is shown.

[0034] Figures 5A to 5C The study compared the long-term killing power of CAR / CCR T cells generated through site-specific integration driven by the EFS promoter or the PGK SSI-1 promoter with that of CD19 CAR / CCR T cells generated through LVV transduction. Figure 5A ) or single-stimulation efficacy assay based on flow cytometry ( Figures 5B to 5C The efficacy of the drug on target Raji cells.

[0035] Figures 6A to 6B The in vivo efficacy of CD19 CAR / CCR T cells generated through site-specific integration driven by the EFS promoter, CypA300 promoter, or PGK SSI-1 promoter was compared with that of CD19 CAR / CCR T cells generated via LVV, using two different CAR T cell doses: 2x10 6 Cells ( Figure 6A ) or 4x10 6 Cells ( Figure 6B ).

[0036] Figures 7A to 7H illustrate the interactions between allogeneic CAR T cells, allogeneic reactive host T cells, and tumor cells. The allogeneic CAR T cells either express CD19CAR / CCR and CD70-binding protein (CD70-BP, or CD70 CAR containing only the CD3z signaling domain and CD70-binding domain, CD70z) (CD70z.CD19CAR / CCR) (Figures 7E to 7H) or do not express CD70-binding protein (CD19CAR / CCR) (Figures 7A to 7D). CD19 CAR / CCR T cells expressing CD70-binding protein (Figure 7E) effectively reduced host T cells (Figure 7F), killed tumor cells (Figure 7G), and exhibited greater CAR T cell expansion than CD19 CAR / CCR T cells not expressing CD70-binding protein (Figure 7H).

[0037] Figures 8A to 8D This indicates that CD19 CAR / CCR T cells co-expressing CD70 binding protein (CD70z.CD19CAR / CCR) prevent the escape of CD19 antigen from Raji cells. Figure 8BThe results showed that after four days of co-culturing Raji cells with CAR T cells, CD19 expression was downregulated, and CD19-negative / CD70 wild-type Raji cells were eliminated by CD19 CAR / CCR T cells co-expressing CD70 binding protein. Figure 8C and Figure 8D The absolute number of total Raji cells (CD70 wild-type or CD70 knockout (KO)) and the absolute number of CD19-negative Raji cells (CD70 wild-type or CD70 KO) are shown separately. In vivo data are presented in... Figures 8E to 8F As shown in the image.

[0038] Figure 9A This study demonstrates the interaction between CD19 CAR / CCR T cells co-expressing CD70 binding protein and cells expressing only CD19 CAR / CCR or only CD70 binding protein. Figure 9B Flow cytometry data were presented to show that CD19 CAR / CCR T cells co-expressing CD70 binding protein may cause CD19 CAR / CCR single-positive T cells to self-kill, and that CD70 binding protein cis-binding to surface CD70 protein (cis-masking) protects CD70 binding protein single-positive T cells from self-killing from days 9 to 14 of the production process.

[0039] Figure 10A The design of different CD19 CAR constructs is described. Figures 10B to 10I The results of CAR T cell generation and characterization are shown.

[0040] Figures 11A to 11B The results show the survival of CAR T cell transplantation against allogeneic reactive T cells in a mixed lymphocyte response (MLR) assay.

[0041] Figures 12A to 12D The study depicted the effect of CAR T cells on CD70 wild-type Raji cells ( Figure 12A ) or CD70 KO Raji cells ( Figure 12C ) cytotoxicity data, and CAR T cell expansion data (respectively) Figure 12B and Figure 12D ).

[0042] Figures 13A to 13B The data show the long-term killing effect of CAR T cells on CD70 KO Raji cells.

[0043] Figures 14A to 14B Average data are presented to demonstrate the in vivo antitumor activity of CAR T cells with different designs at two different CAR T cell doses. Figures 14C to 14G It shows the application of 5x106 Data from individual mice using CAR T cells with different CAR designs.

[0044] Figures 15A to 15G Data on the generation and characterization of CD19 / CD70 dual CAR T cells or PGK SSI CD19 CAR T cells engineered using CRISPR gene editing reagents are presented. NTD: Untransduced control T cells. Figures 15H to 15K Data on T cell phenotypes in CAR+ T cell populations (PGK SSI CD19 / CD70 dual, PGK SSI CD19) or untransduced T cell populations (NTD) are presented, with data gated to CAR+ cells.

[0045] Figures 16A to 16B Cytotoxicity data of CD19 / CD70 dual CAR T cells engineered with CRISPR reagents in cells from two different donors against CD19+ Raji cells are presented. In a short-term killing assay, the cytotoxic activity of the CAR T cells against each target CD19 and CD70 was independently tested. Figure 16C ), using wild-type Raji RGL cells (RGL) or CD19KO or CD70KO Raji RGL cells ( Figure 16D This was demonstrated in a mixed tumor model of CD19KO RGL and CD70KO RGL cells, where the cytotoxic activity of CAR T cells against each target CD19 and CD70 was tested. Figure 16E ),and Figure 16F The results showed that CD19-negative RGL cells could only expand when treated with control CD19 CAR T cells, but could not expand when treated with CD19 / CD70 dual CAR T cells. Figure 16G and Figure 16H The results of in vivo killing of CD19+ tumor cells by CD19 / CD70 dual CAR T cells and the results of in vivo expansion of dual CAR T cells are shown in an animal model. Figure 16I The study showed that CD19 / CD70 dual CAR T cells did not expand in cell culture in the absence of IL-2.

[0046] Figures 17A to 17B Data from the study showed that CD19 / CD70 dual CAR T cells effectively targeted primary CD19+ B cells from healthy donor PBMCs or PBMCs from SLE patients.

[0047] Figures 18A to 18BThe data shown indicate that CD19 / CD70 dual CAR T cells effectively target allogeneic reactive CD70+ primary T cells from healthy or SLE donors in mixed lymphocyte responses (MLR).

[0048] Figure 19A Experimental protocols for establishing in vivo humanized mouse models of PBMCs under different conditions are described. Figure 19B This demonstrates evidence of successful human B cell and T cell transplantation, and Figure 19C The expression level of CD70 on transplanted cells is shown.

[0049] Figure 20A The experimental protocol for establishing a humanized PBMC mouse model under irradiation conditions and for conducting kinetic analysis of B cell remodeling and differentiation is shown. Figures 20B to 20D Data demonstrating the proliferation and differentiation of transplanted B cells in a mouse model are presented. Figures 20E to 20F Data demonstrating the dose-dependent generation of human IgG and IgM on PBMCs are presented.

[0050] Figure 21A An experimental protocol for testing the in vivo efficacy of CD19 / CD70 dual CAR in a PBMC humanized mouse model is presented. Figures 21B to 21C Data on the exhaustion of host CD19+ B cells were shown. Figure 21B and Figure 21C The above figure) and data on the reduction in host antibody production ( Figure 21C (See the image below), and Figure 21D The study showed a reduction in host T cells in the spleen of mice, all of which occurred 7 days after CAR T treatment (i.e., 10 days after human PBMC transfer). Figure 21E This demonstrates the clearance of CD70+ T cells in the spleen of mice 7 days after CAR T therapy, and Figure 21F The results of CAR T treatment at 0, 4, and 7 days (shown) Right now The number of CD70+ T cells in the spleen of mice decreased on days 3, 7 and 10. Figures 21G to 21H The mouse spleen is shown. Figure 21G and Figure 21H (left image) or blood ( Figure 21H (Right figure) Dose-dependent expansion of CD19 / CD70 dual CAR T cells.

[0051] Figure 22A The experimental protocol for the CD34+ humanized NSG mouse model was described. Figure 22B and Figure 22C The figure above shows that on day 7 after CAR T-cell therapy, CD19 / CD70 dual CAR T-cell therapy depleted human B cells in CD34+ humanized mice, and Figure 22C The figure below shows that on day 7 after CAR T treatment, CAR T treatment had little effect on human T cells in mice. Figure 22D Data on CAR T amplification on day 7 post-treatment are shown, and Figure 22E The kinetics of CAR T amplification are shown.

[0052] Figure 23A It is a chart showing the assay of virus-specific T cells. Figures 23B to 23C The results show that CD19 / CD70 dual T cells do not affect the clearance of virus-specific T (VST) cells from viral peptide-presenting antigen-presenting cells, nor do they affect VST cell counts.

[0053] Figure 24A As shown in Figure B, ruxolitinib inhibits the cytotoxic activity of CD19 / CD70 dual CAR T cells against target CD19+CD70 KORaji cells. Figure 24A ) and the proliferation of CD19 / CD70 dual CAR T cells ( Figure 24B ). Detailed Implementation

[0054] Unless otherwise specified, the practice of this disclosure will employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the scope of the art. These techniques are well explained in the literature, such as *Molecular Cloning: A Laboratory Manual, 2nd Edition* (Sambrook et al., 1989), Cold Spring Harbor Press; *Oligonucleotide Synthesis* (MJ Gait, ed., 1984); *Methods in Molecular Biology*, Humana Press; *Cell Biology: A Laboratory Notebook* (JE Cellis, ed., 1998), Academic Press; *Animal Cell Culture* (RI Freshney, ed., 1987); *Introduction to Cell and Tissue Culture* (JP Mather and PE Roberts, 1998), Plenum Press; *Cell and Tissue Culture: Laboratory Procedures* (A. Doyle, JB Griffiths, and DG Newell, ed., 1993–1998), J. Wiley and Sons; *Methods in Enzymology* (Academic Press, Inc.); and *Handbook of Experimental Immunology* (DM Weir and CC). Blackwell (ed.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos (ed., 1987); Current Protocols in Molecular Biology (FM Ausubel et al. (ed., 1987); PCR: The Polymerase Chain Reaction (Mullis et al. (ed., 1994); Current Protocols in Immunology (JE)Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1991); (1995). For example, gene editing techniques using TALENs, CRISPR / Cas9, Cas12, and megaTAL nucleases are within the scope of the art and are well explained in the literature, such as T. Gaj et al. people , Genome-EditingTechnologies: Principles and Applications, Cold Spring Harb Perspective Biol 2016;8:a023754 and its citations.

[0055] As used herein, the terms “a” and “an” are used to mean one or more. For example, references to “cell,” “immune cell,” or “antibody” mean “one or more cells,” “one or more immune cells,” or “one or more antibodies.”

[0056] As used herein, “autologous” means that the cells, cell lines, or cell populations used to treat the subject are obtained from the subject.

[0057] As used herein, "allogeneic" means that the cells or cell populations used to treat the subject were not obtained from the subject, but from a donor.

[0058] As used herein, the term “endogenous” means any material that originates from or is produced within an organism, cell, tissue, or system.

[0059] As used herein, the term "exogenous" means any material introduced or generated from outside an organism, cell, tissue, or system. In some embodiments, the exogenous or recombinant sequence or protein is not a naturally occurring sequence or protein and is not endogenous or native to the cell, tissue, or organism.

[0060] As used herein, “immune cells” refers to hematopoietic cells that are functionally involved in the initiation and / or execution of innate and / or adaptive immune responses. Examples of immune cells include T cells (e.g., α / β T cells and γ / δ T cells, regulatory T (Treg) cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes.

[0061] As used herein, the term “expression” refers to the transcription and / or translation of a specific nucleotide sequence driven by a promoter.

[0062] As used herein, “expression vector” means a vector containing a recombinant polynucleotide that includes an expression control sequence operatively linked to a nucleotide sequence to be expressed. Expression vectors include all those known in the art, including but not limited to visceral particles, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating recombinant polynucleotides.

[0063] As used herein, an "expression cassette" refers to one or more expression units of one or more coding sequences operatively linked to a promoter. In some embodiments, the expression of multiple coding sequences may be driven by a single promoter, and more than one coding sequence may be linked by a sequence encoding a 2A peptide (e.g., P2A or T2A). In some embodiments, the expression of multiple coding sequences is carried out via ribosome jumping. As an example, a bicistronic expression cassette allows the expression of two proteins from the same RNA transcript driven by a single promoter. In some embodiments, the expression of multiple coding sequences may be driven by multiple promoters, each operatively linked to a coding sequence.

[0064] As used herein, a "functionally expressed" gene means a gene that is expressed and that expression produces a functional genetic end product. For example, if a gene encodes a protein, then the cell functionally expresses the gene if the expression of the gene ultimately produces a protein with an appropriate function. Therefore, for example, if a gene is not transcribed, or if the expression of the gene ultimately produces untranslated or non-functional RNA (e.g., the protein is not properly folded or is not transported to its site of action (e.g., the membrane, for membrane-bound proteins)), then the gene is not functionally expressed. Functional expression can be measured directly (e.g., by measuring the gene product itself) or indirectly (e.g., by measuring the function of the gene product).

[0065] As used herein, "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment or molecule such that the function of one nucleic acid sequence is influenced by another. For example, a promoter is operably linked to a coding sequence when it can influence the expression of that sequence (i.e., the coding sequence is under the transcriptional control of the promoter). The first sequence element can be operably linked to the second sequence element in a contiguous or discontinuous manner. When two nucleic acid elements are operably linked, their coding regions remain within the same correct reading frame.

[0066] As used herein, “expression control sequence” refers to the nucleic acid sequence that directs nucleic acid transcription. The expression control sequence can be a promoter (such as a constitutive or inducible promoter), an enhancer, or any other transcriptional regulatory element. The expression control sequence is operatively linked to the nucleic acid sequence to be transcribed.

[0067] The terms "promoter" and "promoter sequence" are used interchangeably and refer to the DNA sequence that controls the expression of a coding sequence or functional RNA. Functional RNA can be a protein-coding mRNA transcript or a non-mRNA, such as miRNA, shRNA, or other types of interfering RNA. Generally, the coding sequence is located at the 3' end of the promoter sequence. Those skilled in the art will understand that different promoters can direct gene expression in different tissues or cell types, at different developmental stages, or in response to different environmental or physiological conditions.

[0068] The terms “DNA sequence,” “nucleic acid sequence,” “nucleotide sequence,” and “polynucleotide sequence” used herein are interchangeable. Similarly, the terms “polynucleotide,” “DNA molecule,” and “nucleic acid molecule” used herein are interchangeable.

[0069] As used herein, the term "extracellular ligand-binding domain" refers to an oligopeptide or polypeptide capable of binding ligands. Preferably, the domain will be able to interact with cell surface molecules. For example, an extracellular ligand-binding domain may be selected to recognize ligands used as cell surface markers on target cells associated with specific disease states. The terms "stalk domain" or "hinge domain" are used herein to refer to any oligopeptide or polypeptide that acts to link a transmembrane domain to an extracellular ligand-binding domain. Specifically, stalk or hinge domains are used to provide greater flexibility and accessibility to the extracellular ligand-binding domain.

[0070] The term "intracellular signal transduction domain" refers to the part of a protein that transduces effector signals and functional signals, guiding the cell to perform specialized functions.

[0071] When this document refers to a value or parameter “about”, it includes (and describes) embodiments involving adding or subtracting 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% from that value or parameter itself. For example, a description of “about X” includes a description of “X”. Numerical ranges include numbers that define the range.

[0072] It should be understood that wherever the implementation is described in the language "includes", similar implementations are also provided for other aspects described in the terms "comprises of" and / or "substantially constitutes of".

[0073] This article provides engineered immune cells, such as engineered T cells, that contain and / or express anti-CD19 or CD19-specific chimeric antigen receptor (CAR) and CD70-binding protein, and optionally contain and / or express chimeric cytokine receptor (CCR). This article also provides engineered immune cells (e.g., engineered T cells) that contain and / or express anti-CD19 (or CD19-specific) chimeric antigen receptor (CAR) and chimeric cytokine receptor (CCR), and optionally contain and / or express CD70-binding protein.

[0074] This document further provides polynucleotides comprising one or more coding sequences encoding CD19-specific CARs and CD70-binding proteins and / or CCRs. In some embodiments, the CD19-specific CARs and CD70-binding proteins or CCRs are expressed by bicistronic expression cassettes and linked via P2A or T2A self-cleaving peptides. In some aspects, CD19-specific immune cells (e.g., CD19-specific CAR T cells) exhibit enhanced cytotoxicity and potency against CD19-positive and / or CD19-negative / CD70-positive hematologic malignancies or other pathogenic cells compared to the same CD19-specific immune cells (e.g., CD19-specific CAR T cells) that do not express CD70-binding proteins and / or CCRs, with reduced rejection by host or patient allogeneic reactive immune cells and / or increased persistence and cell proliferation. In some embodiments, the expression of one or more coding sequences or bicistronic expression cassettes is driven by a PGK promoter, particularly a truncated PGK promoter. In some implementations, CD19-specific immune cells, such as CD19-specific CAR T cells, exhibit enhanced cytotoxicity and potency against CD19-positive pathological or pathogenic cells associated with autoimmune indications.

[0075] In some aspects, the polynucleotides provided herein are cloned into lentiviral vectors (LVVs) and introduced into engineered immune cells, such as peripheral blood mononuclear cells (PBMCs) or T cells, via lentiviral transduction. Transducing the LVV construct into, for example, PBMCs or T cells produces engineered cells with randomly integrated transgenes present in the host cell genome. Alternatively, transgenes can be introduced into cells via site-specific integration (SSI) into one or more predetermined genetic loci. Another benefit of site-specific integration compared to LVV-guided random integration is ensuring consistency of the insertion site of the transgene in the genome and reducing the number of integration events. Therefore, in some aspects, the polynucleotides provided herein are integrated into predetermined loci in the genome of engineered immune cells, such as CD19-specific CAR T cells, CD19-specific CAR T cells expressing CD70 binding protein or CD70 CAR (or CD19 / CD70 dual CAR T cells). In some embodiments, the predetermined genetic locus is the T cell receptor α chain constant region (TRAC) locus. In some embodiments, the predetermined genetic locus is the CD52 locus.

[0076] Surprisingly, PGK promoters, or truncated PGK promoters as described herein (e.g., PGK SSI promoters), have been shown to provide benefits to engineered immune cells (e.g., CAR T cells), such as enhanced CAR T cell expansion and / or enhanced in vitro or in vivo cytotoxicity against target cells, including target tumor cells, when the transgene is introduced via site-specific integration. Therefore, in some embodiments, the expression of a transgene (e.g., one or more coding sequences) is driven by a PGK promoter, particularly a truncated PGK promoter as described herein. In some embodiments, one or more coding sequences are present in a bicistronic or polycistronic expression cassette. In some embodiments, the transgene includes, for example, CAR, CCR, CD70 binding protein, dominant or negative receptor, or other sequences or sequences encoding other proteins or recombinant proteins.

[0077] In some embodiments, engineered immune cells generated through site-specific integration exhibit surprisingly improved in vivo cytotoxic activity against target pathogenic cells (including tumor cells) when the expression of a transgene (e.g., a CD19-specific CAR) is driven by a PGK promoter (particularly a truncated PGK promoter, such as the PGKSSI promoter described herein), compared to, for example, an EF1α short (EFS) promoter. In some embodiments, engineered immune cells generated through site-specific integration exhibit surprisingly improved in vivo cytotoxic activity against target pathogenic cells (including tumor cells) when the expression of a transgene (e.g., CD19-specific CAR and CCR) is driven by a PGK promoter (such as the PGKSSI promoter described herein), compared to an EFS promoter. In some embodiments, engineered immune cells generated through site-specific integration exhibit surprisingly improved in vivo antitumor activity against target pathogenic cells (including tumor cells) when the expression of a transgene (e.g., a CD19-specific CAR and CD70-binding protein or a CD70-specific CAR) is driven by a PGK promoter (such as the PGKSSI promoter described herein), compared to an EFS promoter. In some embodiments, engineered immune cells generated through site-specific integration exhibit surprisingly improved in vivo cytotoxic activity compared to EFS promoters when the expression of transgenes (e.g., CD19-specific CARs and CCRs and / or CD70-binding proteins or CD70-specific CARs) is driven by a PGK promoter (e.g., the PGK SSI promoter described herein). In some embodiments, the polynucleotide is integrated into the TRAC locus. In some embodiments, the polynucleotide is integrated into the CD52 locus.

[0078] PGK promoter In one aspect, the present invention provides polynucleotide or nucleic acid constructs or molecules comprising one or more coding sequences or transgenes. In some embodiments, the one or more coding sequences or transgenes encode polypeptides, such as CARs or other recombinant proteins, and / or nucleic acid inhibitors, such as RNA interference agents. In some embodiments, the one or more coding sequences or transgenes are driven by a truncated PGK promoter to control the expression of one or more coding sequences or transgenes. In other embodiments, the truncated PGK promoter comprises one or more deletions in a promoter sequence from a corresponding promoter sequence of a genomic region containing a PGK gene (e.g., the human PGK gene). Table 1 provides exemplary PGK promoter sequences.

[0079] Table 1 – Promoters ( Bold, underlined text (Indicates the transcription start site)

[0080] In one embodiment, the PGK promoter is derived from a genomic region containing the PGK gene. For example, the PGK promoter may be derived from a genomic region containing the PGK gene in the major assembly of Homo sapiens X chromosome GRCh38.p14 (NCBI reference sequence: NC_000023.11). In another embodiment, the PGK promoter is derived from a polynucleotide sequence corresponding to approximately 78,103,669 to 78,104,334 nucleotide positions in the genomic region containing the PGK gene in the major assembly of Homo sapiens X chromosome GRCh38.p14 (NCBI reference sequence: NC_000023.11).

[0081] Additionally, the truncated PGK promoters described herein contain one or more nucleic acid sequences that bind to or are predicted to bind to one or more transcription factors. Alternatively, the truncated PGK promoters described herein may a) lack one or more nucleic acid sequences that bind to or are predicted to bind to one or more transcription factors, or b) be characterized by the absence of one or more nucleic acid sequences that bind to or are predicted to bind to one or more transcription factors. The promoter sequences described herein can be analyzed using a transcription factor binding site prediction database called JASPAR. JASPAR is an open-access database that stores an artificially compiled transcription factor (TF) binding spectrum in the form of a position frequency matrix (PFM), which summarizes the occurrence of each nucleotide at each position in an observed set of TF-DNA interactions. PFM can be used to scan any DNA sequence to predict TF binding sites.

[0082] In one embodiment, the truncated PGK promoter comprises a nucleic acid sequence that binds to or is predicted to bind to a transcription factor selected from the group consisting of: ZNF692, ZNF257, YY1, TEAD4, TEAD1, TCF4, TCF3, TBX3, SP1, SOX18, RFX7, MYOG, MYF5, KLF9, KLF6, KLF4, KLF10, HIF1, FIGLA, DUXA, DUX4, CTCFL, BHLHE22, BHLHA15, ASCL1, and ARNT::HIF1A. In another embodiment, the truncated PGK promoter comprises the nucleotide sequence of SEQ ID NO: 35, or has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 35.

[0083] In another embodiment, the truncated PGK promoter is characterized by the absence of a nucleic acid sequence that binds to or is predicted to bind to a transcription factor selected from the following groups: ZNF281, ZNF148, VEZF1, TLX2, TFAP2E, TFAP2C, TFAP2B, TFAP2A, SP8, SOX15, RELB, RBPJ, PRDM1, PITX2, NKX3-2, NKX2-8, NKX2-3, NFKB1, NFATC2, MSX2, MEIS1, KLF16, KLF15 The sequence numbers are: KLF11, ISL2, HOXD4, HOXD12, HOXD11, HOXD10, HOXC9, HOXC4, HOXC12, HOXC11, HOXC10, HOXB9, HOXB7, HOXB4, HOXA9, HOXA4, HOXA1, GSX2, FOXD2, FOXC1, EN2, EGR1, EBF1, E2F8, E2F6, E2F4, E2F1, DRGX, CEBPE, CEBPD, CEBPB, BARHL2, and BARHL1. In another embodiment, the truncated PGK promoter comprises the nucleotide sequence of SEQ ID NO: 35, or has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 35.

[0084] In one implementation, the truncated PGK promoter comprises a nucleic acid sequence that binds to or is predicted to bind to a transcription factor selected from the group consisting of: ZNF692, ZNF257, YY1, TEAD4, TEAD1, TCF4, TCF3, TBX3, SPI1, SP1, SOX18, SOX10, RUNX2, RFX7, PLAGL2, OSR2, OSR1, MYOG, MYF5, KLF9, KLF6, KLF4, KLF10, IKZF1, HSF4, HIF1A, FLI1, FIGLA, FEV, ETV5, ETV4, ETS2, ERG, ELK4, ELK1, ELF5, ELF1, DUXA, DUX4, CTCFL, BHLHE22, BHLHA15, ASCL1, and ARNT::HIF1A. In another embodiment, the truncated PGK promoter contains the nucleotide sequence of SEQ ID NO: 36, or has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 36.

