CD25 CHIMERIC ANTIGEN RECEPTORS AND USES THEREOF

Abstract

Provided herein are CD25 chimeric antigen receptors and compositions and methods for using the same. Methods for using CD25 chimeric antigen receptors provided herein include, for example, methods of treatment, methods of enhancing the immune system in a subject, and methods of killing a target cell or a population of target cells.

Claims

1. A chimeric antigen receptor (CAR) comprising: a CD25 targeting component; and a non-specific CAR complex comprising a hinge region, a transmembrane region, and an intracellular signaling domain.

2. The CAR of claim 1, wherein the non-specific CAR complex further comprises one or more costimulatory domains.

3. The CAR of claim 1 or 2, wherein the CD25 targeting component is an IL-2 receptor ligand or a portion thereof.

4. The CAR of claim 3, wherein the IL-2 receptor ligand comprises human IL-2 (SEQ ID NO:21) or a portion thereof.

5. The CAR of any one of claims 1-4, wherein the hinge region is an IgG based hinge region, or a CD8 hinge region, or a CD28 hinge region.

6. The CAR of claim 5, wherein the IgG based hinge region comprises SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or a portion thereof; the CD8 hinge region comprises SEQ ID NO:4, SEQ ID NO:5, or a portion thereof; or the CD28 hinge region comprises SEQ ID NO:6 or a portion thereof.

7. The CAR of any one of claims 1-6, wherein the transmembrane region is a CD3 transmembrane region, a CD28 transmembrane region, or a CD8 transmembrane region.

8. The CAR of claims 7, wherein the CD3 transmembrane region comprises SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or a portion thereof; the CD28 transmembrane region comprises SEQ ID NO:11 or a portion thereof; or the CD8 transmembrane region comprises SEQ ID NO:12, SEQ ID NO:13, or a portion thereof.

9. The CAR of any one of claims 1-8, wherein intracellular signaling domain is a CD3-zeta (CD3) intracellular signaling domain.

10. The CAR of claim 9, wherein the CD3 intracellular signaling domain comprises SEQ ID NO: 14 ora portion thereof.

11. The CAR of any one of claims 2-10, wherein the one or more costimulatory domains is a CD28 costimulatory domain, an inducible costimulatory molecule (ICOS), a 4-1BB costimulatory domain, an 0×40 costimulatory domain, or a CD27 stimulatory domain.

12. The CAR of claim 11, wherein the CD28 costimulatory domain comprises SEQ ID NO:15 or a portion thereof; the inducible costimulatory molecule (ICOS) comprises SEQ ID NO:16 or a portion thereof; the 4-1BB costimulatory domain comprises SEQ ID NO:17 or a portion thereof; the 0X40 costimulatory domain comprises SEQ ID NO:18 or a portion thereof; or the CD27 stimulatory domain comprises SEQ ID NO:19 or a portion thereof.

13. The CAR of any one of claims 1-3, wherein the CAR comprises an amino acid sequence of SEQ ID NO:22.

14. A nucleotide sequence encoding the CAR of any one of claims 1-13.

15. The nucleotide sequence of claim 14, wherein the nucleotide sequence comprises SEQ ID NO:23.

16. An expression cassette comprising a nucleotide sequence encoding the CAR of any one of claims 1-13.

17. The expression cassette of claim 16, wherein the nucleotide sequence comprises SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25.

18. A retroviral vector comprising the expression cassette of claim 16 or claim 17, wherein the retroviral vector is a y-retroviral vector or a lentiviral vector.

19. A T cell or population of T cells that express the CAR or any one of claims 1-13.

20. A method of treating a disease or condition comprising administering a population of T cells that express the CAR of any one of claims 1-13 to a subject having the disease or condition.

21. The method of claim 20, wherein the population of cells are autologous to the subject.

22. The method of claim 20, wherein the population of cells are allogenic to the subject.

23. The method of any one of claims 20-22, wherein the population of T cells are administered by intravenous infusion.

24. The method of any one of claims 20-23, wherein the disease or condition is a lymphoma selected from Adult T-cell Leukemia Lymphoma, Peripheral T-cell Lymphoma, Cutaneous T-cell Lymphoma, Diffuse large B-cell Lymphoma, Follicular Lymphoma, Burkitt's Lymphoma, Anaplastic large cell Lymphoma, or Angioimmunoblastic T-cell Lymphoma.

25. The method of any one of claims 20-23, wherein the disease or condition is an autoimmune or transplant-associated condition selected from graft-versus-host-disease, scleroderma, psoriasis, rheumatoid arthritis, or systemic lupus erythematosus.

26. A method of activating the immune system comprising administering a population of T cells that express the CAR of any one of claims 1-13 to a subject.

27. The method of claim 26, wherein the population of cells are autologous to the subject.

28. The method of claim 26, wherein the population of cells are allogenic to the subject.

29. The method of any one of claims 26-28, wherein the population of T cells are administered by intravenous infusion.

30. The method of any one of claims 26-29, wherein administering the population of T cells that that express the CAR of any one of claims 1-13 increases the release of a cytokine from a target cell.

31. The method of claim 30, wherein the cytokine is Interleukin-2 (IL-2) or Interferon gamma (IFNγ).

32. A method of killing a target cell comprising administering a population of T cells that express the CAR of any one of claims 1-13 to a subject.

33. The method of claim 32, wherein the population of cells are autologous to the subject.

34. The method of claim 32, wherein the population of cells are allogenic to the subject.

35. The method of any one of claims 32-34, wherein the population of T cells are administered by intravenous infusion.

36. The method of any one of claims 32-35, wherein administering the population of T cells that that express the CAR of any one of claims 1-13 is cytotoxic to the target cell.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1 is a schematic of the construction of an MSGV retroviral vector having a CD25-CAR gene insert (pMSGV-IL-2-ligand-h(28BBζ)-3rd-CAR).

[0020] FIG. 2 is a certificate of analysis for the pMSGV-IL-2-ligand-h(28BBζ)-3rd-CAR construct, including a quality control (QC) analysis and a restriction digestion map confirming expected fragment sizes of the construct.

[0021] FIG. 3 is a sequence alignment resulting from Sanger sequencing confirming that the sequence of the constructed plasmid pMSGV-IL-2-ligand-h(28BBζ)-3rd-CAR was in accordance with the design.

[0022] FIG. 4 is a schematic of the construction of lentiviral vector having a CD25-CAR gene insert (pCDH-EF1a-IL-2-ligand-h(28BBζ)-3rd-CAR).

[0023] FIG. 5 is a certificate of analysis for the pCDH-EF1a-IL-2-ligand-h(28BBζ)-3rd-CAR construct, including a quality control (QC) analysis and a restriction digestion map confirming expected fragment sizes of the construct.

[0024] FIG. 6 is a sequence alignment resulting from Sanger sequencing confirming that the sequence of the constructed plasmid pCDH-EF1a-IL-2-ligand-h(28BBζ)-3rd-CAR was in accordance with the design.

[0025] FIG. 7 shows Albumin (ALB) (left panel) and Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE) (right panel) qPCR standard curves used to calculate lentivirus titer.

[0026] FIG. 8 shows pCDH-EF1a-IL-2-ligand-h(28BBζ)-3rd-CAR expression in Jurkat cells.

[0027] FIG. 9 shows pCDH-EF1a-IL-2-ligand-h(28BBζ)-3rd-CAR expression in primary T cells.

[0028] FIG. 10 shows two FACS plots showing expression of CD25 in control Chinese hamster ovary (CHO) cells (left) and CHO-CD25-Luciferase-EGFP target cells (right).

[0029] FIG. 11 shows two fluorescence-activated cell sorting (FACS) plots showing positive CAR-T cells for primary T cells transfected with a control lentiviral vector (Con-T+PE-anti-IL-2, left) and a lentiviral vector expressing the pMSGV-IL-2-ligand-h(28BBζ)-3rd-CAR construct (W122618-1BCAR-T+PE-anti-IL-2, right).

[0030] FIG. 12 shows two bar graphs quantifying cytotoxicity in co-culture experiments using target cells having CD25 (left, Target Cell:CHO-CD25-Luciferase) and control CHO cells without CD25 (right, Target Cell:CHO-S-Luciferase). Cells were co-transfected with control T cells (Con-T) or T cells expressing the pMSGV-IL-2-ligand-h(28BBζ)-3rd-CAR construct (W122618-1BCAR-T).

[0031] FIG. 13 shows experimental results quantifying cytokine release in experimental and control (Con-T) cells. Two standard curves for cytokines IL-2 (upper left) and Interferon gamma (IFNγ, upper right) are shown measuring optical density (O.D.) versus protein concentration. Two bar graphs show quantification of IL-2 (lower left) and IFNγ (lower right) in target cells when co-transfected with control T cells (Con-T) or T cells expressing the pMSGV-IL-2-ligand-h(28BBζ)-3rd-CAR construct (W122618-1BCAR-T).

[0032] FIG. 14 depicts a graph quantifying cell proliferation in cells following lentiviral transfection with either the control lentiviral vector (Con-T) or the experimental lentiviral vector expressing the pMSGV-IL-2-ligand-h(28BBζ)-3rd-CAR construct (W122618-1B CAR-T). Quantification of T cell proliferation (as measured by number of viable cells) is displayed in a table below on Days 1 to 8 (D1-D8) (n.s.).

DETAILED DESCRIPTION

[0033] Chimeric antigen receptors (CAR) that target the alpha chain of the interleukin-2 (IL-2) receptor (also known as CD25) (CD25-CAR), nucleotides that encode CD25-CARs, T cells expressing CD25 CAR, and methods for treating conditions using the same are provided herein.

[0034] Chimeric antigen receptors are artificially engineered fusion proteins that form receptors having specificity to a target cell, for example, cancer cells or immune cells. Grafting the specificity of a monoclonal antibody onto a T cell gives a CAR-T. The basic principle of CAR-T cell design involves recombinant receptors that combine antigen-binding and T-cell activating functions. The general premise of CAR-T cells is to artificially generate T-cells targeted to markers found on cancer cells. CAR-Ts can be derived from either a patient's own blood (autologous) or derived from another healthy donor (allogenic). The expressed artificial T cell receptor is MHC independent and thus the targeting efficiency is greatly increased. During therapy, the CAR-T cells destroy cancer cells through mechanisms such as extensive stimulated cell proliferation, increasing the degree to which the cell is toxic to other living cells i.e. cytotoxicity, and by causing the increased production of factors that are secreted from cells in the immune system that have an effect on other cells in the organism.

[0035] According to some embodiments the CD25-CARs described herein include a non-specific CAR complex (nsCAR complex) and a CD25 targeting component.

[0036] The non-specific CAR complex of the CD25-CAR is designed to include one or more of the following components: a hinge region, a transmembrane region, a costimulatory domain, and an intracellular signaling domain. The components of the non-specific CAR complex form a fusion protein that is fused to the CD25 targeting component.

[0037] According to the embodiments described herein, the hinge region separates the CD25 targeting component from the transmembrane region of the non-specific CAR complex and is based on immunoglobulin (Ig)-like domain hinges. In some embodiments, the hinge region is an IgG based hinge region derived from an IgG1, IgG2, or IgG4, which may include all or a portion of the IgG hinge regions below:

TABLE-US-00001 IGG1 hinge: EPKSCDKTHTCP (SEQ ID NO: 1; full sequence information found at www.uniprot.org/uniprot/P01857) IgG2 hinge: ERKCCVECPPCP (SEQ ID NO: 2; full sequence information found at www.uniprot.org/uniprot/P01859) IgG4 hinge: ESKYGPPCPSCP (SEQ ID NO: 3; full sequence information found at www.uniprot.org/uniprot/P01861)

[0038] In certain embodiments, a hinge region derived from an IgG may also include a CH2 region of the IgG, a CH3 region of the IgG, both a CH2 and a CH3 region of the IgG, or the hinge region may be a short hinge lacking a CH2 or CH3 region (see full sequence information above). In certain aspects, the CH2 or CH3 region, when used as part of the hinge region, may be mutated to alter or improve CAR T cell functionality.

[0039] In other embodiments, the hinge region may be an Ig-based hinge from a native T cell molecule, in which case the hinge may include a CD28 hinge or a CD8 hinge. In some embodiments, the CD28 or CD8 hinge may include a portion (e.g., 40-50 amino acids) of the C terminal of the extracellular domains below:

TABLE-US-00002 CD8a extracellular domain: SQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRGAAASPTFL LYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALS NSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAA GGAVHTRGLDFACD (SEQ ID NO: 4; full sequence information found at www.uniprot.org/uniprot/P01732) CD8b extracellular domain: LQQTPAYIKVQTNKMVMLSCEAKISLSNMRIYWLRQRQAPSSDSHHEFL ALWDSAKGTIHGEEVEQEKIAVFRDASRFILNLTSVKPEDSGIYFCMIV GSPELTFGKGTQLSWDFLPTTAQPTKKSTLKKRVCRLPRPETQKGPLCS P (SEQ ID NO: 5; full sequence information found at www.uniprot.org/uniprot/P10966) CD28 extracellular domain: NKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCV VYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEV MYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 6; full sequence information found at www.uniprot.org/uniprot/P10747)

[0040] The transmembrane region of the non-specific CAR complex is a hydrophobic region of the CAR that spans the cell membrane. In certain embodiments, the transmembrane region includes a CD3 transmembrane domain or portion thereof, a CD28 transmembrane domain or portion thereof, or a CD8 transmembrane domain or portion thereof. In some embodiments, the transmembrane domain may include all or a portion of one of the transmembrane domains (TMDs) below:

TABLE-US-00003 CD3δ TMD: GIIVTDVIATLLLALGVFCFA (SEQ ID NO: 7; full sequence information found at www.uniprot.org/uniprot/P04234) CD3ϵ TMD: VMSVATIVIVDICITGGLLLLVYYWS (SEQ ID NO: 8; full sequence information found at www.uniprot.org/uniprot/P07766) CD3γ TMD: GFLFAEIVSIFVLAVGVYFIA (SEQ ID NO: 9; full sequence information found at www.uniprot.org/uniprot/P09693) CDζ TMD: LCYLLDGILFIYGVILTALFL (SEQ ID NO: 10; full sequence information found at www.uniprot.org/uniprot/P20963) CD28 TMD: FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 11; full sequence information found at www.uniprot.org/uniprot/P10747) CD8a TMD: IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 12; full sequence information found at www.uniprot.org/uniprot/P01732) CD8b TMD: ITLGLLVAGVLVLLVSLGVAI (SEQ ID NO: 13; full sequence information found at www.uniprot.org/uniprot/P10966)

[0041] According to the embodiments described herein, the non-specific CAR complex includes an intracellular signaling domain that includes a cytoplasmic signaling domain of a T cell Receptor (TCR), e.g., an CD3-gamma intracellular signaling domain, a CD3-delta intracellular signaling domain, a CD3-epsilon intracellular signaling domain, or a CD3-zeta intracellular signaling domain (see sequence information above). In certain embodiments, the intracellular signaling domain includes a CD3-zeta (CD3) intracellular signaling domain, which may include all or a portion of its cytoplasmic domain sequence RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDP EMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPR (SEQ ID NO:14; full sequence information found at https://www.uniprot.org/uniprot/P20963).

[0042] In certain embodiments, the non-specific CAR complex also includes one or more costimulatory domains in addition to the intracellular signaling domain. Suitable costimulatory domains that may be used in accordance with the embodiments described herein may include, but are not limited to, a CD28 costimulatory domain, which may include all or a portion of its cytoplasmic domain sequence RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:15; full sequence information found at https://www.uniprot.org/uniprot/P10747), an inducible costimulatory molecule (ICOS), which may include all or a portion of its cytoplasmic domain sequence CWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL (SEQ ID NO:16; full sequence information found at www.uniprot.org/uniprot/Q9Y6W8), a 4-1BB costimulatory domain (also known as CD137), which may include all or a portion of its cytoplasmic domain sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEE EEGGCEL (SEQ ID NO:17; full sequence information found at www.uniprot.org/uniprot/Q07011), an OX40 costimulatory domain (also known as CD134) which may include all or a portion of its cytoplasmic domain sequence ALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO:18; full sequence information found at www.uniprot.org/uniprot/P43489), or a CD27 costimulatory domain which may include all or a portion of its cytoplasmic domain sequence QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP (SEQ ID NO:19; full sequence information found at www.uniprot.org/uniprot/P26842). In some embodiments, the non-specific CAR complex includes no costimulatory domains, in other embodiments, the non-specific CAR complex includes one costimulatory domain, and in other embodiments, the non-specific CAR complex includes two costimulatory domains.