[0085] In another embodiment, the truncated PGK promoter is characterized by the absence of a nucleic acid sequence that binds to or is predicted to bind to a transcription factor selected from the following groups: ZNF281, ZNF148, VEZF1, TLX2, TFAP2C, TFAP2B, TFAP2A, SP8, SOX15, RELB, RBPJ, PRDM1, PITX2, NKX3-2, NKX2-8, NKX2-3, NFKB1, NFATC2, M SX2, KLF16, KLF15, KLF11, ISL2, HOXD4, HOXD12, HOXD11, HOXD10, HOXC9, HOXC4, HOXC12, HOXC11, HOXC10, HOXB9, HOXB7, HOXB4, HOXA9, HOXA4, HOXA1, GSX2, FOXD2, FOXC1, EN2, EGR1, EBF1, E2F8, E2F6, E2F4, E2F1, DRGX, CEBPE, CEBPD, CEBPB, BARHL2, and BARHL1. In another embodiment, the truncated PGK promoter comprises the nucleotide sequence of SEQ ID NO: 36, or has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 36.

[0086] In one implementation, the truncated PGK promoter contains a nucleic acid sequence that binds to or is predicted to bind to a transcription factor selected from the group consisting of: ZNF692, ZNF257, YY1, TEAD4, TEAD1, TCF4, TCF3, TBX3, STAT3, STAT1, SPI1, SPDEF, SP1, SOX18, SOX10, RUNX2, RHOXF1, RFX7, PLAGL2, OSR2, OSR1, MYOG, MYF5, KLF9, KLF6, KLF4, KLF3, KLF10, IKZF1, HSF4, HIF1A, FLI1, FIGLA, FEV, ETV5, ETV4, ETS2, ERG, ELK4, ELK1, ELF5, ELF1, DUXA, DUX4, CTCFL, CREB1, BHLHE22, BHLHA15, ASCL1, and ARNT::HIF1A. In another embodiment, the truncated PGK promoter contains the nucleotide sequence of SEQ ID NO: 37, or has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 37.

[0087] In another embodiment, the truncated PGK promoter is characterized by the absence of a nucleic acid sequence that binds to or is predicted to bind to a transcription factor selected from the following groups: ZNF281, ZNF148, VEZF1, TLX2, TFAP2C, TFAP2B, TFAP2A, SOX15, RELB, RBPJ, PRDM1, PITX2, NKX3-2, NKX2-8, NKX2-3, NFKB1, NFATC2, MSX2, KLF16, KLF15, ISL2. HOXD4, HOXD12, HOXD11, HOXD10, HOXC9, HOXC4, HOXC12, HOXC11, HOXC10, HOXB9, HOXB7, HOXB4, HOXA9, HOXA4, HOXA1, GSX2, FOXD2, FOXC1, EN2, EGR1, EBF1, E2F8, E2F6, E2F4, E2F1, DRGX, CEBPE, CEBPD, CEBPB, BARHL2, and BARHL1. In another embodiment, the truncated PGK promoter comprises the nucleotide sequence of SEQ ID NO: 37, or has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 37.

[0088] In one embodiment, the truncated PGK promoter includes one or more deletions, insertions, or substitutions in the PGK promoter, as shown in SEQ ID NO: 34. In some embodiments, one or more deletions in the truncated PGK promoter include one or more 5' region deletions and / or one or more 3' region deletions. In other embodiments, the 5' region deletion includes deletions from SEQ ID NO: The 5' end of 34 is missing approximately 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, and 345. The deletion of approximately 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, or approximately 390 nucleotides, and related to SEQ The nucleotide sequence of SEQ ID NO: 35 has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity. In some embodiments, the truncated PGK promoter comprises, is substantially composed of, or is composed of the nucleotide sequence of SEQ ID NO: 35.

[0089] In other embodiments, the 5' region deletion includes deletions of approximately 210, approximately 211, approximately 212, approximately 213, approximately 214, approximately 215, approximately 216, approximately 217, approximately 218, approximately 219, approximately 220, approximately 221, approximately 222, approximately 223, approximately 224, approximately 225, approximately 226, approximately 227, approximately 228, approximately 229, approximately 230, approximately 231, approximately 232, approximately 233, approximately 234, approximately 235, approximately 236, approximately 237, approximately 238, approximately 239, approximately 240, approximately 241, approximately 242, approximately 243, approximately 244, approximately 245, approximately 246, approximately 247, approximately 248, approximately 249, and approximately 250 from the 5' end of SEQ ID NO: 34. The deletion of approximately 251, approximately 252, approximately 253, approximately 254, approximately 255, approximately 256, approximately 257, approximately 258, approximately 259, approximately 260, approximately 261, approximately 262, approximately 263, approximately 264, approximately 265, approximately 266, approximately 267, approximately 268, approximately 269, approximately 270, approximately 271, approximately 272, approximately 273, approximately 274, approximately 275, approximately 276, approximately 277, approximately 278, approximately 279, approximately 280, approximately 281, approximately 282, approximately 283, approximately 284, approximately 285, approximately 286, approximately 287, approximately 288, approximately 289, or approximately 290 nucleotides, and related to SEQ The nucleotide sequence of SEQ ID NO: 36 has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity. In some embodiments, the truncated PGK promoter comprises, is substantially composed of, or is composed of the nucleotide sequence of SEQ ID NO: 36.

[0090] In other implementations, the 5' region deletion includes from SEQ ID NO: The 5' end of 34 is missing approximately 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155. The deletion of approximately 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or approximately 200 nucleotides, and related to SEQ The nucleotide sequence of SEQ ID NO: 37 has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity. In some embodiments, the truncated PGK promoter comprises, is substantially composed of, or is composed of the nucleotide sequence of SEQ ID NO: 37.

[0091] In other embodiments, the 5' region deletion includes the deletion of about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, or about 100 nucleotides from the 5' end of SEQ ID NO: The nucleotide sequence of SEQ ID NO: 38 has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity. In some embodiments, the truncated PGK promoter comprises, is substantially composed of, or is composed of the nucleotide sequence of SEQ ID NO: 38.

[0092] In some embodiments, the truncated PGK promoter is substantially composed of or constitutes a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the nucleotide sequence of SEQ ID NO: 35, 36, 37, or 38.

[0093] On the other hand, the polynucleotide, nucleic acid constructs, or molecules described herein contain a donor template. The donor template may contain one or more transgenic or coding sequences. In other embodiments, the donor template contains a promoter, such as a truncated PGK promoter, to control the expression of one or more transgenic or coding sequences.

[0094] In other embodiments, the polynucleotide, nucleic acid construct, or molecule includes a donor template that can be integrated into a double-strand break site in the host cell genome via homologous recombination. In one embodiment, the donor template includes one or more, at least two, or more coding sequences or transgenes. In one embodiment, the expression of one or more, at least two, or more coding sequences or transgenes is controlled by a PGK promoter (e.g., a truncated PGK promoter).

[0095] In other embodiments, the polynucleotide, nucleic acid construct, or molecule further comprises one or more homologous sequences that are homologous to a portion of the integration site in the engineered immune cell genome (for site-specific integration). In one embodiment, the integration site is a gene (target site or target gene) involved in the function or activity of T cell receptor (TCR) αβ. In another embodiment, the gene is the TCR α gene constant region (TRAC) gene. In yet another embodiment, the integration site is located in the TRAC (a component of TCR), HLA, TCRα, TCRβ, β2-microglobulin (“β2m”), CD52, GR, deoxycytidine kinase (DCK), PD-1, and CTLA-4 genes.

[0096] In other embodiments, the polynucleotide, nucleic acid construct, or molecule includes a 5' homologous arm having a sequence homologous to the 5' sequence of a double-strand break located at the integration site of the genome. In another embodiment, the polynucleotide, nucleic acid construct, or molecule further includes a 3' homologous arm having a sequence homologous to the 3' sequence of a double-strand break located at the integration site of the host genome. Nucleotide sequence homology exists in upstream and downstream flanking regions of the double-strand break site and the genome integration site, and the nucleic acid sequence to be introduced (e.g., one or more transgenes) is located between two homologous arms. In some embodiments, in the context of site-specific integration, the terms 5' homologous arm and 3' homologous arm, as well as the terms upstream and downstream flanking regions, are used to refer to the orientation of the target site or target gene for genome integration.

[0097] In some implementations, double-strand breaks can be generated by rare cleaving endonucleases, such as, but not limited to, zinc finger endonucleases, TALE (transcription activator-like effector) nucleases (TALEN), or CRISPR. Polynucleotides, nucleic acid constructs, or molecules can be integrated into the desired integration site of the genome via homologous recombination between the 5' and 3' homologous arms and between homologous sequences at the integration site. Exemplary 5' and 3' homologous sequences are provided in Table 3, for example, SEQ ID NO: 42-45.

[0098] In one embodiment, the polynucleotide, nucleic acid construct, or molecule comprises a donor template including a 5' homologous arm, a 3' homologous arm, a transgene, and a promoter, such as a truncated PGK promoter. In other embodiments, the polynucleotide, nucleic acid construct, or molecule comprises a 5' homologous arm, one or more coding sequences or transgenes, a promoter (such as a truncated PGK promoter), and a 3' homologous arm.

[0099] In some implementations, the polynucleotide, nucleic acid construct, or molecule includes a donor template comprising a 5' homologous arm, a 3' homologous arm, one or more coding sequences or transgenes, and a promoter, such as a truncated PGK promoter.

[0100] In some embodiments, the orientation of one or more coding sequences or transgenes is the same as the orientation of the target site for genome integration. In some embodiments, the polynucleotide, nucleic acid construct, or molecule includes a donor template comprising a 5' homologous arm, a promoter, one or more coding sequences or transgenes, and a 3' homologous arm. In some embodiments, the promoter comprises a truncated PGK promoter, such as the PGK SSI-1, PGK SSI-2, or PGK SSI-3 promoters as described herein.

[0101] In some embodiments, the orientation of one or more coding sequences or transgenes is opposite to the orientation of the target site for genome integration. In some embodiments, the polynucleotide, nucleic acid construct, or molecule includes a donor template comprising a 5' homologous arm, one or more coding sequences or transgenes, a promoter, and a 3' homologous arm. In some embodiments, the promoter comprises a truncated PGK promoter, such as the PGK SSI-1, PGK SSI-2, or PGK SSI-3 promoters as described herein.

[0102] The polynucleotide may contain a native sequence (e.g., an endogenous sequence or endogenous promoter sequence encoding an antibody or a portion thereof) or a variant of such a sequence. Polynucleotide variants contain one or more substitutions, additions, deletions, and / or insertions such that the activity of the encoded polypeptide is not reduced relative to the native molecule. As described herein, the effect on the immunoreactivity of the encoded polypeptide can generally be assessed. The variant preferably has at least about 70% identity with the polynucleotide sequence, more preferably, at least about 80% identity, even more preferably, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, and most preferably, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or about 99% identity.

[0103] Two polynucleotide or polypeptide sequences are said to be "identical" if the nucleotide or amino acid sequences are identical when best aligned as described below. Typically, comparisons between two sequences are performed by comparing sequences within a comparison window to identify and compare local regions of sequence similarity. As used herein, a "comparison window" refers to a segment of at least about 20 consecutive positions, typically 30 to about 75 positions, or 40 to about 50 positions, within which a sequence can be compared to a reference sequence having the same number of consecutive positions, provided that the two sequences have been best aligned.

[0104] Using the Megalign program in the Lasergene Bioinformatics Software Suite (DNASTAR, Inc., Madison, Wl), with default parameters, optimal sequence alignment can be performed for comparison. This procedure embodies several alignment schemes described in the following references: Dayhoff, MO, 1978, A model of evolutionary change in proteins - Matrices for detecting distant relationships. In Dayhoff, MO (edited) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC, Vol. 5, Supplement 3, pp. 345-358; Hein J., 1990, Unified Approach to Alignment and Phylogenes, pp. 626-645; Methods in Enzymology, Vol. 183, Academic Press, Inc., San Diego, CA; Higgins, DG and Sharp, PM, 1989, CABIOS 5:151-153; Myers, EW and Muller W., 1988, CABIOS 4:11-17; Robinson, ED, 1971, Comb. Theor. 11:105; Santou, N., Nes, M., 1987, Mol. Biol. Evol. 4:406-425; Sneath, PHA and Sokal, RR, 1973, Numerical Taxonomythe Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, WJ and Lipman, DJ, 1983, Proc. Natl. Acad. Sci. USA80:726-730.

[0105] In some implementations, the "sequence identity percentage" is determined by comparing two best-aligned sequences within a comparison window of at least 20 positions. The portion of the polynucleotide or polypeptide sequence in the comparison window may contain 20% or less, typically 5% to 15% or 10% to 12%, of additions or deletions (i.e., vacancies) compared to a reference sequence (excluding additions or deletions) for best-alignment of the two sequences. The percentage is calculated as follows: the number of positions in both sequences where identical nucleic acid residues or amino acid bases are present is determined to produce the number of matching positions; the number of matching positions is divided by the total number of positions in the reference sequence (i.e., the window size); and the result is multiplied by 100 to produce the sequence identity percentage.

[0106] Variants can also, or alternatively, be substantially homologous to the natural gene or a portion or complement thereof. Such polynucleotide variants are capable of hybridizing with naturally occurring DNA sequences encoding natural antibodies (or complementary sequences) under moderately stringent conditions.

[0107] Suitable “moderately stringent conditions” include: prewashing in a solution of 5X SSC, 0.5% SDS, and 1.0 mM EDTA (pH 8.0); hybridization overnight at 50-65°C with 5X SSC; followed by washing twice at 65°C with 2X, 0.5X, and 0.2X SSC containing 0.1% SDS, for 20 minutes each time.

[0108] The “highly stringent conditions” or “highly stringent conditions” used in this article are: (1) washing with low ionic strength and high temperature, for example, 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50°C; (2) using denaturing agents such as formamide during hybridization, for example, 50% (v / v) formamide, 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer (pH 6.5) containing 750 mM sodium chloride and 75 mM sodium citrate at 42°C; or (3) using salmon sperm DNA (50 mM sodium phosphate) treated with 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt's solution, and sonication at 42°C. The sample was washed with 0.2×SSC (sodium chloride / sodium citrate) at 42°C, followed by washing with 50% formamide at 55°C, and then a high-rigidity wash with 0.1×SSC containing EDTA at 55°C. Skilled technicians will know how to adjust the temperature, ionic strength, etc., as needed to accommodate factors such as probe length.

[0109] Those skilled in the art will understand that, due to the degeneracy of the genetic code, there are many nucleotide sequences that encode polypeptides as described herein. Some of these polynucleotides have very low homology to the nucleotide sequences of any natural gene. However, this disclosure specifically considers polynucleotides that differ due to codon usage. Furthermore, alleles of genes containing the polynucleotide sequences provided herein are within the scope of this disclosure. An allele is an endogenous gene altered due to one or more mutations (such as nucleotide deletions, additions, and / or substitutions). The resulting mRNA and protein structure or function may (but not necessarily) be altered. Alleles can be identified using standard techniques such as hybridization, amplification, and / or database sequence comparison.

[0110] The polynucleotides disclosed herein can be obtained using chemical synthesis, recombination methods, or PCR. Chemical polynucleotide synthesis methods are well known in the art and need not be described in detail herein. Those skilled in the art can use the sequences provided herein and commercial DNA synthesizers to generate the desired DNA sequences.

[0111] For the preparation of polynucleotides using recombinant methods, a polynucleotide containing the desired sequence can be inserted into a suitable vector, and the vector can then be introduced into a suitable host cell for replication and amplification, as further described herein. Polynucleotides can be inserted into host cells by any method known in the art. Cells are transformed by introducing exogenous polynucleotides through direct uptake, endocytosis, transfection, F-crossing, or electroporation. Once introduced, the exogenous polynucleotide can be maintained intracellularly as a non-integrating vector (such as a plasmid) or integrated into the host cell genome. The polynucleotides thus amplified can be isolated from the host cell using methods well-known in the art. See, for example, Sambrook et al., 1989.

[0112] Alternatively, PCR allows for the replication of DNA sequences. PCR technology is well known in the art and is described, for example, in U.S. Patent Nos. 4,683,195, 4,800,159, 4,754,065, and 4,683,202, and PCR: The Polymerase Chain Reaction, edited by Mullis et al., Birkauswer Press, Boston, 1994.

[0113] Therefore, this document provides a polynucleotide comprising a promoter and at least two coding sequences, one of which encodes an anti-CD19 CAR. In some embodiments, the other coding sequence encodes a CD70 binding protein or an anti-CD70 CAR.

[0114] Chimeric antigen receptor (CAR) As used herein, chimeric antigen receptors (CARs) are proteins that specifically recognize target antigens (e.g., target antigens on cancer cells). Upon binding to a target antigen, a CAR can activate immune cells to attack and destroy cells carrying that antigen (e.g., cancer cells). CARs can also be incorporated with co-stimulatory or signal transduction domains to increase their potency. See Krause et al., J. Exp. Med., Vol. 188, No. 4, 1998 (619–626); Finney et al., Journal of Immunology, 1998, 161: 2791–2797; Song et al., Blood 119:696-706 (2012); Kalos et al., Sci. Transl. Med. 3:95 (2011); Porter et al., N. Engl. J. Med. 365:725-33 (2011); and Gross et al., Annu. Rev. Pharmacol. Toxicol. 56:59–83 (2016); U.S. Patent Nos. 7,741,465 and 6,319,494.

[0115] Taking an anti-CD19 CAR as an example, the chimeric antigen receptor described herein comprises an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain includes a CD19 antigen-binding domain that specifically binds to CD19. In some embodiments, the CD19-specific CAR comprises, from the N-terminus to the C-terminus, the following elements: a cleavable signal sequence, a CD19 antigen-binding domain (e.g., anti-CD19 scFv), a hinge domain, and a transmembrane domain, and one or more consecutive signal transduction domains. In some embodiments, the CD19-specific CAR disclosed herein comprises, from the N-terminus to the C-terminus, the following elements: a CD8α signal sequence, the CD19 scFv described herein, a CD8α hinge domain and a transmembrane domain, a 41BB cytoplasmic signal transduction domain, and a CD3ζ cytoplasmic signal transduction domain. Exemplary sequences are shown in Table 3.

[0116] a. Antigen-binding domain As discussed above, the CD19 CAR described herein includes an antigen-binding domain. As used herein, "antigen-binding domain" means any polypeptide that binds to a specific target antigen, such as the CD19 protein or a fragment thereof (which may be interchangeably referred to herein as "CD19 antigen," "CD19 target antigen," or "CD19 target"). In some embodiments, the antigen-binding domain binds to CD19 antigens on tumor cells. In some embodiments, the antigen-binding domain binds to CD19 antigens on cells involved in hyperproliferative diseases or hematologic malignancies.

[0117] In some embodiments, the antigen-binding domain comprises a variable heavy chain, a variable light chain, and / or one or more CDRs as described herein. In some embodiments, the antigen-binding domain is a single-chain variable fragment (scFv) comprising light chains CDR1, CDR2, and CDR3, and heavy chain CDRs CDR1, CDR2, and CDR3. Exemplary anti-CD19 antibody CDR sequences are shown in Table 3.

[0118] Variants of the antigen-binding domain (e.g., variants of CDR, VH, and / or VL) are also within the scope of this disclosure, such as variable light chains and / or variable heavy chains, each having at least 70-80%, 80-85%, 85-90%, 90-95%, 95-97%, 97-99%, or greater than 99% identity with the amino acid sequence of the antigen-binding domain described herein. In some cases, such molecules comprise at least one heavy chain and one light chain, while in others, the variant forms contain two variable light chains and two variable heavy chains (or sub-parts thereof). Those skilled in the art will be able to identify suitable variants of the antigen-binding domain as shown herein using well-known techniques. In some embodiments, those skilled in the art can identify suitable regions in the molecule that can be altered without destroying activity by targeting regions considered unimportant to activity.

[0119] In some embodiments, the polypeptide structure of the antigen-binding domain is based on an antibody, including but not limited to monoclonal antibodies, bispecific antibodies, microantibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimics"), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), and fragments thereof. In some embodiments, the antigen-binding domain comprises or is composed of an avimer.

[0120] The CD19 antigen-binding domain is said to be "selective" when it binds more tightly to CD19 than to another target.

[0121] In some embodiments, the CD19 antigen-binding domain is an scFv. In some embodiments, the CD19-specific CAR comprises an scFv containing the amino acid sequence of SEQ ID NO: 4. In some embodiments, the CD19-specific CAR comprises the anti-CD19 antibody CDR sequence of SEQ ID NO: 65-70, and is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 4.

[0122] In some embodiments, the CD19-specific CAR comprises a leader peptide or a signal peptide; in some embodiments, the leader peptide comprises an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the amino acid sequence MALPVTALLLPLALLLHAARP (SEQ ID NO: 1). In some embodiments, the leader (signal) peptide comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the leader (signal) peptide is encoded by a nucleic acid sequence comprising the following: ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGCCGCACGCCCG (SEQ ID NO: 48).

[0123] b. Hinge structural domain The extracellular domain of the CAR disclosed herein may include a "hinge" domain (or hinge region). This term generally refers to any polypeptide that acts to link a transmembrane domain in the CAR to an extracellular antigen-binding domain in the CAR. Specifically, the hinge domain can be used to provide greater flexibility and accessibility to the extracellular antigen-binding domain.

[0124] The hinge domain may contain up to 300 amino acids, with 10 to 100 amino acids in some embodiments and 25 to 50 amino acids in others. The hinge domain may be derived from all or part of a naturally occurring molecule, such as from all or part of an extracellular region of CD8, CD4, CD28, 4-1BB, or IgG (particularly the hinge region of IgG; it will be understood that the hinge region may contain some or all of members of the immunoglobulin family, such as IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, or fragments thereof), or from all or part of a constant region of the antibody heavy chain. Alternatively, the hinge domain may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or it may be a fully synthetic hinge sequence. In some embodiments, the hinge domain is a portion of the human CD8α chain (e.g., NP_001139345.1). In another particular embodiment, the hinge and transmembrane domain comprise a portion of the human CD8α chain. In some embodiments, the hinge domain of the CAR described herein comprises a subsequence of CD8α, CD28, IgG1, IgG4, PD-1, or FcγRIIIα, particularly the hinge region of any one of CD8α, CD28, IgG1, IgG4, PD-1, or FcγRIIIα. In some embodiments, the hinge domain comprises a human CD8α hinge, a human IgG1 hinge, a human IgG4, a human PD-1, or a human FcγRIIIα hinge. In some embodiments, the CAR disclosed herein comprises scFv, a human CD8α hinge and a transmembrane domain, a CD3ζ signaling domain, and a 4-1BB signaling domain. Table 3 provides the amino acid sequences of the exemplary hinge domains provided herein.

[0125] In some embodiments, the hinge region comprises an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the extracellular domain amino acid sequence of SEQ ID NO: 9 as described herein.

[0126] c. Transmembrane domain The CAR disclosed herein is designed to have a transmembrane domain fused to the extracellular domain of the CAR. It can similarly fuse to the intracellular domain of the CAR. In some cases, the transmembrane domain may be selectively modified or modified by amino acid substitution to prevent such domains from binding to the transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex. In some embodiments, short linkers may form a link between any and some of the extracellular, transmembrane, and intracellular domains of the CAR.

[0127] The transmembrane domain of the CAR disclosed herein has the ability to: (a) be expressed on the surface of immune cells, such as, for example but not limited to, lymphocytes, such as T helper (T) cells. h Cellular, cytotoxic T (T) c ) cells, T regulation (T reg (a) cells or natural killer (NK) cells, and / or (b) interact with extracellular antigen-binding domains and intracellular signal transduction domains to guide the cellular response of immune cells against target cells.

[0128] Transmembrane domains can be derived from natural or synthetic sources. In the case of natural sources, the domain can be derived from any membrane-bound or transmembrane protein.

[0129] The transmembrane region specifically used in this disclosure may be derived from (including or correspond to) CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death receptor-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD1-1a / CD18), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Igα (CD79a), DAP-10, Fcγ receptor, MHC Class 1 molecules, TNF receptor protein, immunoglobulins, cytokine receptors, integrins, signal transduction lymphocyte activating molecules (SLAM protein), activating NK cell receptors, BTLA, Toll ligand receptors, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (tactile protein), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, ligands that specifically bind to CD83, or any combination thereof.

[0130] As a non-limiting example, the transmembrane domain may be derived from or a subset of the following: T-cell receptors, such as α, β, γ, or δ; polypeptides constituting the CD3 complex; IL-2 receptor p55 (α chain), p75 (β chain), or γ chain; subunit chains of Fc receptors, particularly Fcγ receptor III; or CD proteins. Alternatively, the transmembrane domain may be synthetic and may primarily contain hydrophobic residues, such as leucine and valine. In some embodiments, the transmembrane domain is derived from the human CD8α chain (e.g., NP_001139345.1).

[0131] In some embodiments, the transmembrane domain in the CAR of this disclosure is a CD8α transmembrane domain. In some embodiments, the transmembrane domain in the CAR of this disclosure is a CD8α transmembrane domain comprising the amino acid sequence IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 10) or SEQ ID NO: 99. In some embodiments, the hinge domain and transmembrane domain in the CAR of this disclosure are a CD8α hinge domain and a CD8α transmembrane domain, respectively.