[0043] In certain embodiments, the non-specific CAR complex includes a hinge region selected from those described herein, a transmembrane region selected from those described herein, and a CD3ζ intracellular signaling domain to form a first generation nsCAR complex (nsCAR complex 1). In certain embodiments, the non-specific CAR complex includes a hinge region selected from those described herein, a transmembrane region selected from those described herein, one or more costimulatory signaling domains selected from those described herein, and a CD3ζintracellular signaling domain to form a second (or third) generation nsCAR complex (nsCAR complex 2). In one embodiment, the non-specific CAR complex includes a hinge region selected from those described herein, a transmembrane region selected from those described herein, two costimulatory signaling domains selected from those described herein, and a CD3ζintracellular signaling domain to form a third generation nsCAR complex (nsCAR complex 3). In the embodiments described above, the components of the non-specific CAR complex are fused to each other and arranged as follows to form nsCAR complexes (H=hinge; TM=transmembrane domain; CD=costimulatory domain; n≥1):

5′-(H)-(TM)-(CD3ζ)-3′   (nsCAR complex 1)

5′-(H)-(TM)-(CD).sub.n-(CD3ζ)-3′   (nsCAR complex 2)

5′-(H)-(TM)-(CD)-(CD)-(CD3ζ)-3′   (nsCAR complex 3)

[0044] In some embodiments, the non-specific CAR complexes described above include a hinge region and a transmembrane region derived from the same molecule. In those embodiments, the hinge and transmembrane regions are effectively a single domain that includes two adjacent regions of the same molecule but may be referred to as separate regions for purposes of describing the non-specific CAR complex.

[0045] In certain embodiments, the non-specific CAR complex includes a hinge region of a CD8 molecule (CD8.sub.H), a transmembrane region of a CD8 molecule (CD8.sub.TM), a CD28 costimulatory domain that includes a cytoplasmic portion of CD28 (CD28.sub.CD), a 4-1BB costimulatory domain (4-1BB.sub.CD) that includes a cytoplasmic portion of a 4-1BB, and a CD3ζ intracellular signaling domain. In some embodiments, the nsCAR complex includes components arranged in the following order to form nsCAR complex 4:

5′-(CD8.sub.H)-(CD8.sub.TM)-(CD28.sub.CD)-(4-1BB.sub.CD)-(CD3ζ)-3′   (nsCAR complex 4)

[0046] In one embodiment, the amino acid sequence of the non-specific CAR complex components includes the following sequences:

TABLE-US-00004 CD8.sub.H and CD8.sub.TM (combined, CD8.sub.H portion underlined) (SEQ ID NO: 20) TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWA PLAGTCGVLLLSLVITLYC CD28.sub.CD (SEQ ID NO: 15) RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS 4-1BB.sub.CD (SEQ ID NO: 17) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL CD3ζ (SEQ ID NO: 14) RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR-

[0047] The CD25 targeting component may be any suitable protein or peptide able to bind CD25 and activate signaling by the components of the non-specific CAR complex. In certain embodiments, the CD25 targeting component is an interleukin-2 receptor ligand. In other embodiments, the CD25 targeting component may be an immune-binding portion of an anti-CD25 antibody (e.g., Fab, scFv, single domain antibody or nanobody, etc.). In some embodiments, the CD25 targeting component is an IL-2 receptor ligand. The IL-2 receptor ligand may be derived from a native IL-2 receptor ligand, or it may be a recombinant or synthetic IL-2 receptor ligand. In one embodiment, the IL-2 receptor ligand is human IL-2 (hIL-2), which may include all or a portion of: MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYK NPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVI VLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO:21; full sequence information found at www.uniprot.org/uniprot/P60568)

[0048] According to the embodiments described herein, the CD25 targeting component and the non-specific CAR complex together form a CD25-CAR. In certain embodiments, the CD25-CAR includes a CD25 targeting component selected from those described herein, a hinge region selected from those described herein, a transmembrane region selected from those described herein, and a CD3ζ intracellular signaling domain to form a first generation nsCAR complex (CD25-CAR 1). In certain embodiments, the CD25-CAR includes a CD25 targeting component selected from those described herein, a hinge region selected from those described herein, a transmembrane region selected from those described herein, one or more costimulatory signaling domains selected from those described herein, and a CD3ζ intracellular signaling domain to form a second (or third) generation nsCAR complex (CD25-CAR 2). In one embodiment, the CD25-CAR includes a CD25 targeting component selected from those described herein, a hinge region selected from those described herein, a transmembrane region selected from those described herein, two costimulatory signaling domains selected from those described herein, and a CD3ζ intracellular signaling domain to form a third generation nsCAR complex (CD25-CAR 3). In the embodiments described above, the components of the CD25 targeting component and the non-specific CAR complex are fused to each other and arranged as follows to form CD25-CARs (CD25.sub.TC=CD25 targeting component; H=hinge; TM=transmembrane domain; CD=costimulatory domain; n≥1):

5′-(CD25.sub.TC)-(H)-(TM)-(CD3ζ)-3′   (CD25-CAR 1)

5′-(CD25.sub.TC)-(H)-(TM)-(CD).sub.n-(CD3ζ)-3′   (CD25-CAR 2)

5′-(CD25.sub.TC)-(H)-(TM)-(CD)-(CD)-(CD3ζ)-3′   (CD25-CAR 3)

[0049] In one embodiment, the CD25-CAR includes hIL-2 fused to nsCAR complex 4 to form CD25-CAR4, the components of which are fused to each other and arranged as follows:

5′-(hIL-2)-(CD8.sub.H)-(CD8.sub.TM)-(CD28.sub.CD)-(4-1BB.sub.CD)-(CD3ζ)-3′   (CD25-CAR 4)

[0050] In one embodiment, the amino acid sequence of CD25-CAR4 is shown below (hIL-2=normal text; CD8.sub.H and CD8.sub.TM=underlined; CD28.sub.CD=bold; 4-1BB.sub.CD=itafics; CD3ζ=italics+underlined):

TABLE-US-00005 (CD25-CAR 4 SEQ ID NO: 22) MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINN YKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHL RPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIIS TLTTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY IWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQ PYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE GGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEM GGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLST ATKDTYDALHMQALPPR

[0051] In certain embodiments, a nucleic acid molecule that encodes a CD25-CAR (CD25-CAR gene) is provided. Nucleic acid molecules (i.e., CD25-CAR genes) that encode a desired CD25-CAR may include a nucleotide sequence corresponding to any sequence of degenerate codons that give rise to the desired CD25-CAR amino acid sequence in accordance with the genetic code. In certain embodiments, a CD25-CAR 1 gene, a CD25-CAR 2 gene, or a CD25-CAR 3 gene has a nucleotide sequence that corresponds to a sequence of degenerate codons giving rise to a CD25-CAR 1, a CD25-CAR 2, or a CD25-CAR 3 (described above), respectively.

[0052] In some embodiments, the CD25-CAR gene includes a nucleic acid sequence that corresponds to a sequence of degenerate codons giving rise to CD25-CAR 4, which includes the following components as described above arranged in the following order to form nsCAR complex 4:

5′-(hIL-2)-(CD8.sub.H)-(CD8.sub.TM)-(CD28.sub.CD)-(4-1BB.sub.CD)-(CD3ζ)-3′   (CD25-CAR 4)

[0053] In one embodiment, the nucleic acid sequence of CD25-CAR4 is shown below (hIL-2=normal text; CD8.sub.H and CD8.sub.TM=underlined; CD28.sub.CD=bold; 4-1BB.sub.CD=italics; CD3ζ=italics+underlined):

TABLE-US-00006 (SEQ ID NO: 23) atgtaccggatgcagctgctgagctgtatcgccctgtctctggccctggt cacaaatagcgcccctaccagcagcagcaccaagaaaacacagctgcaac tggaacacctcctgctggacctgcagatgatcctgaacggcatcaacaac tacaagaaccccaagctgacccggatgctgaccttcaagttctacatgcc caagaaggccaccgagctgaagcacctccagtgcctggaagaggaactga agcccctggaagaagtgctgaatctggcccagagcaagaacttccacctg aggcctagggacctgatcagcaacatcaacgtgatcgtgctggaactgaa aggcagcgagacaaccttcatgtgcgagtacgccgacgagacagctacca tcgtggaatttctgaaccggtggatcaccttctgccagagcatcatcagc accctgaccaccacgacgccagcgccgcgaccaccaacaccggcgcccac catcgcgtcgcagcccctgtccctgcgcccagaagcgtgccggccagcgg cggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctac atttgggcccctctggctggtacttgcggggtcctgctgctttcactcgt gatcactctttactgtaggagcaagcggagcagactgctgcacagcgact acatgaacatgaccccccggaggcctggccccacccggaagcactaccag ccctacgcccctcccagggatttcgccgcctaccggagcaaacggggca gaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaa actactcaagaggaagatggctgtagctgccgatttccagaagaagaaga aggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccg cgtacaagcagggccagaaccagctctataacgagctcaatctaggacga agagaggagtacgatgttttggacaagagacgtggccgggaccctgagat ggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaac tgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggc gagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtac agccaccaaggacacctacgacgcccttcacatgcaggccctgccccctc gc

[0054] As described above, the CD25-CARs disclosed herein may be designed as a first generation, a second generation, a third generation, or a fourth or later next generation CAR (see Guedan et al. 2018; Smith et al. 2016). Further, the design of the non-specific CAR complex domains or regions described herein may be modified or optimized to incorporate different isoforms of the peptides described above or in accordance with guidance known in the art and/or as discussed in Guedan et al., which is incorporated herein in its entirety, as if fully set forth herein.

[0055] The CD25-CARs described herein may be expressed in a T cell or a population of T cells to generate CAR-T cells that can be administered to a patient suffering from a disease or condition associated with the expression of CD25. Expression of the CD25-CAR in a T cell or population of T cells is accomplished using any suitable expression system. In some embodiments, a CD25-CAR gene is cloned into a plasmid expression vector and transferred into a host T cell or population of T cells by electroporation.

[0056] In certain embodiments, the expression system is a retroviral packaging system. In such embodiments, an expression cassette that includes a CD25-CAR gene is cloned into a retroviral transfer plasmid vector and transferred into a packaging cell line (e.g., 293T cells, or a helper-free packaging cell line such as Phoenix) along with any other necessary plasm ids (e.g., envelope and/or packaging plasm ids) to allow the packaging cell line to produce retrovirus particles containing the desired CD25-CAR gene. Any suitable retroviral packaging system known in the art may be used in accordance with the embodiments described herein including, but not limited to, γ-retroviral packaging systems derived from a γ-retrovirus (e.g., MMLV (Moloney Murine Leukemia Virus), MSCV (Murine Stem Cell Virus), Murine Leukemia Virus (MLV)) or a lentiviral packaging system derived from a lentivirus (e.g., HIV). In certain aspects, the retroviral packaging system may be self-inactivating.

[0057] In certain embodiments, the expression cassette used to generate CAR-T cells as described above may include a nucleic acid sequence (i.e., CD25-CAR gene) that encodes any of the CD25-CARs described herein. In one embodiment, the expression cassette includes a CD25-CAR gene including SEQ ID NO:23. In another embodiment, the expression cassette includes a CD25-CAR gene that includes the nucleotide sequence below:

TABLE-US-00007 (SEQ ID NO: 24) ctcgaggccaccatgtaccggatgcagctgctgagctgtatcgccctgtc tctggccctggtcacaaatagcgcccctaccagcagcagcaccaagaaaa cacagctgcaactggaacacctcctgctggacctgcagatgatcctgaac ggcatcaacaactacaagaaccccaagctgacccggatgctgaccttcaa gttctacatgcccaagaaggccaccgagctgaagcacctccagtgcctgg aagaggaactgaagcccctggaagaagtgctgaatctggcccagagcaag aacttccacctgaggcctagggacctgatcagcaacatcaacgtgatcgt gctggaactgaaaggcagcgagacaaccttcatgtgcgagtacgccgacg agacagctaccatcgtggaatttctgaaccggtggatcaccttctgccag agcatcatcagcaccctgaccaccacgacgccagcgccgcgaccaccaac accggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaagcgt gccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcc tgtgatatctacatttgggcccctctggctggtacttgcggggtcctgct gctttcactcgtgatcactctttactgtaggagcaagcggagcagactgc tgcacagcgactacatgaacatgaccccccggaggcctggccccacccgg aagcactaccagccctacgcccctcccagggatttcgccgcctaccggag caaacggggcagaaagaaactcctgtatatattcaaacaaccatttatga gaccagtacaaactactcaagaggaagatggctgtagctgccgatttcca gaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgc agacgcccccgcgtacaagcagggccagaaccagctctataacgagctca atctaggacgaagagaggagtacgatgttttggacaagagacgtggccgg gaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcct gtacaatgaactgcagaaagataagatggcggaggcctacagtgagattg ggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccag ggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggc cctgccccctcgctaa

[0058] In some embodiments, the expression cassette may also include a promoter. Any suitable promoter may be used in the expression cassette including, but not limited to, human elongation factor 1α-subunit (EF1α), immediate-early cytomegalovirus (CMV), chicken β-actin (CBA) and its derivative CAG, the β glucuronidase (GUSB), or ubiquitin C (UBC). In one embodiment, the expression cassette is inserted cloned into a lentiviral transfer plasmid vector construct and includes an EF1α promoter and a CD25-CAR gene that includes the nucleotide sequence below (EF1α promoter=underlined):

TABLE-US-00008 (SEQ ID NO: 25) gagtaattcatacaaaaggactcgcccctgccttggggaatcccagggac cgtcgttaaactcccactaacgtagaacccagagatcgctgcgttcccgc cccctcacccgcccgctctcgtcatcactgaggtggagaagagcatgcgt gaggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtcccc gagaagttggggggaggggtcggcaattgaaccggtgcctagagaaggtg gcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttc ccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgtt ctttttcgcaacgggtttgccgccagaacacaggtaagtgccgtgtgtgg ttcccgcgggcctggcctctttacgggttatggcccttgcgtgccttgaa ttacttccacgcccctggctgcagtacgtgattcttgatcccgagcttcg ggttggaagtgggtgggagagttcgaggccttgcgcttaaggagcccctt cgcctcgtgcttgagttgaggcctggcttgggcgctggggccgccgcgtg cgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctct agccatttaaaatttttgatgacctgctgcgacgctttttttctggcaag atagtcttgtaaatgcgggccaagatctgcacactggtatttcggttttt ggggccgcgggcggcgacggggcccgtgcgtcccagcgcacatgttcggc gaggcggggcctgcgagcgcggccaccgagaatcggacgggggtagtctc aagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatcgcc ccgccctgggcggcaaggctggcccggtcggcaccagttgcgtgagcgga aagatggccgcttcccggccctgctgcagggagctcaaaatggaggacgc ggcgctcgggagagcgggcgggtgagtcacccacacaaaggaaaagggcc tttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgcc gtccaggcacctcgattagttctcgagcttttggagtacgtcgtctttag gttggggggaggggttttatgcgatggagtttccccacactgagtgggtg gagactgaagttaggccagcttggcacttgatgtaattctccttggaatt tgccctttttgagtttggatcttggttcattctcaagcctcagacagtgg ttcaaagtttttttcttccatttcaggtgtcgtgattcgaattcgccacc atgtaccggatgcagctgctgagctgtatcgccctgtctctggccctggt cacaaatagcgcccctaccagcagcagcaccaagaaaacacagctgcaac tggaacacctcctgctggacctgcagatgatcctgaacggcatcaacaac tacaagaaccccaagctgacccggatgctgaccttcaagttctacatgcc caagaaggccaccgagctgaagcacctccagtgcctggaagaggaactga agcccctggaagaagtgctgaatctggcccagagcaagaacttccacctg aggcctagggacctgatcagcaacatcaacgtgatcgtgctggaactgaa aggcagcgagacaaccttcatgtgcgagtacgccgacgagacagctacca tcgtggaatttctgaaccggtggatcaccttctgccagagcatcatcagc accctgaccaccacgacgccagcgccgcgaccaccaacaccggcgcccac catcgcgtcgcagcccctgtccctgcgcccagaagcgtgccggccagcgg cggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctac atttgggcccctctggctggtacttgcggggtcctgctgctttcactcgt gatcactctttactgtaggagcaagcggagcagactgctgcacagcgact acatgaacatgaccccccggaggcctggccccacccggaagcactaccag ccctacgcccctcccagggatttcgccgcctaccggagcaaacggggcag aaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaa ctactcaagaggaagatggctgtagctgccgatttccagaagaagaagaa ggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgc gtacaagcagggccagaaccagctctataacgagctcaatctaggacgaa gagaggagtacgatgttttggacaagagacgtggccgggaccctgagatg gggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaact gcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcg agcgccggaggggcaaggggcacgatggcctttaccagggtctcagtaca gccaccaaggacacctacgacgcccttcacatg caggccctgccccctcgctaa