[0132] In some embodiments, the transmembrane domain in the CAR of this disclosure is a CD28 transmembrane domain. In some embodiments, the transmembrane domain in the CAR of this disclosure is a CD28 transmembrane domain containing the amino acid sequence FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 56). In some embodiments, the hinge domain and transmembrane domain in the CAR of this disclosure are a CD28 hinge domain and a CD28 transmembrane domain, respectively.

[0133] d. Intracellular domains The intracellular (cytoplasmic) domain of the CAR disclosed herein can provide activation of at least one of the normal effector functions of an immune cell containing the CAR, such as signal 1 / activation and / or signal 2 / co-stimulation. For example, T cell effector functions can refer to cytolytic activity or helper activity, including cytokine secretion.

[0134] In some embodiments, the activating intracellular signal transduction domains used in CARs can be, for example, but not limited to, cytoplasmic sequences of T-cell receptors and helper receptors that work together to initiate signal transduction upon antigen receptor binding, as well as any derivatives or variants of these sequences and any synthetic sequences having the same functional capabilities.

[0135] It will be understood that suitable (e.g., activating) intracellular domains include, but are not limited to, signal transduction domains derived from (or corresponding to) the following: CD3ζ, CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death receptor-1 (PD-1), inducible T cell costimulators (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD1-1a / CD18), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), LIGHT (TNFSF14), NKG2C, Igα (CD79a), DAP-10, Fcγ receptor, MHC Class 1 molecules, TNF receptor protein, immunoglobulins, cytokine receptors, integrins, signal transduction lymphocyte activating molecules (SLAM protein), activating NK cell receptors, BTLA, Toll ligand receptors, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (tactile protein), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, ligands that specifically bind to CD83, or any combination thereof.

[0136] In addition to the aforementioned activating domains, the intracellular domains of the CAR disclosed herein may also incorporate co-stimulatory signal transduction domains (which are interchangeably referred to herein as co-stimulatory molecules or co-stimulatory domains) to enhance their potency. Besides the primary signal provided by the stimulatory molecules as described herein, the co-stimulatory domains can provide signaling.

[0137] As used herein, "co-stimulatory molecules" refer to homologous binding partners on T cells that specifically bind to co-stimulatory ligands, thereby mediating cellular co-stimulatory responses such as, but not limited to, proliferation. Co-stimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors. Examples of co-stimulatory molecules include CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83.

[0138] It will be understood that suitable costimulatory domains within the scope of this disclosure may be derived from (or correspond to), for example, CD28, OX40, 4-1BB / CD137, CD2, CD3 (α, β, δ, ε, γ, ζ), CD4, CD5, CD7, CD9, CD16, CD22, CD27, CD30, CD33, CD37, CD40, CD45, CD64, CD80, CD86, CD134, CD137, CD154, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1 (CD11a / CD18), CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, Igα (CD79a), DAP-10, Fcγ receptor, MHC Class I molecules, TNFR, integrins, signal transduction lymphocyte activating molecules, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1-1d, IT GAE, CD103, ITGAL, CD1-1a, LFA-1, ITGAM, CD1-1b, ITGAX, CD1-1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (tactile protein), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83 ligands, or fragments or combinations thereof. It will be understood that other co-stimulatory molecules or fragments thereof not listed above are within the scope of this disclosure.

[0139] In some embodiments, the intracellular / cytoplasmic domain of the CAR may be designed to include a 41BB / CD137 domain, either alone or in combination with one or more other desired intracellular domains useful in the context of the CAR disclosed herein. The complete native amino acid sequence of 41BB / CD137 is described in NCBI reference sequence: NP_001552.2. The complete native 41BB / CD137 nucleic acid sequence is described in NCBI reference sequence: NM_001561.5.

[0140] In some implementations, the intracellular / cytoplasmic domain of the CAR may be designed to include a CD28 domain, either alone or in combination with one or more other desired intracellular domains useful in the context of the CAR disclosed herein. The complete native amino acid sequence of CD28 is described in NCBI reference sequence: NP_006130.1. The complete native CD28 nucleic acid sequence is described in NCBI reference sequence: NM_006139.1.

[0141] In some embodiments, the intracellular / cytoplasmic domain of the CAR may be designed to include a CD3ζ domain, either alone or in combination with one or more other desired intracellular domains useful in the context of the CAR disclosed herein. In some embodiments, the intracellular signaling domain of the CAR may include a CD3ζ signaling domain having an amino acid sequence identity of at least about 70%, at least 80%, at least 90%, 95%, 97%, or 99% with the amino acid sequence shown in SEQ ID NO: 11 or 91 (see Table 3). For example, the intracellular domain of the CAR may include a CD3ζ chain portion and a portion of a co-stimulatory signaling molecule. The intracellular signaling sequences within the intracellular signaling portion of the CAR of this disclosure may be linked to each other in a random or designated order. In some embodiments, the intracellular domain is designed to include an activating domain of CD3ζ and a signaling domain of CD28. In some embodiments, the intracellular domain is designed to include an activating domain of CD3ζ and a co-stimulatory / signaling domain of 4-1BB.

[0142] In some embodiments, the 4-1BB (intracellular domain) comprises the amino acid sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 12). In some embodiments, the 4-1BB (intracellular domain) is encoded by the following nucleic acid sequence: AAGCGCGGCAGGAAGAAGCTCCTCTACATTTTTAAGCAGCCTTTTATGAGGCCCGTACAGACAACACAGGAGGAAGATGGCTGTAGCTGCAGATTTCCCGAGGAGGAGGAAGGTGGGTGCGAGCTG (SEQ ID NO: 57).

[0143] In some implementations, the intracellular domain in the CAR is designed to include a portion of CD28 and CD3ζ, wherein the intracellular CD28 contains the amino acid sequence of SEQ ID NO: 13 and is encoded by the nucleic acid sequence described in SEQ ID NO: 58.

[0144] In some embodiments, the CD3ζ amino acid sequence may include SEQ ID NO: 11, and the nucleic acid sequence encoding the CD3ζ amino acid sequence may include SEQ ID NO: 59. AGGGTGAAGTTTTCCAGATCTGCAGATGCACCAGCGTATCAGCAGGGCCAGAACCAACTGTATAACGAGCTCAACCTGGGACGCAGGGAAGAGTATGACGTTTTGGACAAGCGCAGAGGACGGGACCCTGAGATGGGTGGCAAACCAAGACGAAAAAAACCCCAGGAG GGTTCCTATAATGAGCTGCAGAAGGATAAGATGGCTGAAGCCTATTCTGAAATAGGCATGAAAGGAGAGCGGAGAAGGGGAAAAGGGCACGACGGTTTGTACCAGGGACTCAGCACTGCTACGAAGGATACTTATGACGCTCTCCACATGCAAGCCCTGCCACCTAGG (SEQ ID NO: 59).

[0145] In some embodiments, the intracellular signaling domain of the CAR of this disclosure includes a domain of a co-stimulatory molecule. In some embodiments, the intracellular signaling domain of the CAR of this disclosure includes a portion of a co-stimulatory molecule selected from the group consisting of fragments of 4-1BB (GenBank: AAA53133.) and CD28 (NP_006130.1). In some embodiments, the intracellular signaling domain of the CAR of this disclosure includes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 12. In some embodiments, the intracellular signaling domain of the CAR of this disclosure includes an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 13.

[0146] In exemplary embodiments, the CAR of this disclosure comprises, from the N-terminus to the C-terminus: a (cleavable) CD8α signaling sequence, an anti-CD19 scFv, a CD8α hinge region and a transmembrane region, a 4-1BB cytoplasmic (co-stimulatory) signaling domain, and a CD3ζ cytoplasmic signaling domain. In some embodiments, the CD19-specific CAR comprises the amino acid sequences of SEQ ID NO: 4, 9, 10, 11, and 12. In some embodiments, the CD19-specific CAR comprises the amino acid sequences of SEQ ID NO: 4, 9, 10, 11, and 12, and has at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 5 or 7. In some embodiments, the CD19-specific CAR comprises an amino acid sequence having one or more conserved amino acid substitutions of the amino acid sequences of SEQ ID NO: 5 or 7. In some implementations, the CD19-specific CAR contains the amino acid sequence of SEQ ID NO: 5 or 7.

[0147] In some respects, this article provides engineered immune cells that contain and / or express CD19-specific CARs, as well as CD70-binding proteins and / or chimeric cytokine receptors (CCRs).

[0148] CD70 binding protein In a related respect, this disclosure provides a CD70-binding protein as described herein, wherein the CD70-binding protein comprises an extracellular ligand or antigen-binding domain (or a CD70-binding domain) that binds to CD70 and a transmembrane domain. The CD70-binding protein may comprise zero to one or more intracellular signaling domains, as described herein. In some embodiments, the CD70-binding protein is a non-naturally occurring or recombinant CD70-binding protein.

[0149] In some embodiments, the CD70 binding protein provided herein comprises an extracellular domain that binds to CD70 (e.g., an anti-CD70 single-stranded variable fragment (scFv)) and a transmembrane domain. In some embodiments, the CD70 binding protein is a CD70 CAR. In some embodiments, the CD70 binding protein or CD70 CAR provided herein comprises an extracellular ligand-binding domain (e.g., scFv), a transmembrane domain, and an intracellular signaling domain. In some embodiments, the anti-CD70 scFv comprises the amino acid sequence of SEQ ID NO: 16, 17, or 18. In some embodiments, the CD70 binding protein comprises the amino acid sequences of SEQ ID NO: 16 and 17 and has at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 18. In some embodiments, the anti-CD70 scFv comprises an amino acid sequence having one or more conserved amino acid substitutions of the amino acid sequence of SEQ ID NO: 18. In some embodiments, the anti-CD70 scFv comprises the amino acid sequence of SEQ ID NO: 18.

[0150] In some embodiments, the CD70 binding protein includes a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 10 or 56.

[0151] In some embodiments, the CD70-binding protein comprises one or more intracellular signaling domains selected from the group consisting of: CD3ζ signaling domain, CD3δ signaling domain, CD3γ signaling domain, CD3ε signaling domain, CD28 signaling domain, CD2 signaling domain, OX40 signaling domain, and 4-1BB signaling domain, or variants thereof. In some embodiments, the intracellular signaling domain comprises an amino acid sequence of one or more of SEQ ID NO: 11, 12, 13, 82, 83, 84, 85, 86, 87, 88, 89, 90, or 91. In some embodiments, the intracellular signaling domain comprises an amino acid sequence of one or more of SEQ ID NO: 11, 88, 89, or 90. In some embodiments, the CD70-binding protein comprises a CD3ζ signaling domain or a variant thereof and does not comprise a co-stimulatory domain, such as a 4-1BB co-stimulatory domain. In some embodiments, the CD70-binding protein or CD70-specific CAR comprises an intracellular CD3ζ signaling domain containing the amino acid sequence of SEQ ID NO: 11 and does not contain a co-stimulatory domain. In some embodiments, the CD70-binding protein comprises a 4-1BB signaling domain or a variant thereof and does not contain a CD3 signaling domain. In some embodiments, the CD70-binding protein comprises both a 4-1BB signaling domain and a CD3ζ signaling domain. In some embodiments, the CD70-binding protein does not contain an intracellular signaling domain. Different intracellular signaling domains or combinations thereof can confer different signaling strengths, which can promote T cell proliferation, potency, survival, persistence, and / or resistance to host immune cell rejection. This document describes CD70-binding proteins comprising zero to one or more intracellular signaling domains. In some embodiments, the CD70-binding protein comprises the amino acid sequences of SEQ ID NO: 18, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, and has at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 19 or 21. In some embodiments, the CD70-binding protein comprises an amino acid sequence having one or more conserved amino acid substitutions of the amino acid sequence of SEQ ID NO: 19 or 21. In some embodiments, the CD70-binding protein comprises the amino acid sequence of SEQ ID NO: 19 or 21.

[0152] CD70 is expressed on T cells, particularly activated T cells, including host allogeneic reactive T cells. In some embodiments, engineered immune cells containing the CD70-binding protein described herein can exhibit varying levels of persistence and / or resistance to host immune cell rejection and can be adapted for in vivo lymphocyte depletion upon administration to a patient. In some embodiments, engineered immune cells containing the CD70-binding protein described herein can inhibit the proliferation and / or activity of host immune cells to varying degrees upon administration to a patient, which can allow for fine-tuning of the depth of in vivo lymphocyte depletion. For example, engineered immune cells containing the CD70-binding protein, exhibiting prolonged expansion and / or inhibition of host immune cell proliferation or activity in MLR assays, can be used to prolong lymphocyte depletion. In contrast, when less thorough or incomplete lymphocyte depletion is required, engineered immune cells containing the CD70-binding protein, which exhibit less prolonged expansion and / or inhibition of host immune cells in the same or similar assays, can be used. In some embodiments, the host immune cells are T cells or NK cells.

[0153] In some respects, engineered immune cells (e.g., CAR T cells) comprising and / or expressing CD19-specific CARs and CD70-binding proteins or CD70-specific CARs, as described herein, can target CD19-positive and CD19-negative / CD70-positive tumor cells or other pathogenic T cells or allogeneic reactive T cells. CD19-specific CAR T cells co-expressing CD70-binding proteins (which may be referred to as CD19 / CD70 dual CAR T cells) advantageously prevent the escape of CD19 antigens from tumor cells or pathogenic target cells. In some respects, CD19-specific CAR T cells co-expressing CD70-binding proteins and / or chimeric cytokine receptors (CCRs) exhibit further enhanced activity.

[0154] Chimeric cytokine receptor (CCR) The CCR disclosed herein includes a transmembrane domain. The transmembrane domain of this disclosure contains sequences that enable the two monomers to constitutively dimerize, thereby allowing constitutive JAK activation on the intracellular portion and constitutive recruitment and phosphorylation of STAT, for example, on the cytoplasmic region of the receptor.

[0155] The transmembrane domain is located at the N-terminus and is coupled to the intracellular / cytoplasmic domain at the C-terminus. In some embodiments, coupling is optionally achieved via a linker.

[0156] The transmembrane domains used herein can be inserted into the cell membrane expressing the transmembrane domain. In some embodiments, the CCR of this disclosure comprises a transmembrane domain spanning the cell membrane, and an extracellular and / or intracellular portion. In some embodiments, the CCR of this disclosure comprises a transmembrane domain spanning the cell membrane, and an extracellular ligand-binding domain and / or an intracellular signal transduction domain. In some embodiments, the CCR of this disclosure comprises a transmembrane domain spanning the cell membrane, and further comprises an intracellular domain, and does not comprise an extracellular ligand-binding domain. In some embodiments, the CCR comprises the amino acid sequence of SEQ ID NO: 27 or 29.

[0157] In some embodiments, the transmembrane domains of this disclosure are engineered (synthetic) and do not resemble any naturally occurring transmembrane domains; for example, they are non-natural.

[0158] In other embodiments, the transmembrane domains of this disclosure are derived from naturally occurring receptors.

[0159] In some embodiments, the transmembrane domain and / or JAK binding / activation domain (or simply JAK binding domain) of this disclosure are derived from one or more of the following receptors: erythropoietin receptor (EpoR), interleukin-6 signal transducer (GP130 or IL6ST), prolactin receptor (PrlR), growth hormone receptor (GHR), granulocyte colony-stimulating factor receptor (GCSFR), and thrombopoietin receptor / myeloproliferative leukemia protein receptor (TPOR / MPLR). When derived from naturally occurring receptors, the entire receptor or the entire transmembrane sequence of the receptor may not be necessary to achieve constitutive activation and constitutive JAK binding / activation at the intracellular portion. In some embodiments, the transmembrane domain of this disclosure is derived from a truncated version of the naturally occurring TPOR / MPLR (myeloproliferative leukemia protein) receptor, as shown in SEQ ID NO: 51. Therefore, fragments of naturally occurring receptors can be utilized. Furthermore, certain mutations can be introduced into the transmembrane domain derived from naturally occurring receptors to further modulate downstream signaling. In some embodiments, the transmembrane domains of this disclosure are derived from TPOR / MPLR and have H499L / S505N / W515K substitutions as shown in SEQ ID NO: 25.

[0160] The CCR of this disclosure includes a cytoplasmic recruitment domain. The recruitment domain may be a STAT recruitment domain, an AP1 recruitment domain, a Myc / Max recruitment domain, or an NFkB recruitment domain. In some embodiments, the recruitment domain is a signal transducer and transcription activator (STAT) recruitment (STAT activation) domain, for example, derived from a receptor tail (cell tail) or a cytokine receptor tail. These intracellular recruitment domains of the CCR of this disclosure allow signal transduction to propagate in immune cells containing CARs and chimeric cytokine receptors (e.g., CAR-T cells with chimeric cytokine receptors of this disclosure). Cytokine signaling propagates through the STAT recruitment domain, thereby achieving cytokine-based immune enhancement of the cell. In some embodiments, the immune enhancement is homeostatic, for example, the signal transduction leads to an increase in CAR-carrying immune cells. In some embodiments, the immune enhancement is inflammatory, for example, the signal transduction leads to an increase in the potency of CAR-carrying immune cells. In some embodiments, the immune enhancement can prevent exhaustion, for example, the signal transduction can maintain the long-term function of CAR-carrying immune cells.

[0161] In some embodiments, the recruitment domain of this disclosure is synthetic and does not resemble any naturally occurring receptor fragment. In some embodiments, immune enhancement can prevent depletion; for example, signaling can maintain the long-term function of CAR-carrying immune cells. In some embodiments, the Stat recruitment domain of this disclosure is synthetic and does not resemble any naturally occurring receptor fragment.

[0162] In other embodiments, the Stat recruitment domain of this disclosure is derived from the cytoplasmic tail of a naturally occurring receptor, such as a naturally occurring cytokine receptor. These cytoplasmic tails of naturally occurring receptors may be regions downstream of the JAK-binding domain of a transmembrane domain of the receptor. The Stat recruitment domain of a chimeric cytokine receptor includes at least one STAT recruitment domain derived from at least one receptor. In some embodiments, the Stat recruitment domain includes at least one STAT1 recruitment domain. In some embodiments, the Stat recruitment domain includes at least one STAT2 recruitment domain. In some embodiments, the Stat recruitment domain includes at least one STAT3 recruitment domain. In some embodiments, the Stat recruitment domain includes at least one STAT4 recruitment domain. In some embodiments, the Stat recruitment domain includes at least one STAT5 recruitment domain. In some embodiments, the Stat recruitment domain includes at least one STAT6 recruitment domain. In some embodiments, the Stat recruitment domain includes at least one STAT7 recruitment domain.

[0163] In some implementations, the naturally occurring receptor from which the Stat recruitment domain originates is not a cytokine receptor.

[0164] In some embodiments, the naturally occurring receptor from which the Stat recruitment domain is derived is a cytokine receptor. Exemplary cytokine receptors that enhance cytokine signaling in T-cell immunity include, but are not limited to, IL-2, IL-7, IL-15, and IL-21 receptors. In alternative embodiments, the receptor from which the Stat recruitment domain is derived is not a cytokine receptor. By selecting the Stat recruitment domain of the CCR, the receptor can be redirected to the selected signal transduction pathway. In some embodiments, the recruitment domain comprises the amino acid sequence of SEQ ID NO: 26.

[0165] In some embodiments, the CCR of this disclosure comprises a recruitment domain linked to the C-terminus of a transmembrane / JAK2 binding domain, with or without a linker. In some embodiments, the linker comprises one or more amino acid residues. In some embodiments, the CCR comprises the amino acid sequences of SEQ ID NO: 25 and 26. In some embodiments, the CCR comprises the amino acid sequences of SEQ ID NO: 25 and 26 and has at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 27 or 29. In some embodiments, the CCR comprises an amino acid sequence having one or more conserved amino acid substitutions of the amino acid sequence of SEQ ID NO: 27 or 29. In some embodiments, the CCR comprises the amino acid sequence of SEQ ID NO: 27 or 29.

[0166] In some embodiments, the CCR of this disclosure comprises an extracellular ligand-binding domain. In some embodiments, the extracellular ligand-binding domain binds to an immunosuppressive molecule, such as PDL1. In some embodiments, the extracellular ligand-binding domain of the CCR comprises an antibody that binds to PDL-1. In some embodiments, the extracellular ligand-binding domain of the CCR comprises the extracellular domain of PD-1 (PD-1 CCR). In some embodiments, the binding of the PD-1 extracellular domain of the PD-1 CCR to PDL-1 can convert an inhibitory signal of PDL-1 into a stimulatory signal through signal transduction by an intracellular cytoplasmic recruitment domain. In some embodiments, the PD-1 CCR comprises a TPOR / MPLR transmembrane / JAK binding domain comprising the amino acid sequence of SEQ ID NO: 50, and an intracellular recruitment domain of SEQ ID NO: 26. In some embodiments, the PD-1 extracellular domain comprises the wild-type PD-1 extracellular domain and comprises the amino acid sequence of SEQ ID NO: 52.

[0167] In some embodiments, the PD-1 extracellular domain contains a mutation targeting the wild-type PD-1 extracellular domain sequence. In some embodiments, the PD-1 extracellular domain sequence is a high-affinity PD-1 extracellular domain. In some embodiments, the PD-1 extracellular domain sequence is a high-affinity PD-1 extracellular domain and contains the amino acid sequence of SEQ ID NO: 53. In some embodiments, the PD-1 extracellular domain contains one or more tandem repeat sequences of the wild-type or high-affinity PD-1 extracellular domain.

[0168] dominant negative receptor On the other hand, this document provides a dominant-negative PD-1 receptor comprising a PD-1 extracellular domain and a transmembrane domain, but without a functional intracellular signaling domain. In some embodiments, the PD-1 extracellular domain comprises a wild-type PD-1 extracellular domain. In some embodiments, the PD-1 extracellular domain comprises a mutant PD-1 extracellular domain. In some embodiments, the PD-1 extracellular domain comprises a high-affinity PD-1 extracellular domain. In some embodiments, the PD-1 extracellular domain comprises the amino acid sequence of SEQ ID NO: 53.

[0169] In other embodiments, the transmembrane domain comprises a PD-1 transmembrane domain. In some embodiments, the transmembrane domain comprises a CD8a transmembrane domain. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 10, 56, or 60. Conserved amino acid substitutions of the peptides disclosed herein are also considered.

[0170] In some embodiments, the PD-1 dominant-negative receptor comprises the amino acid sequence of SEQ ID NO: 52 or 53. In some embodiments, the PD-1 dominant-negative receptor comprises the amino acid sequence of SEQ ID NO: 52 or 53 and has at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 55. In some embodiments, the PD-1 dominant-negative receptor comprises an amino acid sequence having one or more conserved amino acid substitutions of the amino acid sequence of SEQ ID NO: 54 or 55. In some embodiments, the PD-1 dominant-negative receptor comprises the amino acid sequence of SEQ ID NO: 54 or 55.

[0171] This disclosure covers modifications of peptides disclosed herein, such as CARs (e.g., CD19-specific CARs), CD70-binding proteins, CCRs, and dominant / negative receptors, that do not significantly affect their properties, and variants that enhance or reduce activity and / or affinity as needed. Peptide modification is common practice in the art and need not be described in detail herein. Examples of modified peptides include peptides with conserved substitutions of amino acid residues, deletions or additions of one or more amino acids, where the modification does not significantly and harmfully alter functional activity, or where the modification matures (enhances) the peptide's affinity for its ligands, or the use of chemical analogs.

[0172] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions of peptides ranging in length from one residue to one hundred or more residues, as well as intra-sequence insertions of one or more amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue or antibodies fused to an epitope tag. Other insertional variants of antibody molecules include fusions of enzymes or peptides with the N-terminus or C-terminus of the antibody, which increase the antibody's half-life in blood circulation.

[0173] Substitutional variants remove at least one amino acid residue from the antigen-binding domain and insert a different residue at its position. In some embodiments, the substitutional mutagenic site of interest includes the hypervariable region / CDR, but FR alterations are also considered. Conservative substitutions are shown under the heading “Conservative Substitutions” in Table 2. If such substitutions result in a change in biological activity, more substantial changes (named “Exemplary Substitutions” in Table 2, or further described below with respect to amino acid categories) can be introduced and products screened.

[0174] Table 2: Amino Acid Substitutions

[0175] In some aspects, this disclosure provides immune cells or engineered immune cells comprising polynucleotides, said polynucleotides comprising more than one coding sequence or a first coding sequence and a second coding sequence, wherein the first coding sequence or the second coding sequence encodes an anti-CD19 CAR. In some embodiments, the engineered immune cells further comprise additional polynucleotides comprising one or more coding sequences.

[0176] In some embodiments, one or more coding sequences of the additional polynucleotide include a coding sequence encoding the PD-1 dominant-negative receptor. In some embodiments, the PD-1 dominant-negative receptor comprises a wild-type PD-1 extracellular domain. In some embodiments, the PD-1 dominant-negative receptor comprises a high-affinity PD-1 extracellular domain. In some embodiments, the PD-1 dominant-negative receptor does not comprise a functional intracellular signaling domain. In some embodiments, the PD-1 dominant-negative receptor comprises the amino acid sequence of SE ID NO: 54 or 55.

[0177] In some embodiments, one or more coding sequences of the additional polynucleotide encode a CCR. In some embodiments, the CCR comprises the amino acid sequence of SEQ ID NO: 27 or 29. In some embodiments, one or more coding sequences encode a CD70-binding protein. In some embodiments, the CD70-binding protein comprises the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 21. In some embodiments, one or more coding sequences encode a PD-1 CCR. In some embodiments, the PD-1 CCR comprises a PD-1 extracellular domain comprising the amino acid sequence of SEQ ID NO: 54 or SEQ ID NO: 55; a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 50; and an intracellular signal transduction domain comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, one or more coding sequences encode a PD-1 dominant / negative receptor. In some embodiments, the PD-1 dominant / negative receptor comprises the amino acid sequence of SEQ ID NO: 54 or SEQ ID NO: 55.

[0178] In some embodiments, the additional polynucleotide from 5' to 3' contains a promoter operatively linked to both the third and fourth coding sequences. In some embodiments, the third coding sequence encodes a CCR or CD70-binding protein. In some embodiments, the fourth coding sequence encodes a PD-1 CCR or a PD-1 dominant / negative receptor. In some embodiments, the third coding sequence encodes a PD-1 CCR or a PD-1 dominant / negative receptor. In some embodiments, the fourth coding sequence encodes a CCR or CD70-binding protein.

[0179] In other respects, this article provides engineered immune cells, such as CAR T cells expressing CD19-specific CAR and CD70 binding proteins and / or CCRs. In some embodiments, engineered immune cells, such as CD19 CAR T cells, further express PD-1 dominant-negative receptors.

[0180] In some embodiments, the engineered immune cell comprises a polynucleotide comprising, from 5' to 3', a first coding sequence and a second coding sequence driven by a truncated PGK promoter, wherein the first coding sequence encodes a CD19-specific CAR and the second coding sequence encodes a CD70-binding protein, as described herein; or wherein the first coding sequence encodes a CD70-binding protein and the second coding sequence encodes a CD19-specific CAR, as described herein. In some embodiments, the engineered immune cell further comprises an additional polynucleotide comprising one or more coding sequences, wherein the one or more coding sequences encode a CCR and optionally encode a PD-1 dominant / negative receptor, as described herein. In some embodiments, the additional polynucleotide comprises, from 5' to 3', a third coding sequence and a fourth coding sequence, wherein the third coding sequence encodes a CCR and the fourth coding sequence encodes a PD-1 dominant / negative receptor, as described herein; or wherein the third coding sequence encodes a PD-1 dominant / negative receptor and the fourth coding sequence encodes a CCR, as described herein.

[0181] In some embodiments, the engineered immune cell comprises a polynucleotide comprising, from 5' to 3', a first coding sequence and a second coding sequence driven by a truncated PGK promoter, wherein the first coding sequence encodes a CCR and the second coding sequence encodes a CD19-specific CAR, as described herein; or wherein the first coding sequence encodes a CD19-specific CAR and the fourth coding sequence encodes a CCR, as described herein. In some embodiments, the engineered immune cell further comprises an additional polynucleotide comprising one or more coding sequences, wherein the one or more coding sequences encode a CD70-binding protein and optionally encode a PD-1 dominant / negative receptor, as described herein. In some embodiments, the additional polynucleotide comprises, from 5' to 3', a third coding sequence and a fourth coding sequence, wherein the third coding sequence encodes a CD70-binding protein and the fourth coding sequence encodes a PD-1 dominant / negative receptor, as described herein; or wherein the third coding sequence encodes a PD-1 dominant / negative receptor and the fourth coding sequence encodes a CD70-binding protein, as described herein.

[0182] Also provided are populations of engineered immune cells, such as CAR T cells. In some embodiments, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the engineered immune cell population expresses the CD19 CAR described herein. In some embodiments, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the engineered immune cell population expresses the CD19 CAR and CD70 binding protein described herein. In some embodiments, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the engineered immune cell population expresses the CD19 CAR, CD70 binding protein, and CCR described herein. In some embodiments, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the engineered immune cell population expresses the CD19 CAR, CD70 binding protein, and CCR described herein.

[0183] Engineered immune cells This article provides engineered immune cells (e.g., CAR-T cells) expressing the CAR of this disclosure. For the subject receiving the engineered immune cells, the engineered immune cells can be allogeneic or autologous.

[0184] In some embodiments, the engineered immune cells are T cells (e.g., inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes (Tregs), helper T lymphocytes, tumor-infiltrating lymphocytes (TILs)), natural killer (NK) cells, natural killer T cells (NKTs), TCR-expressing cells, dendritic cells, killer dendritic cells, mast cells, or B cells. In some embodiments, the cells may be derived from the group consisting of CD4+ T lymphocytes and CD8+ T lymphocytes. In some exemplary embodiments, the engineered immune cells are T cells. In some exemplary embodiments, the engineered immune cells are γδ T cells. In some exemplary embodiments, the engineered immune cells are macrophages. In some exemplary embodiments, the engineered immune cells are natural killer (NK) cells.

[0185] In some implementations, engineered immune cells may be derived from, for example, but not limited to, stem cells. Stem cells may be adult stem cells, non-human embryonic stem cells (more specifically, non-human stem cells), umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells (iPSCs), totipotent stem cells, or hematopoietic stem cells.

[0186] In some embodiments, the cells are obtained or prepared from peripheral blood. In some embodiments, the cells are obtained or prepared from peripheral blood mononuclear cells (PBMCs). In some embodiments, the cells are obtained or prepared from bone marrow. In some embodiments, the cells are obtained or prepared from umbilical cord blood. In some embodiments, the cells are human cells.

[0187] In some implementations, cells are transfected or transduced via nucleic acid vectors using methods selected from the group consisting of: electroporation, acoustic pore effect, gene gun method (e.g., gene gun), lipid transfection, polymer transfection, nanoparticles, viral transfection or transduction (e.g., retrovirus, lentivirus, AAV), or polymeric complexes.

[0188] In some embodiments, engineered immune cells expressing the CD19-specific CAR and CD70 binding proteins and / or CCRs of this disclosure at their cell surface membranes contain greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of stem cell memory cells and central memory cells. In some embodiments, engineered immune cells expressing the CD19-specific CAR and CD70 binding protein and / or CCR of this disclosure at their cell surface membrane comprise about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, about 15% to about 100%, about 15% to about 90%, about 15% to about 80%, about 15% to about 70%, about 15% to about 60%, about 15% to about 50%, about 15% to about 40%, about 15% to about 30%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 20% to about 30%, about 30% to about 1% 0.00%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, about 30% to about 50%, about 30% to about 40%, about 40% to about 100%, about 40% to about 90%, about 40% to about 80%, about 40% to about 70%, about 40% to about 60%, about 40% to about 50%, about 50% to about 100%, about 50% to about 90%, about 50% to about 80%, about 50% to about 80%. The percentage of stem cell memory cells and central memory cells is approximately 70%, approximately 50%, approximately 60%, approximately 100%, approximately 60%, approximately 90%, approximately 60%, approximately 80%, approximately 60%, approximately 70%, approximately 70%, approximately 90%, approximately 70%, approximately 80%, approximately 80%, approximately 100%, approximately 80%, approximately 90%, approximately 90%, approximately 100%, approximately 25%, approximately 50%, approximately 75%, approximately 100%, or approximately 50%, approximately 75%.

[0189] In some embodiments, the immune cells are inflammatory T lymphocytes expressing the CD19-specific CAR and CD70-binding protein described herein. In some embodiments, the immune cells are cytotoxic T lymphocytes expressing either the CD19-specific CAR or the CD70-binding protein described herein. In some embodiments, the immune cells are regulatory T lymphocytes expressing either the CD19-specific CAR or the CD70-binding protein described herein. In some embodiments, the immune cells are helper T lymphocytes expressing any of the CARs described herein.

[0190] Prior to amplification and genetic modification, cell sources can be obtained from donors or subjects using various non-limiting methods. Cells can be obtained from many non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, any number of T cell lines available and known to those skilled in the art can be used. In some embodiments, cells can be derived from healthy donors or from patients, such as those diagnosed with cancer or infections. In some embodiments, cells can be part of a mixed population of cells exhibiting different phenotypic characteristics.

[0191] This document also provides cell lines obtained from transformed immune cells (e.g., T cells) according to any of the methods described above. This document also provides modified cells resistant to immunosuppressive therapy. In some embodiments, the isolated cells according to this disclosure contain a polynucleotide encoding a CAR.

[0192] The immune cells of this disclosure can be activated and expanded using well-known methods before or after genetic modification of the immune cells. Typically, the engineered immune cells of this disclosure can be expanded, for example, by contacting agents that stimulate the CD3 TCR complex and co-stimulatory molecules on the surface of T cells to generate T cell activation signals. For example, chemicals such as the calcium ion carrier A23187, phorbol 12-myristate 13-acetate (PMA), or mitogen lectins (such as phytohemagglutinin (PHA)) can be used to generate T cell activation signals.

[0193] In some embodiments, it can be achieved by contacting, for example, an anti-CD3 antibody (such as an OKT3 antibody) or its antigen-binding fragment, or an anti-CD2 antibody immobilized on a surface, or by contacting a protein kinase C activator (e.g., ， Bryostatin is contacted with a calcium ionocarrier to stimulate T cell populations in vitro. Ligands binding to helper molecules on the T cell surface are used for co-stimulation. For example, under conditions suitable for stimulating T cell proliferation, T cell populations can be contacted with anti-CD3 antibodies (e.g., OKT3 antibodies) and anti-CD28 antibodies. The anti-CD3 and anti-CD28 antibodies can be set on beads (such as plastic or magnetic beads), plates, or other substrates. Suitable conditions for T cell culture include appropriate culture media (e.g., [missing information - likely related to a specific culture medium]). ， Minimal essential culture medium or RPMI Media 1640 or X-vivo 15 (Lonza) may contain factors required for proliferation and survival, including serum (e.g., serum). ，Fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-2, IL-15, TGFβ, and TNF, or any other cell growth additives known to the craftsman. Other additives for cell growth include, but are not limited to, surfactants, plasma salts, and reducing agents such as N-acetylcysteine ​​and 2-mercaptoethanol. Culture media may include RPMI 1640, A1M-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, which contain added amino acids, sodium pyruvate, and vitamins, and may be serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined group of hormones, and / or a sufficient amount of cytokines (e.g., IL-7 and / or IL-15) to promote T cell growth and expansion. Antibiotics, such as penicillin and streptomycin, are contained only in the experimental culture and not in the cell culture to be infused into the subject. The target cells are maintained under conditions necessary to support growth, such as a suitable temperature (e.g., 37°C) and atmosphere (e.g., air with 5% CO2). T cells exposed to different stimulation times may exhibit different characteristics. In some embodiments, the cells of this disclosure can be expanded by co-culturing with tissues or cells. The cells can also be expanded in vivo, for example, by expanding in the blood of a subject after the cells have been administered to the subject.

[0194] In some embodiments, the engineered immune cells according to this disclosure may contain one or more disrupted or inactivated genes. In some embodiments, the engineered immune cells according to this disclosure contain one disrupted or inactivated gene selected from the group consisting of CD52, GR, PD-1, CD70, CTLA-4, LAG3, TIM3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, 2B4, HLA, TCRα, and TCRβ, and / or express CAR, multi-chain CAR, and / or pTα transgenes. In some embodiments, the isolated cells contain polynucleotides encoding a polypeptide comprising a multi-chain CAR. In some embodiments, the isolated cells according to this disclosure contain two disrupted or inactivated genes selected from the group consisting of: CD52 and GR, CD52 and TCRα, CD52 and TCRβ, GR and TCRα, GR and TCRβ, TCRα and TCRβ, PD-1 and TCRα, PD-1 and TCRβ, CTLA-4 and TCRα, CTLA-4 and TCRβ, LAG3 and TCRα, LAG3 and TCRβ, TIM3 and TCRα, Tim3 The method includes expressing TCRβ, BTLA and TCRα, BTLA and TCRβ, BY55 and TCRα, BY55 and TCRβ, TIGIT and TCRα, TIGIT and TCRβ, B7H5 and TCRα, B7H5 and TCRβ, LAIR1 and TCRα, LAIR1 and TCRβ, SIGLEC10 and TCRα, SIGLEC10 and TCRβ, 2B4 and TCRα, 2B4 and TCRβ, and / or expressing CAR, multi-stranded CAR, and pTα transgenes. In some embodiments, the method comprises destroying or inactivating one or more genes by introducing a nuclease capable of selectively inactivating the gene through selective DNA cleavage into the cell. In some embodiments, the endonuclease may be, for example, a zinc finger nuclease (ZFN), a megaTAL nuclease, a mega-nuclease, a transcription activator-like effector nuclease (TALE nuclease / TALEN), or a CRISPR endonuclease (e.g., Cas9, Cas12, Cas12a, Cas12b, or Cas12i), or a CRISPR reagent or system. See, for example, US11,168,324, US20230332119, US20230193243, and US20230235305.

[0195] In some embodiments, TCRs are rendered nonfunctional in cells according to this disclosure by disrupting or inactivating the TCRα gene and / or one or more TCRβ genes. Modified cells capable of proliferating independently of the TCRα signaling pathway are covered within the scope of this disclosure. In some embodiments, a method is provided for obtaining modified cells derived from an individual, wherein said cells are capable of proliferating independently of the major histocompatibility complex (MHC) signaling pathway. Modified cells readily obtainable by this method that can proliferate independently of the MHC signaling pathway are covered within the scope of this disclosure. The modified cells disclosed herein can be used for patients in need of treatment against host-vG rejection and graft-versus-host disease (GvHD); therefore, the scope of this disclosure includes methods for treating patients in need of treatment against host-vG rejection and graft-versus-host disease (GvHD) comprising treating the patient by administering to the patient an effective amount of modified cells containing disrupted or inactivated TCRα and / or TCRβ genes.

[0196] In some embodiments, immune cells are engineered to be resistant to one or more chemotherapeutic agents. The chemotherapeutic agents may be, for example, purine nucleotide analogs (PNAs), thereby adapting the immune cells for cancer treatment in combination with adoptive immunotherapy and chemotherapy. Exemplary PNAs include, for example, clofarabine, fludarabine, cyclophosphamide, and cytarabine, alone or in combination. PNAs are metabolized by deoxycytidine kinase (dCK) to monophosphate, diphosphate, and triphosphate PNAs. Their triphosphate forms compete with ATP for DNA synthesis, act as pro-apoptotic agents, and are potent inhibitors of ribonucleotide reductases (RNRs) involved in trinucleotide production. This document provides CD19-specific CAR-T cells containing a destroyed or inactivated dCK gene. In some embodiments, dCK knockout cells are prepared by transfecting T cells with a polynucleotide encoding a specific TAL nuclease targeting the dCK gene, for example, by transfecting T cells with mRNA encoding a specific TAL nuclease via electroporation. dCK knockout CD19-specific CAR-T cells are resistant to PNA, including, for example, clofarabine and / or fludarabine, and retain T-cell cytotoxicity against CD19-expressing cells.

[0197] In some embodiments, the isolated cells or cell lines of this disclosure may contain pTα or a functional variant thereof. In some embodiments, the isolated cells or cell lines may be further genetically modified by disrupting or inactivating the TCRα gene.

[0198] This disclosure also provides engineered immune cells comprising any of the CAR polynucleotides described herein. In some embodiments, the CAR can be introduced into immune cells as a transgene via a plasmid vector. In some embodiments, the plasmid vector may also contain, for example, a selection marker that provides identification and / or selection of cells receiving the vector.

[0199] CAR peptides can be synthesized in situ within cells after the introduction of a polynucleotide encoding the CAR peptide into the cells. Alternatively, CAR peptides can be generated extracellularly and then introduced into the cells. Methods for introducing polynucleotide constructs into cells are known in the art. In some embodiments, the polynucleotide construct can be integrated into the cellular genome using a stable transformation method (e.g., using a lentiviral vector). In other embodiments, the polynucleotide construct, as well as the polynucleotide construct not integrated into the cellular genome, can be transiently expressed using a transient transformation method. In other embodiments, a virus-mediated method can be used. Polynucleotides can be introduced into cells by any suitable means, such as, for example, recombinant viral vectors (e.g., retroviruses, adenoviruses), liposomes, etc. Transient transformation methods include, for example, but not limited to, microinjection, electroporation, or particle bombardment. Polynucleotides can be included in a vector (e.g., a plasmid vector or a viral vector).

[0200] In some implementations, the nucleic acid construct is contained within a viral vector. In some implementations, the viral vector is selected from the group consisting of: retroviral vectors, murine leukemia virus vectors, SFG vectors, adenovirus vectors, lentiviral vectors, adeno-associated virus (AAV) vectors, herpesvirus vectors, and vaccinia virus vectors. In some implementations, the nucleic acid is contained within a plasmid.

[0201] Methods for manufacturing engineered immune cells This article provides methods for preparing the CAR and CAR-containing immune cells disclosed herein.

[0202] A variety of known techniques can be used in the preparation of polynucleotides, peptides, carriers, antigen-binding domains, immune cells, compositions, etc., according to the present disclosure. Prior to the in vitro manipulation or genetic modification of the immune cells described herein, cells can be obtained from a subject (e.g., a patient) or from a healthy donor.

[0203] a. Source materials In some implementations, the immune cells include T cells. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some implementations, T cells can be obtained from a unit of blood collected from the subject using any number of techniques known to the art (such as FICOLL™ isolation).

[0204] Cells can be obtained from an individual's circulating blood via apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In some embodiments, the cells collected via apheresis can be washed to remove the plasma fraction and placed in a suitable buffer or medium for subsequent processing.

[0205] In some embodiments, T cells are isolated from PBMCs by lysing red blood cells and depleting monocytes (e.g., using centrifugation via a PERCOLL™ gradient). Specific T cell subsets (e.g., CD28+, CD4+, CDS+, CD45RA-, CD45RO+, CDS+, CD62-, CD95-, CD95+, IL2R®+, IL2R®-, CCR7+, CCR7-, CDL-, CD62L+, and combinations thereof) can be further isolated using positive or negative selection techniques known in the art. For example, T cell subsets are CD45RA+, CD95-, IL-2R®-, CCR7+, and CD62L+. For example, T cell subsets are CD45RA+, CD95+, IL-2R®+, CCR7+, and CD62L+. For example, T cell subsets are CD45RO+, CD95+, IL-2R®+, CCR7+, and CD62L+. For example, T cell subsets include CD45RO+, CD95+, IL-2R®+, CCR7-, and CD62L-. For example, T cell subsets include CD45RA+, CD95+, IL-2R®+, CCR7-, and CD62L-. For example, T cell populations can be enriched by negative selection by conjugating antibodies against surface markers specific to negatively selected cells. One method used herein is cell sorting and / or selection or flow cytometry via negative magnetic immunoadsorption, using a mixture of monoclonal antibodies against cell surface markers present on negatively selected cells. For example, to enrich CD4+ cells by negative selection, the monoclonal antibody mixture typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. Flow cytometry and cell sorting can also be used to isolate cell populations of interest for use in this disclosure.

[0206] PBMCs can be directly used for genetic modification with immune cells (such as CARs or TCRs) as described herein. In some embodiments, after PBMC isolation, before or after genetic modification and / or expansion, T lymphocytes can be further isolated, and both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subsets.

[0207] In some embodiments, CD8+ cells are further sorted into naive, stem cell memory, central memory, and effector cells by identifying characteristic cell surface antigens associated with each of these types of CD8+ cells. In some embodiments, the expression of phenotypic markers for central memory T cells includes CD45RO, CD62L, CCR7, CD28, CD3, and CD127, and they are negative for granzyme B. In some embodiments, stem cell memory T cells are CD45RO-, CD62L+, and CD8+ T cells. In some embodiments, central memory T cells are CD45RO+, CD62L+, and CD8+ T cells. In some embodiments, effector T cells are negative for CD62L, CCR7, CD28, and CD127, and positive for granzyme B and perforin.

[0208] In some implementations, CD4+ T cells are further sorted into subsets. For example, CD4+ T helper cells can be sorted into naive, central memory, and effector cells by identifying cell populations with characteristic cell surface antigens.

[0209] b. Immune cells derived from stem cells In some implementations, immune cells may be derived from stem cells, such as progenitor cells, bone marrow stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem cells, and mesenchymal stem cells. iPS cells and other types of stem cells may be cultured immortalized cell lines or isolated directly from a patient. Various methods for isolating, developing, and / or culturing stem cells are known in the art and may be used in the practice of this invention.

[0210] In some embodiments, the immune cells are induced pluripotent stem cells (iPSCs) derived from reprogrammed T cells. In some embodiments, the source material can be induced pluripotent stem cells (iPSCs) derived from T cells or non-T cells. Alternatively, the source material can be B cells, or any other cells derived from peripheral blood mononuclear cell isolates, hematopoietic progenitor cells, hematopoietic stem cells, mesenchymal stem cells, adipose-derived stem cells, or any other somatic cell type.

[0211] c. Genetic modification of isolated cells This document provides methods for preparing engineered cells (e.g., engineered immune cells) for immunotherapy. In some embodiments, the methods include obtaining cells from a donor (e.g., donor immune cells), introducing the polypeptides described herein into the donor cells, and expanding the cells.

[0212] Immune cells (such as T cells) can be genetically modified after isolation using known methods, or immune cells can be activated and expanded in vitro (or differentiated in the case of progenitor cells) prior to genetic modification. In some embodiments, isolated immune cells are genetically modified to reduce or eliminate the expression of endogenous TCRα and / or CD52. In some embodiments, the cells are genetically modified using gene-editing technologies (e.g., CRISPR / Cas9, CRISPR / CAS12, zinc finger nucleases (ZFNs), TALEN or TALE-nucleases, MegaTAL, mega-nucleases) to reduce or eliminate the expression of endogenous proteins (e.g., TCRα and / or CD52). In another embodiment, immune cells (such as T cells) are optionally further genetically modified with chimeric antigen receptors as described herein (e.g., transduced with a viral vector containing one or more nucleotide sequences encoding a CAR), followed by in vitro activation and / or expansion.

[0213] Methods for activating and expanding T cells are known in the art and are described, for example, in U.S. Patent Nos. 6,905,874, 6,867,041, 6,797,514, and PCT WO2012 / 079000, the contents of which are hereby incorporated herein by reference in their entirety. Typically, such methods involve contacting PBMCs or isolated T cells with stimulating and co-stimulating molecules (such as anti-CD3 and anti-CD28 antibodies) in a culture medium containing appropriate cytokines (such as IL-2), which are typically attached to plastic beads, magnetic beads, or other surfaces. The anti-CD3 and anti-CD28 antibodies attached to the same beads serve as “surrogate” antigen-presenting cells (APCs). One example is the Dynabeads® system, a CD3 / CD28 activator / stimulator system for the physiological activation of human T cells. In other embodiments, methods such as those described in U.S. Patent Nos. 6,040,177, 5,827,642, and WO2012129514 can be used to activate and stimulate T cell proliferation in the presence of feeder cells and appropriate antibodies and cytokines, the contents of which are hereby incorporated by reference in their entirety.

[0214] Certain methods for preparing the constructs and engineered immune cells of this disclosure are described in PCT application PCT / US15 / 14520, the contents of which are hereby incorporated by reference in their entirety.

[0215] It will be understood that PBMCs may also include other cytotoxic lymphocytes, such as NK cells or NKT cells. Expression vectors carrying coding sequences for chimeric receptors as disclosed herein can be introduced into populations of human donor T cells, NK cells, or NKT cells. In addition to cell activation using anti-CD3 antibodies and IL-2 or other methods known in the art as described elsewhere herein, successfully transduced T cells carrying the expression vector can be sorted by flow cytometry to isolate CD3-positive T cells, which are then further proliferated to increase the number of these CAR-expressing T cells. CAR-expressing T cells are cryopreserved using standard procedures for storage and / or preparation for use in human subjects. In one embodiment, in vitro transduction, culture, and / or expansion of T cells are performed in the absence of products of non-human animal origin, such as fetal calf serum and fetal bovine serum. In one embodiment, cryopreservation may include freezing in suitable media, such as CryoStor® CS10, CryoStor® CS2, or CryoStor® CS5 (BioLife Solutions).