[0059] In certain embodiments, the expression cassette includes a pair of inverted terminal repeats (ITR) or long terminal repeats (LTR). In certain embodiments, the expression cassette also includes a polyadenylation signal (polyA).

[0060] A retroviral transfer plasmid vector construct that includes an expression cassette is provided in accordance with the embodiments described herein. In certain embodiments, the retroviral transfer plasmid vector construct includes an expression cassette that includes any CD25-CAR sequence in accordance with the embodiments described herein and in the examples below. The retroviral transfer plasmid vector construct, along with the rest of the retroviral expression system described above (if any) produce retroviral particles (e.g., γ-retrovirus particles or lentiviral particles) when introduced into the packaging cell line. The retroviral particles are then transfected into a host T cell or population of T cells, which results in expression of the CD25-CAR sequence in the host T cell or population of T cells.

[0061] In certain embodiments, a T cell or a population of T cells that express a CD25-CAR encoded by a CD25-CAR gene is provided in accordance with the embodiments described herein. In certain embodiments, the T cell or population of T cells express a CD25-CAR according to any embodiment described herein, which is encoded by a CD25-CAR gene in accordance with the embodiments described above. The T cell or population of T cells may be a primary T cell line or may be derived from any suitable commercially available T cell line. In one embodiment, the T cell or population of T cells is obtained from a human subject. In some embodiments, the T cell or population of T cells obtained from the subject are contacted with retroviral particles including a CD25-CAR gene that in turn cause the T cell or population of T cells to express a CD25-CAR encoded by the CD25-CAR gene. The CD25-CAR and corresponding CD25-CAR gene may be any such CD25-CAR or CD25-CAR gene in accordance with the embodiments described herein.

[0062] The T cell or population of T cells that express a CD25-CAR as described herein (also referred to herein is a CD25 CAR-T cell) may be used to treat a disease or condition that is associated with expression of CD25 or the IL-2 receptor. In certain embodiments, a method for treating the disease or condition includes a step of administering a population of T cells that express a CD25-CAR to a subject having the disease or condition. The population of T cells may be a population of T cells that expresses any CD25-CAR in accordance with the embodiments described herein.

[0063] In certain embodiments, the population of T cells administered to the subject are allogenic to the subject, i.e., the native T cells that are engineered to express the CD25-CAR are obtained from a donor subject that is different from the subject receiving the treatment. In other embodiments, the population of T cells administered to the subject are autologous to the subject, i.e., the native T cells that are engineered to express the CD25-CAR are obtained from the same subject that receives the treatment and subjected to an ex vivo procedure to engineer the CD25 CAR-T cells for use as a therapeutic. The population of native T cells may be obtained from the donor or subject receiving the treatment by blood draw, and the T cells are then separated from the other blood cells using any suitable procedure known in the art. The separated T cells are transfected with retroviral particles containing the CD25-CAR gene, thereby causing the T cell population to express the CD25-CAR gene by virtue of retroviral infection. The T-cells expressing the CD25-CAR are isolated, optionally expanded in vitro, and administered to the subject in accordance with the embodiments described herein.

[0064] Administration can be by any suitable method of delivering the T cell or population of T cells to an area where a target tissue or target cells express CD25 or IL-2 receptor. In certain embodiments, administration may be by infusion, and/or can be auricular, buccal, conjunctival, cutaneous, subcutaneous, endocervical, endosinusial, endotracheal, enteral, epidural, via hemodialysis, interstitial, intrabdominal, intraamniotic, intra-arterial, intra-articular, intrabiliary, intrabronchial, intrabursal, intracardiac, intracartilaginous, intracaudal, intracavernous, intracavitary, intracerebral, intracisternal, intracorneal, intracoronary, intradermal, intradiscal, intraductal, intraepidermal, intraesophagus, intragastric, intravaginal, intragingival, intraileal, intraluminal, intralesional, intralymphatic, intramedullary, intrameningeal, intramuscular, intraocular, intraovarian, intraepicardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrasinal, intraspinal, intrasynovial, intratendinous, intratesticular, intrathecal, intrathoracic, intratubular, intratumor, intratympanic, intrauterine, intravascular, intravenous, intraventricular, intravesical, intravitreal, laryngeal, nasal, nasogastric, ophthalmic, oral, oropharyngeal, parenteral, percutaneous, periarticular, peridural, rectal, inhalationally, retrobulbar, subarachnoid, subconjuctival, sublingual, submucosal, topical, transdermal, transmucosal, transplacental, transtracheal, ureteral, uretheral, or vaginal. In certain embodiments the administration is by intravenous infusion into a blood vessel or injection into a target tissue or body cavity.

[0065] Diseases or conditions that may be treated in accordance with the embodiments described herein include, but are not limited to, a T cell malignancy or lymphoma, an autoimmune condition, or a transplant-associated condition associated with expression of CD25 or IL-2 receptors. In certain embodiments, the methods for treating the disease or condition may include a step of determining whether or confirming that the subject's cells express CD25. Lymphomas that may be treated in accordance with the embodiments described herein include, but are not limited to, Adult T-cell Leukemia Lymphoma, Peripheral T-cell Lymphoma, Cutaneous T-cell Lymphoma, Diffuse large B-cell Lymphoma, Follicular Lymphoma, Burkitt's Lymphoma, Anaplastic large cell Lymphoma, or Angioimmunoblastic T-cell Lymphoma. Autoimmune conditions, or transplant-associated conditions that may be treated in accordance with the embodiments described herein include, but are not limited to, graft-versus-host-disease, scleroderma, psoriasis, rheumatoid arthritis, or systemic lupus erythematosus.

[0066] “Treating” or “treatment” of a disease or condition may refer to preventing the condition, slowing the onset or rate of development of the condition, reducing the risk of developing the condition, preventing or delaying the development of symptoms associated with the condition, reducing or ending symptoms associated with the condition, generating a complete or partial regression or remission of the condition, or some combination thereof. “Treating” a tumor also means that one or more hallmarks or markers of a tumor may be eliminated, reduced or prevented by the treatment.

[0067] In certain embodiments, the subject is preconditioned with one or more immunosuppressive chemotherapy drugs prior to CAR-T cell infusion. See, for example, U.S. Pat. No. 9,855,298, which is hereby incorporated by reference as if fully set forth herein.