[0216] For the cloning of polynucleotides, a vector can be introduced into a host cell (an isolated host cell) to allow the vector to replicate itself, thereby amplifying the copy number of the polynucleotides contained in the vector. Cloning vectors may contain sequence components, typically including, but not limited to, origin of replication, promoter sequences, transcription initiation sequences, enhancer sequences, and selectability markers. These elements can be selected by those skilled in the art as appropriate. For example, the origin of replication can be selected to facilitate autonomous replication of the vector in the host cell.

[0217] In some embodiments, this disclosure provides isolated host cells containing the vectors provided herein. Host cells containing the vectors can be used for the expression or cloning of the polynucleotides contained in the vectors. Suitable host cells may include, but are not limited to, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells (such as mammalian cells), and more specifically, human cells.

[0218] The vector can be introduced into host cells using any suitable method known in the art, including but not limited to DEAE-dextran-mediated delivery, calcium phosphate precipitation, cationic lipid-mediated delivery, liposome-mediated transfection, electroporation, microparticle bombardment, receptor-mediated gene delivery, and delivery mediated by polylysine, histones, chitosan, and peptides. Standard methods for viral transfection, transduction, and / or transformation of cells expressing the vector of interest are well known in the art. In another embodiment, a mixture of different expression vectors, each encoding a different CAR or different recombinant protein as disclosed herein, can be used when genetically modifying a donor population of immune effector cells. The resulting transduced immune effector cells form a mixed population of engineered cells, a subset of which express more than one different CAR or recombinant protein.

[0219] In one embodiment, this disclosure provides a method for storing genetically engineered cells expressing a CAR targeting the CD19 protein. In one embodiment, this involves cryopreserving immune cells so that the cells remain viable after thawing. In one embodiment, cryopreservation may include freezing in a suitable medium, such as CryoStor® CS10, CryoStor® CS2, or CryoStor® CS5 (BioLife Solutions). A subset of CAR-expressing immune cells can be cryopreserved using methods known in the art to provide a permanent source of such cells for future treatment of patients with malignant tumors. When needed, the cryopreserved transformed immune cells can be thawed, grown, and expanded to obtain more of these cells.

[0220] In some implementations, cells are prepared by first harvesting them from a culture medium, then washing and concentrating them to a therapeutically effective amount in a suitable medium and container system (“pharmaceutically acceptable” carrier). Suitable infusion media can be any isotonic formulation, typically physiological saline, Normosol™ R (Abbott), or Plasma-Lyte™ A (Baxter), but may also utilize 5% dextran aqueous solution or Ringer's lactate. The infusion media may be supplemented with human serum albumin.

[0221] This invention provides a method for generating a population of engineered cells (e.g., engineered immune cells) that contains and / or expresses the polynucleotides described herein. In one embodiment, a targeting approach can be used to introduce the polynucleotide into cells such that the polynucleotide or transgene is inserted or integrated into a target gene of interest at a specific location in the genome. In one embodiment, the targeting approach or site-specific integration includes using a nuclease (such as a rare-cutting endonuclease) that can cleave at a specific site within the target gene, thereby allowing the insertion of a transgene-containing polynucleotide or nucleic acid construct at the specific cleavage site. In one embodiment, the expression of one or more coding sequences in the polynucleotide or nucleic acid construct is controlled by a PGK promoter, such as the truncated PGK promoter described herein.

[0222] Prior to engineering the cells, cell sources can be obtained from the subject using a variety of non-limiting methods. Cells can be obtained from many non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, any number of T cell lines available and known to those skilled in the art can be used. In some embodiments, the cells can be derived from a healthy donor. In some embodiments, the cells can be part of a mixed population of cells exhibiting different phenotypic characteristics.

[0223] In one aspect, the present invention provides a method for preparing genetically modified immune cells, said genetically modified immune cells comprising one or more genetic modifications to one or more target genes involved in the function or activity of T cell receptor (TCR) αβ. In one embodiment, the target gene is TRAC. In another embodiment, the target gene is CD52. The genetic modification is performed such that the expression and / or activity of one or more target genes is disrupted or inactivated. By disrupting or inactivating the target genes, the aim is to prevent the target genes from being expressed in the form of functional proteins and to impair the activity of the gene products.

[0224] In some embodiments, the method includes inactivating one or more target genes by introducing a rare-cutting endonuclease into cells, said rare-cutting endonuclease being capable of selectively inactivating the target gene by introducing a specific double-strand break at a target sequence within the target gene. In other embodiments, the method includes inactivating or reducing the expression level of one or more genes by introducing a rare-cutting endonuclease capable of selectively inactivating genes through selective DNA cleavage into cells. In some embodiments, the rare-cutting endonuclease may be, for example, a transcription activator-like effector nuclease (TALE nuclease or TALEN), a megaTAL endonuclease, a zinc finger endonuclease, a mega-nuclease, or a CRISPR endonuclease (e.g., Cas9, Cas12, Cas12a, or Cas12i). In one embodiment, the rare-cutting endonuclease is a CRISPR endonuclease. In one embodiment, the rare-cutting endonuclease is not a TALE nuclease.

[0225] In one aspect, target gene inactivation is achieved via the use of CRISPR endonucleases. CRISPR endonucleases are clustered regularly spaced short palindromic repeat (CRISPR)-associated endonucleases, such as Cas9 or Cas12. These endonucleases associate with guide RNA (gRNA), which guides the endonuclease to cleave at a specific site by hybridization to the target region. In this case, one or more gRNA molecules can be designed to hybridize to one or more specific sites within one or more target genes to guide cleavage by at least one CRISPR endonuclease. Exemplary gRNAs targeting the TRAC and CD52 genes are provided in Table 3, SEQ ID NO: 46 and 47, respectively.

[0226] On the other hand, rare-cutting endonucleases can be introduced into cells before, simultaneously with, or after genetic modification of cells, such as immune cells. In one embodiment, a polynucleotide encoding or expressing a rare-cutting endonuclease is introduced into cells by appropriate methods (including, but not limited to, transfection, lipid transfection, transduction, and electroporation). In some embodiments, the polynucleotide encoding a rare-cutting endonuclease according to this disclosure can be mRNA introduced directly into cells, for example, via electroporation. In some embodiments, a pre-formed gRNA and CRISPR endonuclease complex is introduced into cells by appropriate methods (e.g., transfection or electroporation). In some embodiments, cell pulse technology can be used to temporarily permeabilize living cells to deliver substances into the cells. Parameters can be adjusted to determine the conditions for achieving high transfection efficiency with minimal mortality.

[0227] This document also provides methods for transfecting cells (such as immune cells, e.g., T cells). In some embodiments, the method includes: contacting the cells with nucleic acids (e.g., RNA) and applying agile pulse sequences to the cells, said agile pulse sequences comprising: (a) electrical pulses with a voltage range of about 2250 to 3000 V per centimeter; (b) pulse widths of 0.1 ms; (c) pulse intervals of about 0.2 to 10 ms between the electrical pulses of steps (a) and (b); (d) electrical pulses with a voltage range of about 2250 to 3000 V per centimeter, pulse widths of about 100 ms, and pulse intervals of about 100 ms between the electrical pulse of step (b) and the first electrical pulse of step (c); and (e) four electrical pulses with a voltage of about 325 V, pulse widths of about 0.2 ms, and pulse intervals of 2 ms between each of the four electrical pulses.

[0228] In some implementations, the method of transfecting cells (such as immune cells, e.g., T cells) includes contacting the cells with RNA and applying a fast pulse sequence to the cells, said fast pulse sequence comprising: (a) an electrical pulse with a voltage of approximately 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2400, 2450, 2500, 2600, 2700, 2800, 2900, or 3000 V per centimeter; (b) a pulse width of 0.1 ms; (c) a pulse interval between the electrical pulses in steps (a) and (b) of approximately 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ms; and (d) a voltage range of approximately 2250 to 3000 V per centimeter. V, for example, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2400, 2450, 2500, 2600, 2700, 2800, 2900 or 3000 V per centimeter, with a pulse width of 100 ms and a pulse interval of 100 ms between the electrical pulse in step (b) and the first electrical pulse in step (c); and (e) 4 electrical pulses with a voltage of about 325 V, a pulse width of about 0.2 ms, and a pulse interval of about 2 ms between each of the four electrical pulses. This application discloses any values ​​included within the above range. The electroporation medium can be any suitable medium known in the art. In some embodiments, the conductivity of the electroporation medium ranges from about 0.01 to about 1.0 millisiemens.

[0229] In other embodiments, the rare cleavage endonuclease is specific for recognizing nucleic acid sequences within the target gene. In another embodiment, the endonuclease is expressed in the cell upon introduction and generates double-strand breaks or cleavage sites at the nucleic acid recognition sequences within the target gene. The presence of double-strand breaks or cleavage sites allows for site-specific integration of the transgene via a recombinant vector, as further described herein.

[0230] Engineered cells, such as engineered immune cells, can be genetically engineered to express one or more transgenes. Transgenes can be introduced into the cell genome via various methods, including retroviral or lentiviral transduction. Transgenes delivered into cells in retroviral or lentiviral vectors can randomly integrate into the cell's genome after viral transduction. Random integration of transgenes can negatively or adversely affect the activity of engineered cells. Therefore, in some embodiments, one or more transgenes are introduced into predetermined loci in engineered immune cells via site-specific integration.

[0231] On the other hand, a site-specific integration method for producing genetically modified cells (e.g., genetically modified immune cells) is provided, the method comprising using homology-directed repair (HDR). In one embodiment, HDR comprises homology recombination (HR). In another embodiment, the method comprises using a recombinant vector configured to use HDR (e.g., HR) to facilitate transgene integration into cells (e.g., immune cells). In another embodiment, the method comprises using a polynucleotide comprising one or more sequences homologous to a predetermined integration site (e.g., a target gene). In another embodiment, the method does not include the use of non-homologous end joining (NHEJ), and / or is not performed without using a polynucleotide comprising one or more sequences homologous to a predetermined integration site (e.g., a target gene).

[0232] In one aspect, the site-specific integration method includes introducing a recombinant viral vector (e.g., an AAV vector) into a population of cells (e.g., immune cells), wherein the recombinant viral vector targets a double-strand break or cleavage site in a target sequence of a target gene. In some embodiments, the AAV vector is an AAV6 vector. The introduction of the recombinant viral vector into the cells can be performed before, simultaneously with, or after the introduction of a rare cleavage endonuclease into the cells, as further described herein. In one embodiment, the method includes providing a recombinant viral vector containing a donor template. The donor template contains one or more coding sequences or transgenes. In other embodiments, the donor template further includes flanking homologous arms. In another embodiment, the flanking homologous arms include a 5' homologous arm and a 3' homologous arm. In another embodiment, the 5' homologous arm contains a sequence homologous to a sequence 5' upstream of the double-strand break or cleavage site in the target sequence, and the 3' homologous arm contains a sequence homologous to a sequence 3' downstream of the double-strand break or cleavage site in the target sequence. In another embodiment, the donor template is integrated into the genome of a population of cells (e.g., an immune cell population) at the double-strand break or cleavage site. In another embodiment, the method includes culturing cells under conditions sufficient to allow transgene integration after the introduction of the recombinant viral vector. In one embodiment, the expression of the receptor peptide and / or nucleic acid inhibitor is controlled by a PGK promoter (e.g., a truncated PGK promoter).

[0233] In some embodiments, the method includes introducing a donor template containing a transgene into cells (e.g., immune cells) and amplifying the cells. In some embodiments, this disclosure relates to a method of engineering cells (e.g., immune cells) that includes providing cells (e.g., immune cells) and expressing one or more transgenes. In some embodiments, the method includes introducing at least one polynucleotide described herein into cells (e.g., by transfecting cells) and expressing one or more coding sequences or transgenes contained in a polypeptide. In one embodiment, the expression of one or more coding sequences or transgenes is controlled by a PGK promoter (e.g., a truncated PGK promoter).

[0234] In some embodiments, cells generated using SSI methods (e.g., engineered immune cells) express higher levels of one or more introduced transgenes compared to cells introduced via non-site-specific integration (SSI) methods (e.g., retroviral or lentiviral transduction). In some embodiments, engineered cells generated via SSI methods (e.g., engineered immune cells) exhibit improved activity of one or more introduced transgenes compared to cells introduced via non-SSI methods (e.g., retroviral or lentiviral transduction). In some embodiments, engineered cells generated using SSI methods (e.g., engineered immune cells) exhibit fewer genomic translocations of one or more transgenes compared to cells introduced via non-SSI methods (e.g., retroviral or lentiviral transduction).

[0235] In some embodiments, one or more transgenes are introduced into a target locus via site-specific integration. In some embodiments, engineered cells (e.g., engineered immune cells) contain additional genetic modifications, including but not limited to knocking out or knocking down endogenous genes, enhancing endogenous genes, and integrating exogenous genes. In some embodiments, engineered immunotherapy includes a transgene integrated into a first genetic locus and further includes one or more genetic modifications at a second genetic locus. In some embodiments, one or more genetic modifications at the first and / or second genetic locus include integrating a transgene into the first and / or second genetic locus. In one embodiment, the expression of the coding sequence of the transgene (e.g., the coding sequence of a receptor peptide and / or a nucleic acid inhibitor) is controlled by a PGK promoter (e.g., a truncated PGK promoter).

[0236] In some embodiments, the method includes the step of introducing a polynucleotide, nucleic acid construct, or molecule into a cell, wherein the polynucleotide, nucleic acid construct, or molecule contains a polynucleotide template for homologous recombination. In other embodiments, the polynucleotide template contains a sequence homologous to at least a portion of a nucleic acid sequence in a target gene, such that homologous recombination occurs between the target gene nucleic acid sequence and the polynucleotide template. In some embodiments, the polynucleotide template contains a 5' homologous arm homologous to a 5' sequence of a double-strand break in the target sequence of the target gene, a sequence encoding a transgene, and a 3' homologous arm homologous to a 3' sequence of a double-strand break in the target sequence of the target gene. After the target nucleic acid sequence is cleaved, a homologous recombination event occurs between the target nucleic acid sequence and the polynucleotide template.

[0237] d. Allogeneic CAR T cells In short, the process for manufacturing allogeneic CAR T therapy involves harvesting healthy, selected, screened, and tested PBMCs or T cells from a healthy donor. The allogeneic T cells are genetically edited to reduce the risk of graft-versus-host disease (GvHD) and prevent allogeneic rejection. Selected T cell receptor genes (e.g., TCRα, TCRβ) are knocked out to avoid GvHD. The CD52 gene may also be knocked out to make the CAR T product resistant to anti-CD52 antibody therapy. Thus, anti-CD52 antibody therapy can be used to deplete the host immune system through lymphatic depletion and allow the CAR T cells to remain transplanted for complete therapeutic effect. An exemplary anti-CD52 antibody may be, for example, alemtuzumab (SEQ ID NO: 67-74). Next, the T cells are engineered to express a CAR that recognizes certain cell surface proteins (e.g., CD19) expressed in, for example, hematologic malignancies. The engineered T cells are then purified and finally cryopreserved in vials for delivery to the patient.

[0238] e. Autologous CAR T cells Autologous chimeric antigen receptor (CAR) T-cell therapy involves collecting a patient's own cells (e.g., white blood cells, including T cells) and genetically engineering the T cells to express a CAR that recognizes a target antigen expressed on the cell surface of one or more specific cancer cells and kills the cancer cells. The engineered cells are then cryopreserved and subsequently administered to a patient from whom the cells were removed for further engineering.

[0239] Treatment This disclosure includes methods for treating or preventing diseases associated with CD19 and / or CD70, or adverse and / or elevated levels of CD19 and / or CD70 in a patient, the methods comprising administering to a patient in need an effective amount of at least one CAR-containing immune cell, or an immune cell comprising a CAR disclosed herein. In some embodiments, the CD70-binding protein or CD70 CAR targets CD70+ allogeneic reactive T cells in the patient and allows the engineered immune cells to have better persistence and expansion. In some embodiments, the engineered immune cells comprise a chimeric antigen receptor (CAR).

[0240] Methods for treating diseases or conditions, including cancer and autoimmune diseases, are provided. In some embodiments, this disclosure relates to generating a T-cell-mediated immune response in a subject, including administering an effective amount of the engineered immune cells disclosed herein to the subject. In some embodiments, the T-cell-mediated immune response targets one or more target cells.

[0241] The CAR-containing immune cells disclosed herein can be used to treat CD19-related diseases or conditions and / or CD70-related diseases or conditions. In some embodiments, the disease or condition is a CD19-related and / or CD70-related autoimmune disease or condition. CD19 is known to be expressed in plasma cells that produce autoreactive antibodies. CD70 expression is associated with acute or chronic immune activation, including diseases such as rheumatoid arthritis and systemic lupus erythematosus, and autoreactive B cells also express CD70. Studies have also shown that CD70 is involved in the differentiation of pro-inflammatory pathogenic lymphocytes with the ability to infiltrate the CNS.

[0242] Therefore, in some embodiments, the disease or condition can be an autoimmune disease, including but not limited to lupus, systemic lupus erythematosus (SLE), lupus nephritis, rheumatoid arthritis, systemic sclerosis, scleroderma, multiple sclerosis (MS), relapsing-remitting multiple sclerosis (RRMS), secondary progressive multiple sclerosis (SPMS), primary progressive multiple sclerosis (PPMS), neuromyelitis optica spectrum disorder (NMOSD), Sjögren's syndrome, stiff-person syndrome, graft-versus-host disease (GvHD), autoimmune hemolytic anemia (AIHA), immune thrombocytopenic purpura (ITP), and myositis. In some embodiments, the autoimmune disease or condition is a B-cell-related and / or T-cell-related autoimmune disease or condition. In some embodiments, the autoimmune disease or condition is associated with CD19+ and / or CD70+ B cells and / or CD19+ and / or CD70+ T cells. In some embodiments, the autoimmune disease or condition is associated with CD19+ B cells and / or CD70+ T cells. In some embodiments, the autoimmune disease or condition is associated with CD19+, CD70+ B cells and / or CD70+ T cells. In some embodiments, the autoimmune disease or condition is associated with CD19+ B cells and / or CD19+, CD70+ T cells. In some embodiments, the autoimmune disease or condition is associated with CD19+, CD70+ B cells and / or CD19+, CD70+ T cells. In some embodiments, the CAR-containing immune cells of this disclosure can be used to deplete pathogenic B cells and T cells in patients with depleted autoimmune diseases.

[0243] In some embodiments, pathogenic B cells and / or T cells are activated B cells and / or activated T cells. In some embodiments, pathogenic B cells and / or T cells are self-reactive B cells and / or self-reactive T cells. In some embodiments, activated B cells and activated T cells express CD19 and / or CD70. The engineered immune cells disclosed herein deplete or are capable of depleting activated B cells, short-lived and long-lived plasma cells, and / or activated T cells without affecting naive B cells and / or inactivated T cells.

[0244] In some embodiments, the target cells are tumor cells. In some aspects, this disclosure includes a method for treating or preventing malignant tumors, the method comprising administering to a subject in need an effective amount of at least one isolated antigen-binding domain as described herein. In some aspects, this disclosure includes a method for treating or preventing malignant tumors, the method comprising administering to a subject in need an effective amount of immune cells, wherein the immune cells contain at least one chimeric antigen receptor, and a CD70-binding protein and / or CCR as described herein. In some embodiments, the method for treating or preventing malignant tumors includes administering to a subject in need an effective amount of a population of immune cells, wherein the immune cells contain at least one chimeric antigen receptor, and a CD70-binding protein and / or CCR as described herein. The CAR-containing immune cells of this disclosure can be used to treat CD19-associated malignant tumors or malignant tumors involving aberrant CD19 expression. In some embodiments, the CAR-containing immune cells of this disclosure can be used to treat malignant tumors such as non-Hodgkin lymphoma (NHL), refractory and / or relapsed NHL, large B-cell lymphoma (LBCL), follicular lymphoma (FL), T-cell lymphoma (TCL), B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia (TALL), primary central nervous system lymphoma (PCNSL), mantle cell lymphoma (MCL), chronic lymphocytic leukemia (CLL), and peripheral T-cell lymphoma (PTCL). In exemplary embodiments, CAR-containing immune cells, such as the anti-CD19 CAR-T cells of this disclosure, are used to treat LBCL.

[0245] A method for reducing tumor size in a subject is also provided, the method comprising administering engineered cells of the present disclosure to the subject, wherein the cells contain a chimeric antigen receptor containing a CD19 antigen-binding domain and binding to a CD19 antigen on the tumor.

[0246] In some embodiments, the subject has a solid tumor or a hematologic malignancy such as lymphoma or leukemia. In some embodiments, engineered cells are delivered to a tumor bed, such as a tumor bed in small cell lung cancer. In some embodiments, the cancer is present in the subject's bone marrow. In some embodiments, the engineered cells are autologous immune cells, such as autologous T cells. In some embodiments, the engineered cells are allogeneic immune cells, such as allogeneic T cells. In some embodiments, the engineered cells are heterologous immune cells, such as heterologous T cells. In some embodiments, the engineered cells are transfected or transduced in vitro. As used herein, the term "in vitro cell" means any cell cultured in vitro.

[0247] "Effective dose" is any amount that provides the expected or beneficial outcome when used alone or in combination with another agent. For a therapeutic agent (such as engineered CAR T cells), "therapeutic effective dose," "effective dose," or "therapeutic effective dose" refers to any amount that, when used alone or in combination with another therapeutic agent, protects a subject from disease onset or promotes disease remission (manifested as a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom resolution, or prevention of injury or disability caused by the disease). The ability of a therapeutic agent to promote disease remission can be assessed using a variety of methods known to skilled practitioners (e.g., physicians or clinicians), such as assessment in human subjects during clinical trials, assessment in animal model systems predicting efficacy in humans, or assessment by measuring the activity of the agent in an in vitro assay.

[0248] The terms “patient” and “subject” are used interchangeably and include human and non-human animal subjects, as well as people with a formally diagnosed condition, people with an undiagnosed condition, people receiving medical care, people at risk of developing a condition, etc.

[0249] The term "treatment" includes therapeutic treatment, preventative treatment, and applications that reduce the risk of a subject developing a certain condition or other risk factors. Treatment does not necessarily require a complete cure for the condition and encompasses implementation plans that alleviate symptoms or potential risk factors. The term "prevention" does not require a 100% elimination of the likelihood of the event. Rather, it indicates a reduced likelihood of the event occurring in the presence of a compound, immune cell, therapeutic agent, or method.

[0250] The desired therapeutic total number of cells in the composition includes at least two cell types (e.g., at least one CD8+ T cell and at least one CD4+ T cell, or two CD8+ T cells, or two CD4+ T cells), or more typically greater than 10. 2 Cells, and at most 10 6 One, up to 10 8One or 10 9 One cell, and can be 10 10 Or 10 12 One or more cells. The number of cells will depend on the intended use of the composition and the type of cells included in the composition. A cell density greater than 10 is typically desired. 6 10 cells / ml and usually greater than 10 7 cells / ml, typically 10 8 Cells / ml or greater. Clinically relevant numbers of immune cells can be allocated to multiple infusions, accumulating to 10 or more. 5 10 6 10 7 10 8 10 9 10 10 10 11 Or 10 12 Cells. In some aspects of this disclosure, particularly because all infused cells will be redirected to a specific target antigen (e.g., CD19), a lower number of cells can be administered, in 10 6 / kg (10 6 -10 11 CAR therapy can be administered multiple times at doses within these ranges. For patients undergoing therapy, the cells can be autologous, allogeneic, or xenogeneic.

[0251] In some implementations, the therapeutically effective dose of CAR T cells is approximately 1 x 10⁻⁶. 5 cells / kg, approximately 2 x 10 5 Cells / kg, approximately 3 x 10 5 Cells / kg, approximately 4 x 10 5 1 cell / kg, approximately 5 x 10 5 Cells / kg, approximately 6 x 10 5 1 cell / kg, approximately 7 x 10 5 Cells / kg, approximately 8 x 10 5 Cells / kg, approximately 9 x 10 5 cells / kg, 2 x 10 6 Cells / kg, approximately 3 x 10 6 Cells / kg, approximately 4 x 10 6 1 cell / kg, approximately 5 x 10 6 Cells / kg, approximately 6 x 10 6 1 cell / kg, approximately 7 x 10 6 Cells / kg, approximately 8 x 10 6Cells / kg, approximately 9 x 10 6 cells / kg, approximately 1 x 10 7 cells / kg, approximately 2 x 10 7 Cells / kg, approximately 3 x 10 7 Cells / kg, approximately 4 x 10 7 1 cell / kg, approximately 5 x 10 7 Cells / kg, approximately 6 x 10 7 1 cell / kg, approximately 7 x 10 7 Cells / kg, approximately 8 x 10 7 1 cell / kg or approximately 9 x 10 7 Cells / kg

[0252] In some implementations, the target dose range for CAR+ / CAR-T+ cells is approximately 1 × 10⁻⁶. 6 To approximately 1×10 10 cells / kg, for example, about 1×10⁻⁶ 6 cells / kg, approximately 1×10 7 cells / kg, approximately 1×10 8 cells / kg, approximately 1×10 9 Cells / kg or approximately 1×10⁻⁶ 10 Cells / kg. It should be understood that doses higher or lower than this range may be suitable for some subjects, and healthcare providers may determine the appropriate dose level as needed. Furthermore, multiple doses of cells may be provided according to this disclosure.

[0253] In some aspects, this disclosure includes a pharmaceutical composition comprising at least one CD19-specific CAR as described herein and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition further comprises an additional active agent.

[0254] The CAR-expressing cell populations disclosed herein can be administered alone or as pharmaceutical compositions in combination with diluents and / or other components (such as IL-2 or other cytokines or cell populations). The pharmaceutical compositions of this disclosure may comprise CAR-expressing cell populations (such as T cells) as described herein, said cell populations in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may comprise buffers, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose, or dextran, mannitol; proteins; peptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of this disclosure can be formulated for intravenous administration.

[0255] Pharmaceutical compositions (solutions, suspensions, etc.) may include one or more of the following: sterile diluents, such as water for injection, saline solutions (preferably physiological saline, Ringer's solution, isotonic sodium chloride), non-volatile oils (such as synthetic monoglycerides or diglycerides) that can be used as solvents or suspension media, polyethylene glycol, glycerol, propylene glycol, or other solvents; antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates, or phosphates; and agents for adjusting tonicity, such as sodium chloride or dextran. Parenteral preparations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic. For therapeutic applications, injectable pharmaceutical compositions are preferably sterile.

[0256] In some embodiments, when administered to a patient, engineered immune cells expressing any of the CD19-specific CARs described herein on their cell surface can reduce, kill, or lyse the patient's endogenous CD19-expressing cells. In one embodiment, the percentage reduction or lysis of endogenous CD19-expressing cells or CD19-expressing cell lines by engineered immune cells expressing any of the CD19-specific CARs described herein is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In one embodiment, the percentage reduction or lysis of endogenous CD19-expressing cells or CD19-expressing cell lines expressed by engineered immune cells expressing any of the CD19-specific CARs described herein is about 5% to about 95%, about 10% to about 95%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 25% to about 75%, or about 25% to about 60%. In one embodiment, the endogenous CD19-expressing cells are endogenous CD19-expressing bone marrow cells.

[0257] In one embodiment, the assays disclosed herein can be used to measure engineered immune cells expressing the CD19-specific CAR of this disclosure on their cell surface membranes, target cells (e.g., ， The percentage reduction or lysis of cell lines expressing CD19.

[0258] The method may further include administering one or more chemotherapeutic agents to the patient prior to the administration of the engineered cells provided herein. In some embodiments, the chemotherapeutic agent is a lymphodepleting (preconditioning) chemotherapeutic agent. For example, a method of preconditioning a patient requiring T-cell therapy includes administering a specific beneficial dose of cyclophosphamide (200 mg / m²) to the patient. 2 / day up to 2000 mg / m 2 / day, approximately 100 mg / m 2 / day to approximately 2000 mg / m 2 / day; for example, approximately 100 mg / m² 2 / day, approximately 200 mg / m 2 / day, approximately 300mg / m 2 / day, approximately 400 mg / m 2 / day, approximately 500 mg / m 2 / day, approximately 600 mg / m 2 / day, approximately 700 mg / m 2 / day, approximately 800 mg / m 2 / day, approximately 900 mg / m 2 / day, approximately 1000 mg / m 2 / day, approximately 1500 mg / m 2 / day or approximately 2000 mg / m 2 ( / day) and a specific dose of fludarabine (20 mg / m²) 2 / day up to 900 mg / m 2 / day, approximately 10 mg / m² 2 / day to approximately 900 mg / m 2 / day; for example, approximately 10mg / m² 2 / day, approximately 20 mg / m 2 / day, approximately 30 mg / m 2 / day, approximately 40 mg / m 2 / day, approximately 40 mg / m 2 / day, approximately 50 mg / m 2 / day, approximately 60 mg / m 2 / day, approximately 70 mg / m 2 / day, approximately 80 mg / m 2 / day, approximately 90 mg / m 2 / day, approximately 100 mg / m 2 / day, approximately 500mg / m 2 / day or approximately 900 mg / m 2 / day). An exemplary dosing regimen involves treating a patient and includes administering approximately 300 mg / m² to the patient daily. 2 / day of cyclophosphamide and approximately 30 mg / m2 Fludarabine per day, or cyclophosphamide administered to the patient before or after fludarabine administration for three days, followed by a therapeutically effective dose of engineered T cells.

[0259] In some embodiments, particularly when the engineered cells provided herein have been genetically edited to eliminate or minimize the surface expression of CD52, lymphocyte depletion further includes administration of an anti-CD52 antibody, such as alemtuzumab. In some embodiments, the CD52 antibody is administered at a dose of about 1-20 mg / day IV, such as about 13 mg / day IV, such as about 20 mg / day IV, such as about 30 mg / day IV, for 1, 2, 3 days or longer. The antibody may be administered in combination with other elements of the lymphocyte depletion regimen (e.g., cyclophosphamide and / or fludarabine), before or after the use of these components. Exemplary anti-CD52 antibody sequences, such as SEQ ID NO: 67-74, are provided in Table 3.

[0260] In other implementations, the antigen-binding domain, transducing (or other engineered) cells, and chemotherapeutic agents are each administered in an amount effective in treating the subject's disease or ailment.

[0261] In some embodiments, the compositions disclosed herein comprising CAR-expressing immune effector cells can be administered in combination with any number of chemotherapeutic agents. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; and ethyleneimine and methylmelamine derivatives, including altretamine, triethylenemelamine, and triethylenylphosphamide. ephosphoramide, triethylenethiophosphoramide, and trimethylomelamine; nitrogen mustards, such as chlorambucil, chlomaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, and mechlorethamine hydrochloride. Oxygen hydrochloride, melphalan, novombhichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitroureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimnustine;Antibiotics, such as aclacinomysin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin C, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin D, daunorubicin, detorubicin, 6-diazo-5-oxo-L-leucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, and mycophenolic acid. Folic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, such as methotrexate and fluorouracil (5-FU); folic acid analogues, etc. Examples include denopterin, methotrexate, pteropterin, and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; and pyrimidine analogs such as ancitabine, azacitidine, 6-azouridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, fluxuridine, and 5-FU.Androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; anti-adrenergics, such as aminoglutethimide, mitotane, and trilostane; folic acid supplements, such as frolinic acid; aceglatone; aldophosphamide glycoside; and aminolevulinic acid. acid); amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; mitoguazone; mitoxantrone; mopidamnol; nitraerine; pentostatin; phenamet; pirarubicin; podophyllinic acid acid); 2-ethylhydrazine; procarbazine; PSK®; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2”-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactalol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa;Taxanes, such as paclitaxel (TAXOL™, Bristol-Myers Squibb) and docetaxel (TAXOTERE®, Rhone-Poulenc Rorer); chloranmbucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C. C); mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RF S2000; difluoromethylornithine (DMFO); retinic acid derivatives such as Targretin™ (bexarotene), Panretin™ (alitretinoin); ONTAK™ (denileukin diftitox); esperamicins; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the above. This definition also includes anti-hormonal agents that modulate or inhibit the effects of hormones on tumors, such as anti-estrogens, including, for example, tamoxifen, raloxifene, aromatase inhibitors 4(5)-imidazole, 4-hydroxytamoxifen, trioxifene, raloxifene hydrochloride, LY117018, onapristone, and toremifene (Fareston); and anti-androgens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing. Chemotherapy agents, including but not limited to the CHOP regimen, may also be administered in combination where appropriate, namely cyclophosphamide (Cytoxan®), doxorubicin (hydroxydoxorubicin), vincristine (Oncovin®), and prednisone.

[0262] In some embodiments, the chemotherapeutic agent is administered concurrently with or within one week of the engineered cells, peptides, or nucleic acids. In other embodiments, the chemotherapeutic agent is administered approximately 1–7 days, approximately 1 to approximately 4 weeks, approximately 1 week to approximately 1 month, approximately 1 week to approximately 2 months, approximately 1 week to approximately 3 months, approximately 1 week to approximately 6 months, approximately 1 week to approximately 9 months, or approximately 1 week to approximately 12 months after the administration of the engineered cells, peptides, or nucleic acids. In other embodiments, the chemotherapeutic agent is administered at least 1 month prior to the administration of the cells, peptides, or nucleic acids. In some embodiments, the method further includes administering two or more chemotherapeutic agents.

[0263] A variety of additional therapeutic agents can be used in combination with the compositions described herein. For example, potentially useful additional therapeutic agents include PD-1 inhibitors such as nivolumab (Opdivo®), pembrolizumab (Keytruda®), pembrolizumab, pidilizumab, and atezolizumab.

[0264] Additional therapeutic agents suitable for use in combination with this disclosure include, but are not limited to: ibrutinib (Imbruvica®), ofatumumab (Arzerra®), rituximab (Rituxan®), bevacizumab (Avastin®), trastuzumab (Herceptin®), trastuzumab emtansine (KADCYLA®), imatinib (Gleevec®), cetuximab (Erbitux®), and panitumumab.Vectibix®, catumaxomab, ibritumomab, oftatumumab, tositumomab, brentuximab, alemtuzumab, gemtuzumab, erlotinib, gefitinib, vandetanib, afatinib, lapatinib, and more. Latinib, Axitinib, Masitinib, Pazopanib, Sunitinib, Sorafenib, Toceranib, Lestaurtinib, Axitinib, Cedilanib, Lenvatinib, Nintedanib, Pazopanib, Regorafenib nib), semaxanib, sorafenib, sunitinib, tivozanib, toceranib, vandetanib, entrectinib, cabozantinib, imatinib, dasatinib, nilotinib, ponatinib, radotinib, bosutinib Bosutinib, Letatinib, Ruxolitinib, Pacritinib, Cobimetinib, Selumetinib, Trametinib, Binimetinib, Alectinib, Ceritinib, Crizotinib, Aflibercept, Adipotide, Denileukin Diftitox, mTOR inhibitors such as Everolimus and Temsirolimus, Hedgehog inhibitorsInhibitors such as sodegib and vismodegib, and CDK inhibitors such as the CDK inhibitor palbociclib.

[0265] In some embodiments, compositions comprising CAR-containing immune cells as described herein may be administered in conjunction with a treatment regimen to prevent or reduce cytokine release syndrome (CRS) or neurotoxicity. Treatment regimens for preventing CRS or neurotoxicity may include lenzilumab, tocilizumab, atrial natriuretic peptide (ANP), anakinra, and iNOS inhibitors (e.g., L-NIL or 1400W). In other embodiments, compositions comprising CAR-containing immune cells may be administered in conjunction with an anti-inflammatory agent. Anti-inflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), nonsteroidal anti-inflammatory drugs (NSAIDs) (including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide), anti-TNF drugs, cyclophosphamide, and mycophenolate mofetil. Exemplary NSAIDs include ibuprofen, naproxen, naproxen sodium, COX-2 inhibitors, and sialic acid derivatives. Exemplary analgesics include acetaminophen, oxycodone, tramadol, or propoxyphene hydrochloride. Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include molecules targeting cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors such as TNF antagonists (e.g., etanercept (ENBREL®), adalimumab (HUMIRA®), and infliximab (REMICADE®), chemokine inhibitors, and adhesion molecule inhibitors. Biological response modifiers include monoclonal antibodies and recombinant forms of molecules. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, gold preparations (oral (auronoxine) and intramuscular), and minocycline. In some embodiments, the compositions described herein may be used to treat autoimmune diseases or conditions.

[0266] In some embodiments, the compositions described herein are administered in combination with cytokines. Examples of cytokines include lymphokines, monokines, and conventional polypeptide hormones. Among the cytokines are: growth hormones, such as human growth hormone, N-methionylhuman growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; pro-relaxin; glycoprotein hormones, such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); hepatocyte growth factor (HGF); fibroblast growth factor (FGF); prolactin; placental prolactin; Müllerian inhibitory substances; mouse gonadotropin-related peptides; and inhibitory peptides. Inhibitors; activators; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factor (NGF), such as NGF-β; platelet-derived growth factor; transforming growth factor (TGF), such as TGF-α and TGF-β; insulin-like growth factor-I and insulin-like growth factor-II; erythropoietin (EPO); bone-inducing factor; interferons, such as interferon-α, interferon-β, and interferon-γ; colony-stimulating factors (CSF), such as macrophage-CSF. (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs), such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, IL-21; tumor necrosis factor, such as TNF-α or TNF-β; and other polypeptide factors, including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins derived from natural sources or recombinant cell cultures, as well as bioactive equivalents of naturally occurring sequence cytokines.

[0267] reagent kits and products This application provides a kit comprising any of the CD19 CAR-containing immune cells described herein, and pharmaceutical compositions thereof. In some embodiments, the CD19 CAR T cells contain or express CD70-specific CARs. In one embodiment of the kit, engineered CAR cells (e.g., CAR T cells) are frozen in a suitable culture medium, such as CryoStor® CS10, CryoStor® CS2, or CryoStor® CS5 (BioLife Solutions).

[0268] In some exemplary embodiments, the kits disclosed herein include T cells containing allogeneic CD19 CAR for administering lymphocyte depletion and CAR-T regimens to subjects.

[0269] This application also provides articles or kits comprising any of the therapeutic compositions described herein. Examples of articles include bags or vials (e.g., sealed vials).

[0270] Table 3 below shows exemplary sequences.

[0271] Example Experimental methods To prepare lentiviruses encoding the desired constructs, HEK293T cells were seeded at 450,000 cells / mL in 2 mL of DMEM (Gibco) supplemented with 10% FBS (Hyclone) in each well of a 6-well plate the day before transfection. On the day of transfection, lentiviruses were prepared as follows: 1.5 μg psPAX2 lentiviral packaging vector, 0.5 μg pMD2G, and 0.5 μg appropriate transfer CAR vector were mixed together in 250 μL Opti-MEM (Gibco) in each well of a 6-well plate (“DNA mixture”). 10 μL of Lipofectamine 2000 (Invitrogen) in the 250 μL Opti-MEM was incubated at room temperature for 5 minutes, and then added to the DNA mixture. The mixture was incubated at room temperature for 20 minutes, and a total volume of 500 μL was slowly added to the well walls containing HEK293T cells. One day after transfection, the culture medium for HEK293T cells in each well of a 6-well plate was replaced with 2 mL of T cell transduction medium per well, i.e., X-Vivo-15 medium supplemented with 10% FBS. Two days after transfection, the lentiviral supernatant from HEK293T cells was harvested and passed through a 0.45 μm filter (EMD Millipore) to remove cell debris. The supernatant was then concentrated 25-fold using a Lenti-X concentrator (Takara Bio) according to the manufacturer's instructions, aliquoted, and rapidly frozen. Lentiviral titers were determined by thawing aliquots of frozen lentivirus, performing 4-fold serial dilutions, and then performing limiting dilution titrations on JurkaT cells (clone E6-1; ATCC). On day 0, purified T cells were activated in Grex-24 plates (Wilson Wolf, catalog number 80192M) in X-Vivo-15 medium (Lonza) supplemented with 100 IU / mL human IL-2 (Miltenyi Biotec), 10% FBS (Hyclone), and human T TransAct (Miltenyi Biotec, catalog number 130-111-160, 1:100 dilution). On day 2, T cells were resuspended at 500,000 cells / mL in T cell transduction medium and transduced in Grex-24 plates at MOI=5 with the appropriate lentiviral stock solution and 100 IU / mL human IL-2. On day 5 after transduction, cells were harvested and washed to remove residual IL-2. They were then resuspended in T-cell expansion medium, namely X-Vivo-15 supplemented with 5% human AB serum (Gemini Bio), and each sample was divided into two parts, one part receiving 100 IU / mL human IL-2 according to the standard protocol, and the other part receiving a lower concentration of 25 IU / mL human IL-2.Cells were expanded into larger G-Rex containers (Wilson Wolf) as needed using T-cell expansion medium and appropriate concentrations of human IL-2. The absolute number of T cells in each sample was counted on days 5, 9, and 14, and transduction efficiency was determined by detecting the percentage of T cells binding to the FITC-conjugated v5-tagged monoclonal antibody (Thermo Fisher) using flow cytometry. The CAR-T cell products were cryopreserved on day 14 or 15 and thawed as needed for further assays.

[0272] For cells generated via AAV6 site-specific integration, TransAct was removed by centrifugation two days after T cell activation. Cells were then washed with PBS and electroporated using an AMAXA 4D nuclear transfection electroporator at 1 x 10⁻⁶ cells per cell. 6 Cells were electroporated with 0.5 μg of TALEN® mRNA per arm of nuclease. Following the gene editing step, recombinant AAV6 containing one or more transgenes was added to T cells at an MOI of 10,000–15,000. After electroporation, cells were incubated overnight at 30°C. Cells were then returned to 37°C and the above T cell culture conditions. On day 14 post-activation, TCRα / β depletion was performed using the EasySep™ Human TCRα / β Depletion Kit (STEMCELL Technologies) according to the manufacturer's instructions. On day 15 or 16 post-activation, T cells were cryopreserved in 90% FBS / 10% DMSO using a rate-controlled freezer and stored in liquid nitrogen vapor phase.

[0273] AAV-mediated site-specific integration can also be performed using CRISPR endonucleases, such as Cas12i endonuclease, or CRISPR reagents. Pan-T cell donors were thawed and activated for 2 days with GMP-grade TransAct and IL-2. Prior to electroporation, Cas12i nuclease and TRAC-targeting guide RNA (gRNA) were incubated together at room temperature to form a ribonucleoprotein complex (RNP). T cells were then mixed with the RNP and electroporated. Immediately afterward, cells were incubated overnight with AAV containing one or more transgenes at a specified MOI and then transferred to GREX plates in complete T cell culture medium supplemented with IL-2. Cells were subsequently expanded for 12 days and then frozen. Flow cytometry analysis was performed on days 4, 7, and 11 post-electroporation. The transgene cassette was expected to insert into the target sequence GACCCTGCC or ATCCAGAACCCTGACCCTGCCGTGTACCAG (SEQ ID NO: 92) within exon 1 of the TRAC. Standard long-term killing assay (LTKA) was performed. CAR T cells were co-cultured with Raji cells expressing luciferase-GFP at an effector-to-target cell ratio of 8:1. Every 2–3 days, half of the cells were passaged into fresh Raji cells. The remaining half of the cells were then used to measure target cell killing using Bright-glo reagent (Promega).

[0274] Continuous restimulation assay based on flow cytometry In a 24-well G-rex® (Wilson Wolf, catalog number 80192M), place 5x10 5 CAR T cells and 5x10 5 Raji luciferase-GFP cells were co-cultured in RPMI 1640 + 10% FBS medium (Hyclone). A small aliquot of the co-cultured CAR T cells and target cells was stained with antibodies using 123count eBeads™ counting beads (Thermo Fisher Scientific, catalog number 01-1234-42) to determine the baseline phenotype and cell count at the start of the assay. Every 3–4 days, 50% of the supernatant was removed from the G-rex® container, and the remaining cell suspension was thoroughly mixed. A small aliquot was then used for phenotypic analysis and cell counting, as described above. After determining the cell count, fresh Raji cells were added back to each well to restore the E:T ratio to 1:1. Continuous restimulation was repeated until CAR T cells no longer killed Raji target cells.

[0275] Single-stimulation FACS assay Similar to flow cytometry-based continuous stimulation assays, CAR T cells and Raji cells were co-cultured in 24-well G-rex® cells at an E:T ratio of 1:2 or 1:4, rather than a 1:1 E:T ratio. Every 3–4 days, 50% of the supernatant was removed from the G-rex® cells, and the remaining cell suspension was thoroughly mixed and a small aliquot was taken for phenotypic analysis and cell counting, as described above. Fresh Raji cells were not added back; only fresh culture medium was added back. Time points were collected every 3–4 days until Raji cells were completely eliminated by CAR T cells, or until Raji cells outnumbered T cells.

[0276] Sensitized T mixed lymphocyte response (MLR) Human PBMCs (host cells) were sensitized to irradiated, unedited T cells derived from donors used to create the aforementioned gene-edited transplanted T cells to promote the expansion of allogeneic reactive T cell clones. Briefly, transplanted PBMCs were irradiated with 30 Gy and co-cultured with host PBMCs at a 1:1 ratio in R10 + 20 IU / mL IL-2 + 10 ng / mL IL-7 + 10 ng / mL IL-15 (Miltenyi, catalog number 130-095-765) for 4 days. The medium was then replaced with cytokine-free R10, and the cells were cultured for another 3 days. Subsequently, pan-T cells were isolated using MACS negative selection (Miltenyi, Human Pan-T Cell Isolation Kit, catalog number 130-096-535) as recommended by the manufacturer. In 96-well plates, 20,000 gene-edited transplanted T cells were seeded together with 20,000 sensitized host T cells and cultured for 2 days at 37°C and 5% CO2 in R10 + 20 IU / mL IL-2. The viability of the transplanted T cells was determined by flow cytometry using gated absolute counts of live TCRαβ-CAR+ T cells.

[0277] Example 1: Generation of CAR T cells expressing anti-CD19 CAR from lentiviral vector Based on the anti-CD19 scFv, CD3z signaling domain, and 4-1BB co-stimulatory domain of SEQ ID NO: 4, constructs for expressing second-generation anti-CD19 CAR were designed and cloned into lentiviral vectors (LVV). CAR T cells generated from donor PBMCs either expressed CD19 CAR alone or expressed a chimeric cytokine receptor (CCR) and CD19 CAR (CD19 CAR / CCR) from a bicistronic expression cassette linked via a cleavable P2A peptide. The CCR contained a TPOR transmembrane / JAK binding and activation domain and an IL2R intracellular signaling domain, as illustrated in SEQ ID NO: 27. Gene expression in all LVV constructs was driven by the elongation factor 1α (EF-1α) promoter. CAR T cells were generated from donor PBMCs activated by LVV transduction. The cytotoxic activity of CAR T cells with different constructs from three different donors against CD19-positive Raji and Daudi cells was evaluated by a long-term killing assay (LTKA) in vitro. like Figures 1A to 1C As shown, in most cases, co-expression of CCR enhances the cytotoxicity of CD19 CAR T cells.

[0278] In an orthotopic tumor model established by injecting Raji cells into NSG mice, the in vivo cytotoxicity of CAR T cells engineered with a selected construct was evaluated. Briefly, Raji cells expressing luciferase were intravenously injected into mice. CAR T cells were injected 4 days after tumor cell transplantation. Tumor burden was measured using an IVIS Spectrum instrument. Figures 2A to 2B As shown, CD19 CAR T cells co-expressing CCR in 3x10 6 At a dose level of CAR T cells, tumor cells in animal models were effectively reduced.

[0279] Example 2: Site-specific integration of CAR T cells expressing anti-CD19 CAR LVV constructs were transduced into PBMCs to generate engineered cells with randomly integrated transgenes in the host cell genome. Transgenes can be introduced into cells via site-specific integration (SSI) into predetermined genetic loci to ensure consistency of insertion sites in the genome and limit the number of integration events. Site-specific integration using vectors such as adeno-associated virus (AAV) can also accommodate one or more transgenes, which can be larger than those accommodated by lentiviral vectors, while maintaining high transduction efficiency. CD19 CAR / CCR constructs cloned into AAV vectors were inserted into the TRAC locus via homologous recombination, and the expression of CCR and CD19 CAR was driven by a short EF1α promoter (EFS). The resulting CAR T cells were evaluated for in vivo cytotoxicity in a Raji orthotopic tumor model in NSG mice and compared with CAR T cells expressing the same CCR and CD19 CAR generated via LVV transduction (N=8 mice / group, 2.5 x 10⁻⁶ cells / year). 6 CAR + (cells / mouse).

[0280] Figure 3A The results showed, unexpectedly, that CAR T cells generated through site-specific integration exhibited significantly lower tumor control in vivo compared to CAR T cells transduced via LVV. CAR T cells derived from different donors reproduced these results. Consistent with poor in vivo cytotoxicity, CAR T cells generated through site-specific integration failed to expand during the experimental period and did not persist in treated mice, in contrast to CAR T cells transduced via LVV. Figure 3B ).exist Figures 5A to 5CSimilar in vitro results were also observed, in which CAR T cells generated by site-specific integration (SSI) driven by the EFS promoter (SSI EFS CD19 / CCR) showed lower cytotoxicity in standard long-term killing assays and expanded less than CAR T cells generated by LVV transduction.

[0281] Example 3: Optimization of site-specific integrated anti-CD19 CAR construct To enhance the in vitro and in vivo antitumor activity of our SSI-engineered CAR T cells, we tested a range of different promoters to drive CCR and CAR expression. All SSI constructs were designed to integrate into the T cell receptor α constant region (…). TRAC The CD19CAR gene locus was used. Surface expression of CD19CAR was measured by flow cytometry using anti-idiotype (anti-ID) antibodies (ACROBiosystems) to measure the mean fluorescence intensity (MFI) of CAR and to detect the antitumor activity of CAR T cells. Several heterologous promoters were tested, including three derived from human phosphoglycerate kinase 1 (…). PGK1 The promoters of the gene were named PGK-SSI-1, PGK-SSI-2 and PGK-SSI-3.