[0068] From the foregoing, it will be appreciated that specific embodiments of the invention have been described herein for purposes of illustration, but that various modifications may be made without deviating from the scope of the invention. Accordingly, the invention is not limited except as by the appended claims. Additional background and embodiments related to the design, development, and use of the CD25-CARs the CD25-CAR genes, and the CD25 CAR-T cells described herein may be found in Guedan et al. 2018 and Smith et al. 2016, which are hereby incorporated by reference as if fully set forth herein.

Examples

[0069] The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention.

Example 1: Generation of a γ-Retroviral Vector for Expressing a CD25-CAR (pMSGV-IL-2-ligand-h(28BBζ)-3rd-CAR)

[0070] An IL-2 ligand CAR-T (i.e., CD25-CAR T cell) may be constructed using an IL-2 ligand to engage the IL-2R (CD25) on malignant lymphomas that express the IL-2 receptor.

[0071] A CD25-CAR retroviral vector design using an MSGV retroviral vector plasmid was constructed comprising the following components: an MSGV retroviral backbone, an IL-2 ligand, the hinge and transmembrane regions of the CD8 molecule, the cytoplasmic portions of CD28 and 4-1BB, and the cytoplasmic component of the TCR molecule (pMSGV-IL-2-ligand-h(28BBζ)-3rd-CAR). An expression cassette including a nucleic acid sequence encoding IL-2 ligand, CD8, CD28, 4-1BB and TCR components was subcloned into an MSGV retroviral vector to produce the pMSGV-IL-2-ligand-h(28BBζ)-3rd-CAR retroviral vector (Creative Biolabs), the structure of which is shown in FIG. 1. The sequences of the components and expression cassette are shown below:

TABLE-US-00009 Amino Acid Sequence of IL-2-ligand (SEQ ID NO: 21) MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINN YKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHL RPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIIS TLT Nucleotide sequence of the CAR expression cassette (IL-2-ligand-CD8 hinge and TM-CD28-4-1BB-CD3zeta) (SEQ ID NO: 24) ctcgaggccaccatgtaccggatgcagctgctgagctgtatcgccctgtc tctggccctggtcacaaatagcgcccctaccagcagcagcaccaagaaaa cacagctgcaactggaacacctcctgctggacctgcagatgatcctgaac ggcatcaacaactacaagaaccccaagctgacccggatgctgaccttcaa gttctacatgcccaagaaggccaccgagctgaagcacctccagtgcctgg aagaggaactgaagcccctggaagaagtgctgaatctggcccagagcaag aacttccacctgaggcctagggacctgatcagcaacatcaacgtgatcgt gctggaactgaaaggcagcgagacaaccttcatgtgcgagtacgccgacg agacagctaccatcgtggaatttctgaaccggtggatcaccttctgccag agcatcatcagcaccctgaccaccacgacgccagcgccgcgaccaccaac accggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaagcgt gccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcc tgtgatatctacatttgggcccctctggctggtacttgcggggtcctgct gctttcactcgtgatcactctttactgtaggagcaagcggagcagactgc tgcacagcgactacatgaacatgaccccccggaggcctggccccacccgg aagcactaccagccctacgcccctcccagggatttcgccgcctaccggag caaacggggcagaaagaaactcctgtatatattcaaacaaccatttatga gaccagtacaaactactcaagaggaagatggctgtagctgccgatttcca gaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgc agacgcccccgcgtacaagcagggccagaaccagctctataacgagctca atctaggacgaagagaggagtacgatgttttggacaagagacgtggccgg gaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcct gtacaatgaactgcagaaagataagatggcggaggcctacagtgagattg ggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccag ggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggc cctgccccctcgctaa Amino acid sequence of the CAR expression cassette (IL-2-lidand-CD8 hinge and TM-CD28-4-1BB-CD3zeta (SEQ ID NO: 22) MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINN YKNPKLTRMLIFKFYMPKKATELKHLOCLEEELKPLEEVLNLAQSKNFHL RPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIIS TLTTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY IWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQ PYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE GGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEM GGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLST ATKDTYDALHMQALPPR

[0072] The insert encoding IL-2 ligand, CD8, CD28, 4-1BB and TCR components CAR expression was confirmed by Sanger sequencing (FIG. 3) and the expected fragment sizes for the construct were observed upon Restriction Digest (FIG. 2).

[0073] After packaging the pMSGV-IL-2-ligand-h(28BBζ)-3rd-CAR retroviral vector, the transduction efficiency was observed to be very low in HEK293 and primary T cells. Thus, development of the MSGV based retroviral vector above may be optimized or modified to improve transduction efficiency.

Example 2: Generation of a Lentiviral Vector for Expressing a CD25-CAR (pCDH-EF1a-IL-2-ligand-h(28BBζ)-3rd-CAR)

[0074] Because of poor transduction efficiency, a lentiviral vector expressing the CD25-CAR was developed. The CD25-CAR lentiviral vector plasmid was constructed comprising the following components: an IL-2 ligand, the hinge and transmembrane regions of the CD8 molecule, the cytoplasmic portions of CD28 and 4-1BB, and the cytoplasmic component of the TCR molecule (pMSGV-IL-2-ligand-h(28BBζ)-3rd-CAR). An expression cassette including an EF1a promoter and a nucleic acid sequence encoding IL-2 ligand, CD8, CD28, 4-1BB and TCR components was subcloned into a self-inactivating lentivirus vector to produce the pMSGV-IL-2-ligand-h(28BBζ)-3rd-CAR retroviral vector (Creative Biolabs), the structure of which is shown in FIG. 4. The sequences of the components and expression cassette are shown below:

TABLE-US-00010 Sequence of IL-2-ligand (SEQ ID NO: 21) MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINN YKNPKLTRMLIFKFYMPKKATELKHLOCLEEELKPLEEVLNLAQSKNFHL RPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIIS TLT Nucleotide sequence of the CAR expression cassette (EF1a-promoter-IL-2-ligand-CD8 hinge and TM-CD28-4-1BB-CD3zeta) (SEQ ID NO: 25) gagtaattcatacaaaaggactcgcccctgccttggggaatcccagggac cgtcgttaaactcccactaacgtagaacccagagatcgctgcgttcccgc cccctcacccgcccgctctcgtcatcactgaggtggagaagagcatgcgt gaggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtcccc gagaagttggggggaggggtcggcaattgaaccggtgcctagagaaggtg gcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttc ccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgtt ctttttcgcaacgggtttgccgccagaacacaggtaagtgccgtgtgtgg ttcccgcgggcctggcctctttacgggttatggcccttgcgtgccttgaa ttacttccacgcccctggctgcagtacgtgattcttgatcccgagcttcg ggttggaagtgggtgggagagttcgaggccttgcgcttaaggagcccctt cgcctcgtgcttgagttgaggcctggcttgggcgctggggccgccgcgtg cgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctct agccatttaaaatttttgatgacctgctgcgacgctttttttctggcaag atagtcttgtaaatgcgggccaagatctgcacactggtatttcggttttt ggggccgcgggcggcgacggggcccgtgcgtcccagcgcacatgttcggc gaggcggggcctgcgagcgcggccaccgagaatcggacgggggtagtctc aagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatcgcc ccgccctgggcggcaaggctggcccggtcggcaccagttgcgtgagcgga aagatggccgcttcccggccctgctgcagggagctcaaaatggaggacgc ggcgctcgggagagcgggcgggtgagtcacccacacaaaggaaaagggcc tttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgcc gtccaggcacctcgattagttctcgagcttttggagtacgtcgtctttag gttggggggaggggttttatgcgatggagtttccccacactgagtgggtg gagactgaagttaggccagcttggcacttgatgtaattctccttggaatt tgccctttttgagtttggatcttggttcattctcaagcctcagacagtgg ttcaaagtttttttcttccatttcaggtgtcgtgattcgaattcgccacc atgtaccggatgcagctgctgagctgtatcgccctgtctctggccctggt cacaaatagcgcccctaccagcagcagcaccaagaaaacacagctgcaac tggaacacctcctgctggacctgcagatgatcctgaacggcatcaacaac tacaagaaccccaagctgacccggatgctgaccttcaagttctacatgcc caagaaggccaccgagctgaagcacctccagtgcctggaagaggaactga agcccctggaagaagtgctgaatctggcccagagcaagaacttccacctg aggcctagggacctgatcagcaacatcaacgtgatcgtgctggaactgaa aggcagcgagacaaccttcatgtgcgagtacgccgacgagacagctacca tcgtggaatttctgaaccggtggatcaccttctgccagagcatcatcagc accctgaccaccacgacgccagcgccgcgaccaccaacaccggcgcccac catcgcgtcgcagcccctgtccctgcgcccagaagcgtgccggccagcgg cggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctac atttgggcccctctggctggtacttgcggggtcctgctgctttcactcgt gatcactctttactgtaggagcaagcggagcagactgctgcacagcgact acatgaacatgaccccccggaggcctggccccacccggaagcactaccag ccctacgcccctcccagggatttcgccgcctaccggagcaaacggggcag aaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaa ctactcaagaggaagatggctgtagctgccgatttccagaagaagaagaa ggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgc gtacaagcagggccagaaccagctctataacgagctcaatctaggacgaa gagaggagtacgatgttttggacaagagacgtggccgggaccctgagatg gggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaact gcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcg agcgccggaggggcaaggggcacgatggcctttaccagggtctcagtaca gccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcg ctaa Amino acid sequence of the CAR expression cassette (IL-2-ligand-CD8 hinge and TM-CD28-4-1BB-CD3zeta) (SEQ ID NO: 22) MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINN YKNPKLTRMLIFKFYMPKKATELKHLOCLEEELKPLEEVLNLAQSKNFHL RPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIIS TLTTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY IWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQ PYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE GGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEM GGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLST ATKDTYDALHMQALPPR