[0282] CAR MFI is measured at the end of manufacturing. It is derived from human ubiquitin B (…). UBB The CAR construct driven by the promoter (UBB300) exhibited the highest CAR expression at the end of manufacturing. Figure 4A The three PGK SSI promoters, as well as the human elongation factor 1-α1 short (EFS) promoter and the human cyclophilin A (CypA300) promoter, all produced similar CAR expression at the end of manufacturing. Figure 4A Next, in a flow cytometry-based continuous restimulation cytotoxicity assay, CAR T cells were analyzed against the CD19+ tumor cell line Raji at an effector cell to target cell (E:T) ratio of 1:1. Surprisingly, the PGK SSI promoter construct exhibited superior antitumor activity compared to promoters used in all other assays. Figure 4B ).

[0283] To understand the mechanisms underlying the differences in antitumor efficacy, we monitored CAR expression (MFI) during consecutive restimulation assays. We noted that although the CAR MFI was similar between the EFS and PGK SSI promoters at the end of the process ( Figure 4AHowever, the promoters regulated CAR expression differently after exposure to CD19+ tumor cells. On day 3 post-target exposure, all promoters increased CAR expression (except for those controlled by the TRAC endogenous promoter and the UBC300 promoter), with the EFS and UBB promoters reaching the highest expression levels. Figure 4C Another TRAC endogenous promoter was tested, and similar results were observed (data not shown). By day 17, all promoters downregulated CAR expression; however, the EFS and UBB promoters maintained significantly higher expression levels for the remainder of the assay compared to the PGK SSI promoter. Figure 4C Surprisingly, this sustained high level of CAR expression after day 17 was associated with CAR T cell expansion. Figure 4D ) and antitumor activity ( Figure 4B The expression was negatively correlated with CAR expression. In contrast, the PGK SSI promoter maintained low levels of CAR expression from day 17 onwards, but showed the highest peak amplification compared to other constructs. Figure 4D ) and optimal antitumor activity ( Figure 4B Despite the reluctance to be limited to specific mechanisms, the results indicate that the EFS and UBB promoters lead to long-term high CAR expression, which may result in CAR T cell exhaustion and dysfunction; while the PGK SSI promoter leads to low CAR expression after target exposure, thereby maintaining CAR T cell function and resulting in better CAR T cell expansion and better anti-tumor activity.

[0284] Based on these results, we then tested the PGK SSI promoter (e.g., PGK SSI-1) and compared it with the EFS promoter in CAR T cells generated by site-specific integration and CAR T cells generated by LVV transduction. Figure 5A The results showed that, in standard LTKA, when tested against wild-type Raji cells at an E:T ratio of 4:1, CD19 CAR T cells co-expressing CCR under the PGK SSI-1 promoter (but not the EFS promoter) performed comparably to LVV cells expressing the same CD19 CAR and CCR. When tested against wild-type Raji cells at an E:T ratio of 1:2 in a single-stimulation FACS-based power assay, the PGK SSI-1-driven SSI construct outperformed not only the EFS construct but also LVV-transduced CAR T cells, as evidenced by the number of remaining target cells (…). Figure 5B ) and the expansion of CAR T+ cells ( Figure 5C This has been confirmed in all of them. Figure 5A All results shown in Figure C were obtained using CAR T cells derived from cells from the same donor.

[0285] The results of the in vitro killing assay were confirmed in vivo in the Raji orthotopic tumor NSG mouse model. In the animal tumor model, CD19 CAR T cells driven by the PGK SSI-1 promoter were superior to LVV-transduced CAR T cells and other constructs driven by the Cyp300A and EFS promoters at two different cell doses. Figures 6A to 6B ).

[0286] Example 4: CD19 CAR T cells co-express CD70 binding protein to reduce rejection and improve persistence Allogeneic CAR T cells derived from donor cells may be recognized as foreign substances and rejected by the immune system of patients receiving allogeneic CAR T cells. To reduce rejection by allogeneic reactive host T cells and improve persistence and further enhance the efficacy of CAR T cells, CD70-binding protein was introduced into and expressed in CD19 CAR T cells. CD70 is a membrane-bound protein expressed on immune cells, particularly activated T cells, including allogeneic reactive T cells. CAR T cells expressing CD70-binding protein or CD70-binding protein (e.g., CD70-specific CAR in this experiment) can bind to CD70 expressed on the surface of allogeneic reactive host T cells, leading to the death of the allogeneic reactive host T cells.

[0287] To illustrate this, CD19 CAR / CCR T cells generated through site-specific integration into the TRAC locus were tested in an in vitro MLR assay. In the MLR assay, host T cells were first sensitized for 7 days with unedited allogeneic transplanted cells, and then co-cultured with genetically edited CAR T cells and target Raji tumor cells. As shown in Figure 7A, because the CAR T cells do not express CD70-binding protein, they were rejected by allogeneic reactive CD70+ host T cells and could not proliferate (Figure 7D), while allogeneic reactive host T cells proliferated (Figure 7B), and CD19+ Raji tumor cells continued to grow (Figure 7C).

[0288] Conversely, when CD70-binding protein was co-expressed in CD19 CAR / CCR T cells, CAR T cells were able to proliferate and persist (Figs. 7E to 7H), while allogeneic reactive CD70+ host T cells failed to proliferate (Fig. 7F), and CD19+ Raji tumor cells were eliminated (Fig. 7G, comparison squares, no CD70-binding protein; circles, CD70-binding protein present). The CD70-binding protein exemplified in this experiment includes anti-CD70 scFv, a CD8 hinge / transmembrane domain, and a wild-type CD3z signaling domain. In this experiment, the construct expressing CD70-binding protein was inserted into the CD52 locus via site-directed integration. Data were obtained from four distinct host pairs: transplant donors, and two independent experiments are shown.

[0289] Example 5: CD19 CAR / CCR T cells co-expressing CD70 binding protein exhibited enhanced cytotoxic activity by preventing antigen escape.

[0290] Studies have shown that, under low E:T ratios in vitro, Raji lymphoma cells downregulate CD19 expression upon encountering anti-CD19 effector cells (Schneider et al. 2017, J. Immunotherapy of Cancer 5:42). Since CD70 is expressed on CD19+ target tumor cells, dual targeting of CD19 and CD70, compared to CD19 single-targeting, may prevent CD19 antigen escape and enhance anti-tumor activity. To verify this hypothesis, in a single-antigen stimulation FACS assay, engineered Raji cells expressing GFP (CD70 wild-type (WT) or CD70 knockout (KO)) were first incubated with TRAC KO CAR T cells or untransduced TRAC KO (NTD) control cells at a low E:T ratio of 1:3 for 0–4 days. Figure 8A The total number of live Raji cells gated by GFP+ and CD20+ was measured on days 0, 1, 2, and 4. Figure 8B Data showed that on day 4, in Raji CD70 wild-type or Raji CD70 KO cells, when CD19 was targeted alone, Raji cells downregulated CD19 expression. CD19-CD70+ Raji cells were able to evade killing by CD19 CAR / CCR, but could not escape killing by CD19 CAR / CCR T cells co-expressing CD70 binding protein. Figure 8B When counting the total number of viable Raji cells during the experimental period, Figure 8C Data suggests that the Raji cells remaining on day 4 (which may be CD19 negative) can only be cleared in the presence of dual-targeting CD19 CAR / CCR T cells that co-express CD70 binding protein. Figure 8D The results were confirmed by careful examination of CD19-negative Raji cells. The results shown are representative data from two independent experiments.

[0291] The observations were also confirmed in in vivo studies using an orthotopic mouse model. (5×10) 6 One CD19 CAR T cell was administered to NSG mice that had previously received Raji cells (N=10 mice / group). At 5x10 6 At the endpoint of the dose, bone marrow was collected, and Raji cells were analyzed in vitro by flow cytometry. Figures 8E to 8F The data confirmed that co-expression of CD70 binding protein did not negatively affect the cytotoxic activity of CD19 CAR / CCR T cells in vivo, and further demonstrated the additional benefit of preventing the escape of CD19 antigen in vivo.

[0292] Example 6: Analysis of CD19 CAR T cell design expressing CD70 binding protein Next, we investigated the potential impact of co-expression of CD70-binding proteins on CAR T cell production. Since CD70 is expressed on T cells, especially activated T cells, the interaction between CD70-binding proteins on neighboring cell surfaces and CD70 can lead to cannibalism. Figure 9A T cells expressing CD70-binding protein, such as CD70-binding protein-sing T cells or CD19 CAR and CD70-binding protein-double-positive CAR T cells, are protected from cannibalism because the CD70-binding protein cis-binds to the surface CD70 protein on the same cell, thus masking the surface CD70 protein. Figure 9A As shown. T cells that do not express CD70 binding protein, such as CD19 CAR single-positive CAR T cells, will not be immune to cannibalism. Figure 9A ).

[0293] To better understand masking and cannibalism events, we analyzed CD19CAR / CCR T cells that co-expressed CD70-binding protein during cell generation. In this experiment, the CD52 locus of the cells integrated CD70-binding protein, and the TRAC locus integrated a bicistronic expression construct of CCR and CD19CAR. As the generation process progressed from day 9 to day 14, the CD70-binding protein single-positive population and the CD19CAR / CCR and CD70-binding protein double-positive population were enriched, while the CD19CAR / CCR single-positive population decreased, which may be due to cannibalism. Figure 9B ).

[0294] Example 7: Optimization and Analysis of CD19 CAR T Cell Design Expressing CD70 Binding Protein Next, we designed a series of such Figure 10A The constructed organism shown was used to enrich a double-positive population of CD19 CAR and CD70 binding proteins and increase yield. Phenotypic analysis was performed on the generated CAR T cells.

[0295] Using cells from two different donors, the expression of CD19CAR and CD70 binding proteins was analyzed by flow cytometry at the end of the production process (day 14). Figure 10B ), and the quantification results are as follows Figures 10C to 10D As shown (symbols represent individual donors). The results of cell phenotype, exhaustion marker analysis, and activation marker analysis are as follows: Figure 10E , Figure 10F and Figure 10G As shown. Except for the data from day 9 of the activation marker analysis, all data are shown as day 14 of the generation process. CAR T cell expansion data are as follows. Figure 10H As shown, and the CD4:CD8 ratio is as follows: Figure 10I As shown.

[0296] From these experiments, we draw the following conclusions: Figure 10A CAR T cells expressing both CD19 CAR and CD70 binding proteins were successfully generated in the construct. The cell population proved to be homogeneous, primarily composed of cells that were double-positive for both CD19 CAR and CD70 binding proteins. Compared to other constructs tested, the use of tandem and dual constructs (…) Figure 10D (See the image above) A higher frequency of CD19 CAR+ cells was obtained. Among the constructs tested, the PGK SSI-1 CD19 / CD70 dual CAR T cells appeared to have the highest number of double-positive cells and consistently exhibited a good phenotype compared to other designs.

[0297] Next, CAR T cells were detected in an MLR assay targeting allogeneic reactive T cells. Figure 11A ). Figure 11B Data from the study indicate that, on average, EFS CD19 / CD70 dual, PGK SSI CD19 / CD70 dual, and PGK SSICD70z.CD19 / CCR cells exhibit comparable anti-rejection activity and better transplant survival than PGK SSI CD19CAR T cells that do not express CD70-binding proteins. The better transplant survival suggests that CD19 / CD70 dual CAR T cells can exhaust CD70+ host T cells.

[0298] The cytotoxic activity of CAR T cells was tested in a single-stimulation FACS assay. Raji CD70 KO cells were used to investigate the effects of CD19 CAR alone or CD19 CAR / CCR. Figures 12A to 12B Data from the study indicate that, in a single-stimulation FACS assay targeting CD70KO Raji cells, the PGK SSI CD19 / CD70 dual-cell approach outperformed other forms containing CD70-binding proteins in both tumor killing and CAR T cell amplification. For Raji, cells were gated as CD19+ CAR T cells and GFP+ cells. Similar results were observed in a single-stimulation FACS assay targeting CD70 wild-type Raji cells. Figures 12C to 12D ).all Figures 12A to 12D The table shows the average of three replicates, and the data represent two donors.

[0299] same, Figures 13A to 13B Data from the study indicates that, at an E:T ratio of 8:1, the PGK SSI CD19 / CD70 dual-design outperforms other designs in standard long-term lethality assays against CD70KO Raji. Figures 14A to 14G Data from the study showed that, in mouse tumor models, the PGK SSI CD19 / CD70 dual, PGK SSI CD19, and PGK SSI CD70.CD19 / CCR constructs all exhibited the best activity, and the PGK SSI CD70.CD19 / CCR construct showed the best activity at 5 × 10⁻⁶. 5 The design with 1 CAR T cell was superior to other designs (N = 8 NSG mice / group).

[0300] Example 8: Generation and Analysis of PGK SSI CD19 / CD70 Dual CAR T Cells Engineered Using CRISPR Reagents We generated PGK SSI CD19 / CD70 dual CAR T cells engineered using CRISPR gene-editing reagents (e.g., CRISPR endonuclease Cas12i and gRNA) to knock out TRAC and insert the transgene into the TRAC locus via site-specific integration. At the end of the process, the CAR T cell fold expansion between CD19 / CD70 dual CAR T cells and CD19 CAR T cells was significant. Figure 15A ) and CAR+ cell number ( Figure 15BThe fold expansion and CAR+ cell count at the end of the process were comparable between CRISPR-engineered CAR T cells and TALEN-engineered CAR T cells (data not shown). CRISPR-engineered CD19 / CD70 dual CAR T cells showed a higher CAR+ cell percentage and a higher peak MFI compared to CRISPR-engineered CD19 CAR T cells. Figures 15C to 15D ).

[0301] Next, we examined the expression level of CD70 binding protein (i.e., CD70 CAR) in CD19 / CD70 dual CAR T cells. Figures 15E to 15F Data from the study indicate that at the end of the production process, the expression of CD70-binding protein (i.e., CD70 CAR) increases, with a percentage of positive cells (%). Figure 15E ) and MFI ( Figure 15F This was indicated by the detection of anti-CD70 scFv anti-id antibody. No significant differences were observed between CRISPR-engineered CAR T cells and TALEN-engineered CAR T cells. The CD4:CD8 ratio decreased during the manufacturing process in all test groups. Figure 15G Furthermore, depending on the donor, the process completion rate is related to... Figure 10I The ratios shown are comparable or slightly lower. The percentages of CD25+ and 4-1BB+ CD4 or CD8 T cells in CD19 / CD70 dual CAR T cells are slightly higher than those in CD19 CAR T cells (respectively). Figure 15H and Figure 15I This is consistent with what happened before. Figure 10G The situation observed was similar. In CD19 / CD70 dual CAR T cells, the percentages of CD4 and CD8 T cells with Tscm / Tcm were slightly lower than in CD19 CAR T cells (respectively...). Figure 15J and Figure 15K ).

[0302] Next, we examined the activity of CAR T cells against the CD19+ human type B lymphoblast cell line Raji in a flow cytometry-based continuous restimulation assay. Figures 16A to 16B The data in the figure demonstrate cytotoxic activity against the target cell line (top figure) and fold expansion of CAR T cells from two donors within the same time period (bottom figure). Furthermore, no expansion of dual CAR T cells was observed in the absence of exogenous IL-2 stimulation and target cells. Figure 16I ).

[0303] Since the CD19+ Raji cell line also expresses CD70, this cell line can be used to confirm that CRISPR-engineered CD19 / CD70 dual CAR T cells can effectively target both CD19 and CD70 on Raji target cells. In short-term killing assays, target-dependent cytotoxicity was assessed using wild-type (WT) Raji-GFL-Luciferase (RGL), CD19KO RGL, or CD70KO RGL cells to independently study the effect of CD19 / CD70 dual CAR T cells on each target. CD19 / CD70 CAR T cells or control cells were co-cultured with target cells at a defined effector cell to target cell (E:T) ratio for 24 hours. Figure 16C Target cell killing was determined by luciferase assay. The CAR T cells tested were generated from two different donors. CD19 / CD70 dual CAR T cells effectively eliminated WT, CD19KO, and CD70KO RGL cells, indicating that they exhibit target-dependent cytotoxicity against both CD19 and CD70. Figure 16D As expected, control CD19 CAR T cells showed cytotoxicity against WT and CD70KO RGL cells, but not against CD19KO RGL cells. Dual targeting was also evaluated in a mixed tumor model. CD19KO (CD19 阴性 ) and CD70KO (CD70 阴性 RGL cells were mixed at a 1:1 ratio and then co-cultured with CAR T cells at a 1:1 E:T ratio. The growth of RGL cells over time was monitored by flow cytometry. Figure 16E When mixed RGL cells were co-cultured with control CD19 CAR T cells, CD19-positive RGLs (i.e., CD70KO RGLs) were eliminated. Figure 16F (See the bottom right image), while CD19-negative (CD19KO) RGL cells can expand ( Figure 16F (See bottom left image). Conversely, mixed RGL cells were cleared by CD19 / CD70 CAR T cells via dual targeting of CD19 and CD70, and could not proliferate. Figure 16F (See the top and bottom images). When measuring total RGL cells, only CD19 / CD70 dual CAR T cells (not control CD19 CAR T cells) cleared target cells, possibly due to the expansion of CD19-negative RGL cells, which control CD19 CAR T cells were unable to clear. Figure 16F (See the figure above). The data shown represents the combined results of repeated experiments from the same representative donor.

[0304] The ability of CD19 / CD70 dual CAR T cells to kill target-positive cells in vivo was tested in a mouse model. 1 x 10 5 One RGL cell was injected intravenously into NSG mice via the tail vein. One day prior to administration, mice were randomly divided into ten groups, with similar mean whole-body bioluminescence values ​​in each group. On day 4 post-tumor cell engraftment, cryopreserved CD19 / CD70 dual CAR T cells were thawed, washed, and resuspended in phenol red-free RPMI 1640 medium. In the treatment group, mice were intravenously administered 3.5 x 10⁻⁶ RGL cells. 6 Or 10 x 10 6 CAR + Cells / animals. Mice in the control group received no treatment and served as a reference. Tumor bioluminescence, body weight, and overall animal health were recorded twice weekly. Figure 16G As shown, the growth of target cells (as indicated by tumor volume) was reduced in a CD19 / CD70 dual CAR T dose-dependent manner. Figure 16H Data from the study indicate that the expansion of CD19 / CD70 dual CAR T cells in animals is also dose-dependent.

[0305] Example 9: CD19 / CD70 dual CAR T cells effectively target primary CD19+ B cells and CD70+ T cells. In order to test the original CD19 + Target-dependent cytotoxicity of B cells was assessed by co-culturing CD19 / CD70 CAR T cells or control cells with PBMCs from healthy individuals or patients with systemic lupus erythematosus (SLE) and performing short-term killing assays. Co-culture was performed for 24 hours at a defined E:T ratio. After 24 hours, CD19 levels were measured by flow cytometry. + B cell killing. The CAR T cells tested were generated from two different donors. CD19+ B cells from healthy individuals or SLE patients survived and were detected after co-culturing with untransduced (NTD) control cells. Figure 17A However, it was eliminated when co-cultured with control CD19CAR T cells or CD19 / CD70 CAR T cells. Figure 17A and Figure 17B The data shown are composite results from repeated experiments using the same representative donor.

[0306] CD70 is expressed in activated, allogeneic reactive T cells that reject transplanted CAR T cells. To test the anti-allogeneic rejection function of CD70-binding protein expressed in CD19 / CD70 dual CAR T cells (i.e., CD70 CAR), a mixed lymphocyte reaction (MLR) was performed to demonstrate that dual CAR T cells can effectively target and eliminate CD70+ primary T cells. For irradiated allogeneic transplanted T cells, PBMCs from healthy donors or SLE (systemic lupus erythematosus) patients were sensitized for 7 days. Sensitized allogeneic reactive T cells were isolated by magnetic separation (Miltenyi) and then co-cultured at a 1:1 ratio with CAR T cells derived from the same allogeneic transplanted T cells used in the sensitization step. The host allogeneic reactive T cells and transplanted CAR T cells were evaluated over time by flow cytometry. A total of four different host pairs were tested: transplant donor pairs, two of which were healthy host donors and two of which were SLE host donors. Figure 18A As shown in the right panel, NTD or CD19 CAR T cell controls were rapidly rejected by healthy donor-host allogeneic reactive T cells, while CD19 / CD70 dual CAR T cells resisted allogeneic rejection and proliferated over time. Conversely, when co-cultured with NTD controls or CD19 CAR T cells, CD70+ allogeneic reactive host T cells proliferated, but these cells were eliminated when co-cultured with CD19 / CD70 dual CAR T cells, indicating that CD70 CAR enhances the durability of CAR T cell resistance to host T cell rejection. Figure 18A (Left figure). Similar results were observed using allogeneic reactive T cells from SLE donors ( Figure 18B The results also showed that CD19 / CD70 dual CAR T cells effectively eliminated CD70+ primary T cells from both healthy and SLE donors. The data are a composite of technical replicates from representative host:transplant donor pairs.

[0307] Example 10: In vivo efficacy of CD19 / CD70 dual CAR T cells Several in vivo mouse SLE models were studied. For example... Figure 19A As shown, human PBMCs (2x10⁻¹⁰) were administered via intravenous (iv) injection. 7A humanized mouse model of humanized B lymphocytes (PBMCs) was established by adoptively transferring PBMCs (10 μg / mouse) into immunodeficient NSG mice (Jackson Laboratory). To improve B cell survival and engraftment, two conditions were tested in addition to a control group that received only PBMC injections. In one group, mice received human B lymphocyte stimulator (huBlyS, 10 μg / mouse, Peprotech) four times intraperitoneally (ip) over one week. In the second group, mice received 1 Gy irradiation one day before transplantation. Spleens were harvested 10 or 14 days post-transplantation, and human immune cell engraftment and phenotype were analyzed by flow cytometry. Figure 19B As shown, successful transplantation of B cells and T cells was detected in all mice. Under different test conditions, irradiation consistently improved the transplantation rate of different cell types. Figure 19B Therefore, it was selected for subsequent research. Different levels of CD70 expression were detected in both transplanted B cells and T cells. Figure 19C The data is a composite result from three different donors.

[0308] To monitor the dynamics of B cell remodeling and differentiation, we administered healthy donor PBMCs (5 x 10⁻⁶ cells / mL) via intravenous injection. 6 1x10 7 2x10 7 (1 cell / mouse) was transplanted into irradiated NSG mice (Jackson Laboratory) Figure 20A Spleens were harvested at different days post-injection (days 3, 7, and 10) and processed into single-cell suspensions. Human cell transplantation and phenotype were assessed using flow cytometry. Serum was collected at the study endpoint, and human IgG and IgM levels in mouse serum were measured by ELISA (ThermoFisher). Mice receiving different doses of PBMCs exhibited similar transplantation patterns. Within 10 days of PBMC adoptive transfer, transplanted B cells continued to proliferate and differentiate, characterized by a reduction in immature cells and an increase in memory phenotype, which could be indicated by staining for different biomarkers. Figures 20B to 20D A large number of plasmablasts and plasma cells were also detected, indicating that the transplanted B cells were activated and further differentiated after transfer. Figure 20D On day 10 post-adoptive transfer, human antibodies were detected in mouse serum, indicating IgG ( Figure 20E ) and IgM ( Figure 20F It is produced in a dose-dependent manner.

[0309] With the establishment of the PBMC humanized mouse model, according to Figure 21AThe protocol shown evaluated the in vivo efficacy of CD19 / CD70 dual CAR T cells in a model. 7-8 week old NSG mice (Jackson Laboratory) were pre-conditioned with 1 Gy irradiation, then... iv Inject 2x10 7 Healthy donor PBMCs. Three days later, mice were randomly assigned to receive two dose levels of CD19 / CD70 dual CAR T cells (1x10⁻¹²). 6 Or 4x10 6 CAR T cells / mouse, or no treatment. Spleen and blood were collected on days 4 and 7 after CAR T treatment for flow cytometry. HLA-A2 cell surface expression was used to differentiate between graft and host populations. Human IgG and IgM levels in plasma of control and treated mice were measured by ELISA (ThermoFisher). Figure 21B and Figure 21C As shown, administration of CD19 / CD70 dual CAR T cells at both dose levels led to CD19+ B cell depletion in mice. Figure 21B and Figure 21C (See above image) and reduced antibody production ( Figure 21C (See figure below). Furthermore, compared to the NT control group, the total expansion of host CD3+ T cells in the spleen was reduced on day 7 after CAR T therapy / day 10 after metastasis. Figure 21D ), and all CD70+ T cells were eliminated. Figures 21E to 21F Based on the targeted killing effect of CD19+ B cells and CD70+ T cells, the spleen of mice on day 7 post-treatment / day 10 post-metastasis ( Figure 21G ), on day 4 or day 7 after treatment / day 7 or day 10 after transfer, the spleen of mice ( Figure 21H (left image) or mouse blood ( Figure 21H In the right figure, dose-dependent expansion of CAR T cells was observed.

[0310] Next, as Figure 22A As shown, the in vivo efficacy of CD19 / CD70 dual CAR T cells was evaluated in a hematopoietic stem cell (HSC) humanized mouse model. 16-17 week old CD34+ hu-NSG mice (Jackson Laboratory) were used. iv Injection of CD19 / CD70 dual CAR T cells or untransduced T cells (6x10) 6Treatment was administered to mice at a dose of 1 cell / mouse. Changes in circulating B cells and T cells in mouse peripheral blood were monitored weekly by flow cytometry. HLA-A2 cell surface expression was used to differentiate between graft and host populations. CD19 / CD70 dual-CAR T cells depleted human B cells in CD34+ hu-NSG mice on day 7 post-treatment. Figure 22B Simultaneously, CD19 / CD70 dual CAR T cell expansion was observed. Figure 22D Subsequently, B cell depletion persisted until day 28 post-treatment, at which point host B cell recovery was detected. Figure 22B and Figure 22C (See the image above), while CD19 / CD70 dual CAR T cell proliferation decreased ( Figure 22E The host T cells generated in this model were all naive CD70- T cells (data not shown), and were largely unaffected by CD19 / CD70 dual CAR T therapy. Figure 22C (See the image below).

[0311] Example 11 Further evaluation of CD19 / CD70 dual CAR T cells Since T cells can be activated during infection, CD70 CAR activity may suppress the host T cell response to pathogens. For example, virus-specific T cells may be activated during toxin-producing infections and may upregulate CD70, making them susceptible to CD70 CAR targeting. Therefore, we evaluated the potential of dual-CAR T cells to influence virus-specific T cell (VST) activity. In this experiment, CMV pp65-specific T cells (VSTs) were co-cultured with pp65 peptide pulse antigen-presenting cells (APCs) and CD19 CAR T cells, CD19 / CD70 dual-CAR T cells, or untransduced control cells (NTDs) at an 8:1:1 ratio for 48 or 72 hours. APCs alone were also included as controls. See also Figure 23A Data indicate that the presence of CD19 / CD70 dual CAR T cells does not affect the clearance of viral peptide pulsed APCs by VST cells. Figure 23B Furthermore, the presence of CD19 / CD70 dual CAR T cells at the end of the experiment did not affect the VST count. Figure 23C These results are expressed as the average of three technical replicates over 48 hours. Similar results were observed at 72 hours. APC is defined as HLA-A2. + CD3 阴性 TCR 阴性 CD20 + And VST is defined as HLA-A2 + CD3 + TCR + CD20- CAR T cells and NTD cells are gated based on HLA-A2 expression, and the results are derived from a single graft:host donor pair.

[0312] Next, we investigated whether the cytotoxic activity and expansion of CD19 / CD70 dual CAR T cells could be controlled by external agents such as ruxotinib. Ruxotinib is a Janus kinase (JAK) inhibitor that modulates the JAK-STAT pathway in immune cells. See, for example, Heine et al. people , 2013, Blood, 122; 1192-1202; Mantov et al. people ,2022, Front Pharmacol., 13:896167. In this experiment, dual CAR T cells were co-cultured with target Raji RGL cells (CD19+CD70KO) at different E:T ratios and incubated for 24 hours or 96 hours with or without 10 μM ruxolitinib. Figure 24A Data from the study indicate that ruxolitinib inhibited the cytolytic activity of CD19 / CD70 dual CAR T cells in a luciferase-based assay. Figure 24B Data from the study indicated that, in flow cytometry-based assays, ruxolitinib reduced the proliferation of CD19 / CD70 dual CAR T cells after 5 days of co-culture at an E:T ratio of 1:2.

[0313] While the disclosed teachings have been described with reference to various applications, methods, kits, and compositions, it should be understood that various changes and modifications can be made without departing from the teachings herein and the inventions claimed below. The above embodiments are intended to better illustrate the disclosed teachings and are not intended to limit the scope of the teachings presented herein. Although the teachings herein are described through these exemplary embodiments, those skilled in the art will readily understand that many variations and modifications can be made to these exemplary embodiments without requiring extensive experimentation. All such variations and modifications are within the scope of the existing teachings.

Claims

1. A polynucleotide comprising a truncated PGK promoter, a first coding sequence, and a second coding sequence, wherein the truncated PGK promoter comprises a deletion of at least 50 nucleotides from the 5' end of a long PGK promoter having the nucleotide sequence of SEQ ID NO: 34, wherein the truncated PGK promoter is operatively linked to the first coding sequence and the second coding sequence, and wherein the first coding sequence or the second coding sequence encodes an anti-CD19 chimeric antigen receptor (CAR).

2. The polynucleotide of claim 1, wherein the first coding sequence and the second coding sequence are linked by a P2A peptide.

3. The polynucleotide as claimed in any of the preceding claims, wherein the anti-CD19 CAR comprises the amino acid sequence of SEQ ID NO:

4.

4. The polynucleotide of any of the preceding claims, wherein the anti-CD19 CAR further comprises a CD8 hinge domain and a transmembrane domain.

5. The polynucleotide of any of the preceding claims, wherein the anti-CD19 CAR further comprises a CD3 signaling domain and / or a 4-1BB co-stimulatory domain.

6. The polynucleotide of any of the preceding claims, wherein the anti-CD19 CAR comprises the amino acid sequence of SEQ ID NO:5 or 7.

7. The polynucleotide of any of the preceding claims, wherein the coding sequence encoding the anti-CD19 CAR comprises a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 6 or 8.

8. The polynucleotide of any of the preceding claims, wherein the truncated PGK promoter comprises a deletion of at least 100 nucleotides, at least 150 nucleotides, at least 200 nucleotides, at least 250 nucleotides, at least 300 nucleotides, or at least 350 nucleotides from the 5' end of the long PGK promoter having the nucleotide sequence of SEQ ID NO:

34.

9. The polynucleotide of any of the preceding claims, wherein the truncated PGK promoter comprises a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 35, SEQ ID NO: 36, or SEQ ID NO:

37.

10. The polynucleotide of claim 9, wherein the truncated PGK promoter comprises a nucleotide sequence: (a) The nucleotide sequence comprises a deletion of about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380 or about 390 nucleotides from the 5' end of SEQ ID NO: 34, and has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 35; (b) The nucleotide sequence comprises a deletion of about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280 or about 290 nucleotides from the 5' end of SEQ ID NO: 34, and has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 36; (c) The nucleotide sequence comprises a deletion of about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, or about 190 nucleotides from the 5' end of SEQ ID NO: 34, and has at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 37; or (d) The nucleotide sequence comprises a deletion of about 50, about 60, about 70, about 80, about 90 or about 100 nucleotides from the 5' end of SEQ ID NO: 34, and has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the nucleotide sequence of SEQ ID NO:

38.

11. The polynucleotide of any of the preceding claims, wherein the first coding sequence encodes the anti-CD19 CAR and the second coding sequence encodes a CD70 binding protein, or wherein the second coding sequence encodes the anti-CD19 CAR and the first coding sequence encodes a CD70 binding protein.

12. The polynucleotide of claim 11, wherein the CD70 binding protein comprises an anti-CD70 antibody or an antigen-binding fragment thereof and a transmembrane domain, and optionally wherein the CD70 binding protein further comprises a hinge domain, and wherein the hinge domain and the transmembrane domain are a CD8 hinge domain and a CD8 transmembrane domain, or a CD28 hinge domain and a CD28 transmembrane domain.

13. The polynucleotide of claim 11 or 12, wherein the CD70 binding protein further comprises a CD3z signaling domain.

14. The polynucleotide of any one of claims 11 to 13, wherein the CD70 binding protein comprises a CD3z signaling domain and does not comprise a co-stimulatory domain.

15. The polynucleotide of any one of claims 11 to 14, wherein the CD70 binding protein comprises the amino acid sequences of SEQ ID NO: 16 and SEQ ID NO:

17.

16. The polynucleotide of any one of claims 11 to 15, wherein the CD70 binding protein comprises the amino acid sequence of SEQ ID NO:

18.

17. The polynucleotide of any one of claims 11 to 16, wherein the CD70 binding protein comprises the amino acid sequence of SEQ ID NO: 19 or 21.

18. The polynucleotide of any one of claims 11 to 17, wherein the coding sequence encoding the CD70 binding protein comprises a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 20 or 22.

19. The polynucleotide of any one of claims 1 to 10, wherein the first coding sequence encodes a chimeric cytokine receptor (CCR) and the second coding sequence encodes the anti-CD19 CAR, or the first coding sequence encodes the anti-CD19 CAR and the second coding sequence encodes a CCR.

20. The polynucleotide of claim 19, wherein the CCR comprises a thrombopoietin receptor / myeloproliferative leukemia protein receptor (TPOR / MPLR) transmembrane and JAK binding domain and an intracellular recruitment domain.

21. The polynucleotide of claim 20, wherein the TPOR / MPLR transmembrane domain and the JAK binding domain comprise the amino acid sequence of SEQ ID NO: 25 or 51.

22. The polynucleotide of claim 20 or 21, wherein the intracellular recruitment domain comprises the amino acid sequence of SEQ ID NO:

26.

23. The polynucleotide of any one of claims 19 to 22, wherein the CCR comprises the amino acid sequence of SEQ ID NO: 27 or 29.

24. The polynucleotide of any one of claims 19 to 23, wherein the coding sequence encoding CCR comprises a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 28 or 30.

25. The polynucleotide of any of the preceding claims, the polynucleotide further comprising a 5' homologous arm and a 3' homologous arm, wherein the length of each of the 5' homologous arm and the 3' homologous arm is at least 100 nucleotides.

26. The polynucleotide of claim 25, wherein the 5' homologous arm and the 3' homologous arm each comprise a nucleotide sequence homologous to the nucleotide sequence of the T cell receptor α constant region (TRAC) gene.

27. A vector comprising the polynucleotide sequence of any one of claims 1 to 26.

28. The vector of claim 27, wherein the vector is an adeno-associated virus (AAV) vector or a lentiviral vector.

29. An immune cell comprising any one of the polynucleotides of claims 1 to 18.

30. The immune cell of claim 29, wherein the polynucleotide is integrated into the human TRAC gene.

31. The immune cell of claim 30, wherein the integration of the polynucleotide into the human TRAC gene is mediated by an endonuclease.

32. The immune cell of claim 31, wherein the CRISPR endonuclease is Cas9 or Cas12.

33. The immune cell of any one of claims 30 to 32, wherein the CRISPR endonuclease causes a double-strand break to create an integration site in a target sequence within the TRAC gene, and the polynucleotide is integrated into the integration site via AAV-mediated site-specific integration, and optionally wherein the target sequence comprises the nucleic acid sequence of SEQ ID NO:

92.

34. The immune cell of any one of claims 29 to 33, wherein the immune cell further comprises additional polynucleotides, the polynucleotides comprising a promoter operatively linked to one or more coding sequences, optionally wherein the one or more coding sequences encode a chimeric cytokine receptor (CCR).

35. The immune cell of claim 34, wherein the additional polynucleotide further comprises a 5' homologous arm and a 3' homologous arm, wherein the length of the 5' homologous arm and the 3' homologous arm is each at least 100 nucleotides.

36. The immune cell of claim 35, wherein the 5' homologous arm and the 3' homologous arm each comprise a nucleotide sequence homologous to the nucleotide sequence of the CD52 gene.

37. The immune cell of any one of claims 34 to 36, wherein the additional polynucleotide further comprises an EFS promoter or a truncated PGK promoter operatively linked to one or more coding sequences encoding the CCR, wherein the truncated PGK promoter comprises a deletion of at least 50 nucleotides from the 5' end of the long PGK promoter having the nucleotide sequence of SEQ ID NO:

34.

38. The immune cell of claim 37, wherein the additional polynucleotide further comprises a truncated PGK promoter, the truncated PGK promoter comprising the following nucleotide sequence: (a) The nucleotide sequence comprises a deletion of about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380 or about 390 nucleotides from the 5' end of SEQ ID NO: 34, and has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 35; (b) The nucleotide sequence comprises a deletion of about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280 or about 290 nucleotides from the 5' end of SEQ ID NO: 34, and has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 36; (c) The nucleotide sequence comprises a deletion of about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, or about 190 nucleotides from the 5' end of SEQ ID NO: 34, and has at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 37; or (d) The nucleotide sequence comprises a deletion of about 50, about 60, about 70, about 80, about 90 or about 100 nucleotides from the 5' end of SEQ ID NO: 34, and has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the nucleotide sequence of SEQ ID NO:

38.

39. The immune cell of any one of claims 34 to 38, wherein the additional polynucleotide is integrated into the CD52 gene.

40. The immune cell of any one of claims 29 to 39, wherein the immune cell expresses the anti-CD19 CAR comprising the amino acid sequence of SEQ ID NO: 5 or 7, and the CD70 binding protein comprising the amino acid sequence of SEQ ID NO: 19 or 21.

41. The immune cell of any one of claims 29 to 40, wherein the immune cell further expresses the CCR, the CCR comprising the amino acid sequence of SEQ ID NO: 27 or 29.

42. An immune cell comprising any one of the polynucleotides of claims 19 to 26.

43. The immune cell of claim 42, wherein the polynucleotide is integrated into the human TRAC gene.

44. The immune cell of claim 43, wherein the integration of the polynucleotide at the human TRAC gene is performed by a CRISPR endonuclease.

45. The immune cell of claim 44, wherein the CRISPR endonuclease is Cas9 or Cas12.

46. ​​The immune cell of any one of claims 43 to 45, wherein the CRISPR endonuclease cleaves the TRAC gene at a target sequence, and the polynucleotide is integrated into the TRAC gene at the target sequence via AAV-mediated site-specific integration, optionally wherein the polynucleotide is inserted into the TRAC gene at the target sequence of SEQ ID NO:

92.

47. The immune cell of any one of claims 42 to 46, the immune cell further comprising additional polynucleotides, the polynucleotides comprising a promoter operatively linked to one or more coding sequences, wherein the one or more coding sequences encode a CD70 binding protein.

48. The immune cell of claim 47, wherein the additional polynucleotide further comprises a 5' homologous arm and a 3' homologous arm, wherein the length of the 5' homologous arm and the 3' homologous arm is each at least 100 nucleotides.

49. The immune cell of claim 48, wherein the 5' homologous arm and the 3' homologous arm each comprise a nucleotide sequence homologous to the nucleotide sequence of the CD52 gene.

50. The immune cell of any one of claims 47 to 49, wherein the additional polynucleotide further comprises an EFS promoter or a truncated PGK promoter operatively linked to the third coding sequence encoding the CD70 binding protein, wherein the truncated PGK promoter comprises a deletion of at least 50 nucleotides from the 5' end of the long PGK promoter having the nucleotide sequence of SEQ ID NO:

34.

51. The immune cell of claim 50, wherein the additional polynucleotide further comprises a truncated PGK promoter, the truncated PGK promoter comprising the following nucleotide sequence: (a) The nucleotide sequence comprises a deletion of about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380 or about 390 nucleotides from the 5' end of SEQ ID NO: 34, and has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 35; (b) The nucleotide sequence comprises a deletion of about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280 or about 290 nucleotides from the 5' end of SEQ ID NO: 34, and has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 36; (c) The nucleotide sequence comprises a deletion of about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, or about 190 nucleotides from the 5' end of SEQ ID NO: 34, and has at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 37; or (d) The nucleotide sequence comprises a deletion of about 50, about 60, about 70, about 80, about 90 or about 100 nucleotides from the 5' end of SEQ ID NO: 34, and has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the nucleotide sequence of SEQ ID NO:

38.

52. The immune cell according to any one of claims 42 to 51, wherein the immune cell expresses the anti-CD19 CAR comprising the amino acid sequence of SEQ ID NO: 5 or 7, and the CCR comprising the amino acid sequence of SEQ ID NO: 27 or 29.

53. The immune cell according to any one of claims 42 to 52, wherein the immune cell further expresses the CD70 binding protein comprising the amino acid sequence of SEQ ID NO: 19 or 21.

54. The immune cell according to any one of claims 29 to 53, wherein the immune cell is a T cell, NK cell, NK-T cell, tumor-infiltrating lymphocyte (TIL) or a T cell or NK cell derived from an iPSC.

55. An immune cell comprising a recombinant polynucleotide sequence integrated into a human TRAC gene, wherein the recombinant polynucleotide sequence comprises the 5' region of the human TRAC gene, a polynucleotide according to any one of claims 1 to 18, and the 3' region of the human TRAC gene, wherein the integrated recombinant polynucleotide sequence prevents the expression of the human TRAC gene.

56. The immune cell of claim 55, wherein the integration of the recombinant polynucleotide at the human TRAC gene is mediated by a CRISPR endonuclease.

57. The immune cell of claim 56, wherein the CRISPR endonuclease is Cas9 or Cas12.

58. The immune cell of any one of claims 55 to 57, wherein the CRISPR endonuclease causes a double-strand break to create an integration site in a target sequence within the TRAC gene, and the polynucleotide is integrated into the integration site via AAV-mediated site-specific integration, and optionally wherein the target sequence comprises the nucleic acid sequence of SEQ ID NO:

92.

59. The immune cell of any one of claims 55 to 58, the immune cell further comprising an additional recombinant polynucleotide sequence integrated into the human CD52 gene, wherein the additional recombinant polynucleotide sequence comprises the 5' region of the human CD52 gene, a coding sequence encoding CCR, and the 3' region of the human CD52 gene.

60. The immune cell of claim 59, wherein the integration of the additional recombinant polynucleotide at the human CD52 gene is mediated by a CRISPR endonuclease.

61. The immune cell of claim 60, wherein the CRISPR endonuclease is Cas9 or Cas12.

62. The immune cell of any one of claims 55 to 61, wherein the immune cell expresses the anti-CD19 CAR comprising the amino acid sequence of SEQ ID NO: 5 or 7, and the CD70 binding protein comprising the amino acid sequence of SEQ ID NO: 19 or 21, and optionally further expresses the CCR comprising the amino acid sequence of SEQ ID NO: 27 or 29.

63. An immune cell comprising a recombinant polynucleotide sequence integrated into a human TRAC gene, wherein the recombinant polynucleotide sequence comprises the 5' region of the human TRAC gene, a polynucleotide according to any one of claims 19 to 26, and the 3' region of the human TRAC gene, wherein the integrated recombinant polynucleotide sequence prevents the expression of the human TRAC gene.

64. The immune cell of claim 63, wherein the integration of the recombinant polynucleotide at the human TRAC gene is mediated by a CRISPR endonuclease.

65. The immune cell of claim 64, wherein the CRISPR endonuclease is Cas9 or Cas12.

66. The immune cell of any one of claims 63 to 65, wherein the CRISPR endonuclease causes a double-strand break to create an integration site in a target sequence within the TRAC gene, and the polynucleotide is integrated into the integration site via AAV-mediated site-specific integration, and optionally wherein the target sequence comprises the nucleic acid sequence of SEQ ID NO:

92.

67. The immune cell of any one of claims 63 to 66, the immune cell further comprising an additional recombinant polynucleotide sequence integrated into the human CD52 gene, wherein the additional recombinant polynucleotide sequence comprises the 5' region of the human CD52 gene, a coding sequence encoding a CD70 binding protein, and the 3' region of the human CD52 gene.

68. The immune cell of claim 67, wherein the integration of the additional recombinant polynucleotide at the human CD52 gene is mediated by a CRISPR endonuclease.

69. The immune cell of claim 68, wherein the CRISPR endonuclease is Cas9 or Cas12.

70. The immune cell of any one of claims 63 to 68, wherein the immune cell expresses the anti-CD19 CAR comprising the amino acid sequence SEQ ID NO: 5 or 7, and the CCR comprising the amino acid sequence SEQ ID NO: 27 or 29, and optionally further expresses the CD70 binding protein comprising the amino acid sequence SEQ ID NO: 19 or 21.

71. An immune cell comprising a polynucleotide encoding an anti-CD19 CAR and a CD70 binding protein, wherein the anti-CD19 CAR comprises the amino acid sequence of SEQ ID NO: 5 or 6, and the CD70 binding protein comprises the amino acid sequence of SEQ ID NO: 19 or 21.

72. The immune cell of claim 71, wherein the polynucleotide is integrated into the human TRAC gene via a CRISPR endonuclease, wherein the CRISPR endonuclease causes a double-strand break to create an integration site in a target sequence within the TRAC gene, and the polynucleotide is integrated into the integration site via AAV-mediated site-specific integration, and optionally wherein the target sequence comprises the nucleic acid sequence of SEQ ID NO:

92.

73. The immune cells of any one of claims 29 to 72, wherein the immune cells are derived from the patient.

74. The immune cell of any one of claims 29 to 72, wherein the immune cell is derived from a healthy donor.

75. A population of immune cells comprising any of the various immune cells claimed in any one of claims 29 to 74.

76. A pharmaceutical composition comprising the immune cells of any one of claims 29 to 74, or the immune cell population of claim 75, and a pharmaceutically acceptable excipient.

77. A method of treating a disease condition in a subject, the method comprising administering to the subject any of the immune cells of any one of claims 29 to 74, the immune cell population of claim 75, or the pharmaceutical composition of claim 76.

78. The method of claim 77, wherein the immune cells are capable of reducing or depleting B cells, pathogenic B cells, autoreactive B cells, pathogenic T cells, autoreactive T cells, or allogeneic reactive T cells.

79. The method of claim 77 or 78, wherein the disease condition is cancer or an autoimmune disease or condition.

80. The method of any one of claims 77 to 79, wherein the disease condition is non-Hodgkin's lymphoma.

81. The method of claim 79 or 80, wherein the disease condition is large B-cell lymphoma (LBCL), follicular lymphoma (FL), T-cell lymphoma (TCL), B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia (TALL), primary central nervous system lymphoma (PCNSL), mantle cell lymphoma (MCL), chronic lymphocytic leukemia (CLL), and peripheral T-cell lymphoma (PTCL).

82. The method of claim 79, wherein the autoimmune disease or condition is lupus, systemic lupus erythematosus (SLE), lupus nephritis, rheumatoid arthritis, systemic sclerosis, scleroderma, multiple sclerosis (MS), relapsing-remitting multiple sclerosis (RRMS), secondary progressive multiple sclerosis (SPMS), primary progressive multiple sclerosis (PPMS), neuromyelitis optica spectrum disorder (NMOSD), myasthenia gravis, Sjögren's syndrome, and myositis.

83. The method of any one of claims 77 to 82, wherein the immune cells are autologous relative to the subject.

84. The method of any one of claims 77 to 82, wherein the immune cells are allogeneic relative to the subject.

85. A method for manufacturing engineered immune cells, the method comprising introducing an engineered endonuclease that cleaves a target sequence within the human TRAC gene, and a polynucleotide of any one of claims 1 to 26 or a vector of claim 27 or 28 into the immune cells, and culturing the immune cells under suitable conditions.

86. The method of claim 85, wherein the polynucleotide is introduced into the immune cells via transfection, electroporation, LVV transduction, or AAV transduction.

87. The method of claim 85 or 86, wherein the engineered endonuclease is a CRISPR endonuclease, wherein the polynucleotide is integrated into the TRAC gene at the target sequence, and optionally wherein the CRISPR endonuclease is Cas12i.

88. An engineered immune cell comprising or expressing an anti-CD19 CAR and an anti-CD70 CAR, wherein the anti-CD19 CAR comprises the amino acid sequence of SEQ ID NO: 5 or 7, and the anti-CD70 CAR comprises the amino acid sequence of SEQ ID NO: 19 or 21.

89. An engineered immune cell comprising a polynucleotide containing a nucleic acid sequence encoding an anti-CD19 CAR containing an amino acid sequence of SEQ ID NO: 5 or 7, and an anti-CD70 CAR containing an amino acid sequence of SEQ ID NO: 19 or 21.

90. The engineered immune cell of claim 89, wherein the polynucleotide is integrated into the first exon of the TRAC locus.

91. The engineered immune cell of claim 90, wherein the polynucleotide is integrated into the TRAC locus at an integration site comprising the nucleic acid sequence of SEQ ID NO:

92.

92. The engineered immune cell of any one of claims 89 to 91, wherein the polynucleotide is operatively linked to a truncated PGK promoter.

93. A polynucleotide comprising a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:

97.

94. The polynucleotide of claim 93, wherein the polynucleotide comprises the nucleic acid sequence of SEQ ID NO:

96.

95. The polynucleotide of claim 93 or 94, wherein the polynucleotide is operatively linked to a truncated PGK promoter.

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