[0075] The insert encoding IL-2 ligand, CD8, CD28, 4-1BB and TCR components CAR expression was confirmed by Sanger sequencing (FIG. 6) and the expected fragment sizes of the construct were observed upon Restriction Digest (FIG. 5).

[0076] Lentivirus Packaging and Titering. The lentivirus was produced from the pCDH-EF1a-IL-2-ligand-h(28BBζ)-3rd-CAR vector transfected into 293T cells using the lentiviral packaging kit and transfection reagents from Creative Biolabs following a polyethyleneimine (PEI) transfection protocol. The titer of the viral stock was then determined by Real-Time PCR. The cycle threshold (Ct) values are shown in Table 1 below:

TABLE-US-00011 TABLE 1 Ct value Sample WPRE ALB Lenti-CAR virus 21.53 21.4 23.96 23.84

[0077] The number of lentiviral vector copies was measured by the standard curve created automatically by Roche 480 matched software (FIG. 7), and the lentiviral titer was calculated to be 1.62×10.sup.8 TU/mL using the formula below:

[00001]$\mathrm{Lentivirus}\mathrm{titer}\left(\mathrm{TU}/\mathrm{mL}\right)=\frac{\left({\mathrm{Copy}}_{\mathrm{WPRE}}÷{\mathrm{Copy}}_{\mathrm{ALB}}\right)\times 2\times {\mathrm{Cell}}_{\mathrm{NO}}}{{\mathrm{Volume}}_{\mathrm{virus}}}$

[0078] Cell Transfection. 2*10.sup.6 Jurkat cells in logarithmic growth phase and 5*10.sup.6 primary T cells isolated from PBMC were transfected with 100 μL or 50 μL concentrated lentivirus respectively. 6 μg/mL polybrene was added. Cells were cultured for another 48 h before FACS analysis by anti-IL-2 antibody. Transduction efficiency was very high. The positive rate of CAR expression in Jurkat cells was 86% (FIG. 8), and the positive rate of CAR expression in primary T cells was 93.1% (FIG. 9). Control group was cells without lentivirus transfection.

Example 3: Analysis of CAR-T Cells Expressing the pMSGV-IL-2-ligand-h(28BBζ)-3rd-CAR and Effects on Target CD25-Expressing Cells

[0079] To analyze whether the CAR-T cells expressing the CD25-CAR activate target cells in vitro, two stable target cell lines were generated using Chinese hamster ovary (CHO) cells. Control target cells expressing a luciferase construct (CHO-S-Luciferase) and experimental target cells expressing a CD25 luciferase construct (CHO-CD25-Luciferase) were generated as follows.

[0080] Lentivirus Packaging. Fifteen cm culture dishes were prepared and inoculated with 5×10.sup.6 CHO cells per culture dish in complete culture medium (high glucose DMEM, 10% FBS). Culture dishes were incubated at 37° C., 5% CO.sub.2 in an incubator overnight.

[0081] The LVTransm transfection reagent and lentivirus packaging plasmids (Lenti-GOI, Lenti-packaging Mix) were defrosted at room temperature and thoroughly mixed by pipetting up and down. Next, 2 mL PBS was added into a well of a 6-well-plate followed by adding 10 μg Lenti-GOI and 30 μL Lenti-packaging Mix, respectively. Next, 50 μL LVTransm was added to the well and mixed well before incubation at room temperature for 10-15 min to form a complex.

[0082] The complex was added into a 15 cm culture dish dropwise and mixed well by waggling the dish back and forth. The culture dish was then incubated at 37° C., 5% CO.sub.2 in an incubator for 6-8 hours. After 6-8 hours, the culture medium was refreshed. The culture the dish was continuously cultured for 48 hours, the culture medium was harvested every 24 hours.

[0083] After 48 hours, the culture medium containing lentiviral particles was harvested and the supernatant was filtered using a 0.45 μm membrane. Next, the culture medium containing lentiviral particles was centrifuged at 50000×g for 2 hours at 4° C. After centrifugation, the supernatant was removed in a biocabinet, and 500 μL PBS buffer was added to resuspend the pellet. Following resuspension, the lentiviral particles were aliquoted and preserved at −80° C. The titer was detected using Quantitative PCR (Q-PCR).

[0084] Killing Curve. CHO cells were adjusted in logarithmic growth phase after cell recovery and seeded at a concentration of 5×10.sup.4 cells per well in a 24-well plate. The cells were Incubated overnight at 37° C. and 5% CO.sub.2 in an incubator.

[0085] Following incubation, Puromycin was added to the culture medium at concentrations of 0.5 μg/mL, 1 μg/mL, 2 μg/mL, 4 μg/mL, 6 μg/mL, 8 μg/mL, and 10 μg/m L. Cells were observed daily for 3 days. The final Puromycin concentration used for stable cell line selection was 8 μg/mL, the concentration that was able to completely kill the cells after 3 days.

[0086] Stable Cell Line Selection. CHO cells were adjusted to a logarithmic growth phase after cell recovery and were seeded at a concentration of 5×10.sup.6 cells per well in a 6-well plate. The cells were then incubated at 37° C. and 5% CO.sub.2 in an incubator overnight.

[0087] Lenti-Luciferase-EGFP-puro and CD25 lentiviruses were then added to the cells at a multiplicity of infection (MOI) of 2 for transfection. Cells were centrifuged at 800×g for 1 hour at 25° C. Following centrifugation, the cells were incubated for 24 hours at 37° C. and 5% CO.sub.2 in an incubator.

[0088] After 24 hours, the medium was replaced with fresh medium and the cells were cultured for an additional 24 hours. Next, a puromycin medium was used to perform puromycin selection.

[0089] Cells were incubated in the puromycin medium for 5 days to killing untransfected cells via puromycin selection. Following purification, CD25 and luciferase expression were detected using FACS. An APC-anti CD25 antibody was incubated with CHO-CD25-Luciferase-EGFP target cells. FACS results showed that the positive rate of CD25 was 99% and the positive rate of GFP was 38% (FIG. 10). Double positive cells were 35% and could be used as target cells for co-culture experiments (FIG. 10).

[0090] Separation of T Cells from Peripheral Blood. Blood samples from healthy human donor peripheral blood were transferred into 15 mL sterile centrifuge tubes and centrifuged at 800×g for 20 minutes.

[0091] After centrifugation, the tubes were removed from the centrifuge gently without violent shaking or inverting the centrifuge tube. The pale-yellow serum in the upper layer was removed, and an equal volume of saline was added to the lower layer (the peripheral blood layer). The saline and peripheral blood layer were then gently mixed by inverting the centrifuge tube.

[0092] A lymphocyte separation solution was prepared and inverted upside down several times to mix well. Next, 5 mL of lymphocyte separation solution was added to a 15 mL centrifuge tube. The blood sample diluted in the second step was carefully pipetted along the tube wall to the upper layer of the lymphocyte separation reagent using a pipette to avoid mixing of the separation reagent and the blood sample. The tube was then centrifuged at 800×g for 20 minutes.

[0093] After centrifugation, the centrifuge tube was gently removed from the centrifuge. The white mononuclear cell layer in the middle of the tube was then drawn into a new sterile centrifuge tube an equal volume of saline was added to the tube, gently mixed, and centrifuged at 800×g for 5 minutes. After centrifugation, the supernatant was removed, and the PBMCs were washed once again. Cell density was adjusted to 5×10.sup.7 cells/m L, and the cells were transfer into a 2 mL cell cryotube.

[0094] Preparation of CAR-T Cells. First, Dynabeads were washed with PBS twice. The appropriate amount of Dynabeads were added to the PBMC, mixed gently, and incubated for 20 minutes at room temperature.

[0095] Next, a 2 mL cell cryotube was inserted into the magnetic pole and incubated for 1 minute at room temperature. Keeping the cryotube inserted in the hole of the magnetic pole, the tube was gently inverted to pour out the liquid in the tube.

[0096] The cell cryotube was removed from the magnetic pole, 3 mL of X-Vivo 15 medium (containing 200 IU/mL IL-2, 10 ng/mL IL7, 5 ng/mL IL-15) was added, and the cells and beads were resuspended with a pipette. Cell density was adjusted to 0.5-1×10.sup.6 cells/mL.

[0097] Cells were incubated at 37° C. and 5% CO.sub.2 in an incubator for 48 hours. Following incubation, cell density was adjusted to 1×10.sup.6 cells/mL. Cells were transfected using lentivirus expressing the pMSGV-IL-2-ligand-h(28BBζ)-3rd-CAR with MOI=20.

[0098] Polybrene was added to the prepared T cells in a final concentration of 6 μg/m L. Next, lentivirus was added to the prepared T cells and mixed thoroughly with a pipette, the culture vessel was sealed with a parafilm, and centrifuged at 800×g at room temperature for 1 hour.

[0099] After centrifugation, the culture was continued for 24 hours, and the medium was refreshed. After another 48 hours, the positive rate of CAR-T cells was detected by FACS.

[0100] For FACS analysis, primary T cells were transfected with lentivirus and CAR-T cells were collected as described. A PE-anti IL-2 antibody was incubated with the resultant cells. After incubation, a cytometer was used to detect the expression percentage of CAR-T cells. The control T cell (Con-T) group showed very low CAR-T at 0.87%, whereas the cells transfected with lentivirus expressing the pMSGV-IL-2-ligand-h(28BBζ)-3rd-CAR showed a very high percentage of CAR-T cells at 96.1% (FIG. 11). These results show that the prepared CAR-T cell surface can correctly express the constructed CAR and can be used for in vitro pharmacodynamic experiments.

[0101] Cytotoxicity of CAR-T Cells. First, target cells were adjusted into the logarithmic growth phase, and subcultured twice before performing the cytotoxicity experiment.

[0102] Target cells were resuspended in complete medium, and the cell density was adjusted to 2-5*10.sup.5 cells/m L. Using a new 96-well plate, the target cells were inoculated according to the amount of 100 μL/well. Unused wells around the 96-well plate were filled with 100 μL of sterile water per well to prevent evaporation of medium from the middle of the experimental wells. The well plates were placed in a 5% CO.sub.2, 37° C. incubator and cultured overnight.

[0103] Following incubation, prepared CAR-T cells were collected by centrifugation and resuspend in 1640 medium with 10% FBS. The medium was removed from the 96-well plates, and the cells were gently washed once with sterile PBS. Then, the lysis experiment was carried out using 4 E/T ratios (1:1, 1:2.5, 1:5, and 1:10). The well plates were placed in a 5% CO.sub.2, 37° C. incubator, and cultured for 24 hours. In the experiment, target cells were set as Maxi lysis and Mini lysis.

[0104] After the completion of the culture, the well plates were taken out from the incubator, and the ability of the recombinant CAR-T cells to lyse the target cells was examined by detecting the Luciferase activity (FIG. 12). The formula for calculating the percentage of target cell lysis is:

[00002]$\mathrm{Lysis}\%=\left(1-\frac{{\mathrm{RLU}}_{\mathrm{Sample}}}{{\mathrm{RLU}}_{\mathrm{Max}}}\right)\times 100\%$

[0105] Experimental groups cells expressing the CAR (W122618 CAR-T) showed a significant cytotoxic effect when co-cultured with target cells expressing CD25 (FIG. 12). There was no significant cytotoxic effect when control CHO cells were co-cultured with the experimental CAR-T (W122618 CAR-T) or control T (Con-T) cells.

[0106] Cytokine Detection. Target cells were adjusted into the logarithmic growth phase and subcultured twice before performing the experiment.

[0107] The target cells were resuspended in complete medium, and cell density was adjusted to 2-5*10.sup.5 cells/m L. In a new 96-well plate, the target cells were inoculated with 100 μL/well. Unused wells around the 96-well plate were filled with 100 μL of sterile water per well to prevent evaporation of medium from the middle of the experimental wells. The well plates were placed in a 5% CO.sub.2, 37° C. incubator and cultured overnight.

[0108] Prepared CAR-T cells were collected by centrifugation and resuspend in 1640 medium with 10% FBS. The medium was removed from the 96-well plates, and the cells were gently washed with sterile PBS once. Then, the cytokine secretion detection experiment was grouped into experimental and control groups as in the lysis test above. The well plates were placed in a 5% CO.sub.2, 37° C. incubator, and cultured for 24 hours.

[0109] After the completion of the culture, the well plate was taken out of the incubator, centrifuged at 1200×g for 5 minutes at room temperature, and the supernatants were collected to detect the secretion of IL-2 and IFNγ by ELISA.

[0110] For the ELISA, A standard curve was generated measuring optical density (O.D.) compared to cytokine concentration (FIG. 13 upper). Cytokine levels were measured for IL-2 (FIG. 13, lower left) and IFNγ (FIG. 13, lower right) at a series of effector/target (E/T) ratios. These data showed that compared with the control group (Con-T cultured with target cells), all the experimental groups showed increased IL-2 and IFNγ when co-cultured with target cells (FIG. 13).

[0111] T Cell Proliferation. To quantify T cell proliferation, T cells were transfected with the control (Con-T) or the experimental (W122618-1B CAR-T) lentiviral vector. The first day after virus infection was recorded as day 1 (FIG. 14). CAR-T and control T cells were counted using tryptan blue staining, and the number of viable cells were counted. Cell proliferation was analyzed to day 8 (FIG. 14).

REFERENCES

[0112] The references, patents and published patent applications listed below, and all references cited in the specification above are hereby incorporated by reference in their entirety, as if fully set forth herein.

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[0114] Guedan et al. Engineering and Design of Chimeric Antigen Receptors. Mol Ther Methods Clin Dev 12:145-156 (2018).

[0115] Kochenderfer et al. Construction and preclinical evaluation of an anti-CD19 chimeric antigen receptor. J Immunother 32(7):689-702 (2009).

[0116] Matutes. Adult T-cell leukaemia/lymphoma. J Clin Pathol 60(12):1373-1377 (2007).

[0117] Smith et al. Chimeric antigen receptor (CAR) T cell therapy for malignant cancers: Summary and perspective. J Cell Immunother 2(2):59-68 (2016).

[0118] Williams et al. Structure/function analysis of interleukin-2—toxin(DAB486IL-2). Fragment B sequences required for the delivery of fragment A to the cytosol of target cells. J Biol Chem 265(20):11885-11889 (1990).