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Msg  4159 of 4338  at  9/17/2020 1:17:05 AM  by

JBWIN


Building IP: Juno Therapeutics Patent Application re "METHODS OF IDENTIFYING CELLULAR ATTRIBUTES RELATED TO OUTCOMES ASSOCIATED WITH CELL THERAPY"

United States Patent Application20200292526
Kind CodeA1
HAUSE, Jr.; Ronald James ; et al.September 17, 2020

METHODS OF IDENTIFYING CELLULAR ATTRIBUTES RELATED TO OUTCOMES ASSOCIATED WITH CELL THERAPY

Abstract

Provided herein are methods for tracking certain cells associated with a cell therapy, such as from a starting cell composition or a sample prior to administration to a subject and from a sample following administration to a subject. In some aspects, the methods include assessing one or more parameters or attributes of such cells and methods of identifying cellular attributes associated with particular desired cells. The provided methods can be used in connection with cell therapy including adoptive transfer of engineered T cells or T cell precursors.


Inventors:HAUSE, Jr.; Ronald James; (Seattle, WA) ; LEVITSKY; Hyam I.; (Seattle, WA) ; CLOUSER; Christopher R.; (Seattle, WA) ; JOHNSTONE; Timothy G.; (Seattle, WA)
Applicant:
NameCityStateCountryType

Juno Therapeutics, Inc.

Seattle

WA

US
Assignee:Juno Therapeutics, Inc.
Seattle
WA

Family ID:1000004896169
Appl. No.:16/644486
Filed:September 7, 2018
PCT Filed:September 7, 2018
PCT NO:PCT/US2018/050114
371 Date:March 4, 2020

Related U.S. Patent Documents

Application NumberFiling DatePatent Number
62555643Sep 7, 2017

Current U.S. Class:1/1
Current CPC Class:G01N 33/505 20130101
International Class:G01N 33/50 20060101 G01N033/50

Claims



1. A method for identifying a property or attribute of a cell, the method comprising: (a) identifying the clonotype and/or a TCR sequence of all of a portion of a native TCR alpha and/or beta variable region or pair thereof, of at least one T cell from at least one test biological sample from a subject, said test biological sample obtained from the subject following administration of a cell therapy comprising T cells expressing a recombinant receptor, wherein the T cell in the test biological sample is genetically engineered with and/or expresses the recombinant receptor; (b) identifying, from a T cell composition, a cell that has the same clonotype or the same TCR sequence as the at least one T cell identified in (a), thereby identifying an originator T cell, wherein the T cell composition comprises T cells that are, or have been derived from, T cells previously obtained from the subject prior to administering the cell therapy to the subject; and (c) determining at least one property or attribute of the originator T cell.

2. A method for identifying a property or attribute of a cell, the method comprising: (a) identifying the clonotype and/or a TCR sequence of all of a portion of a native TCR alpha and/or beta variable region or pair thereof, of at least one T cell from at least one test biological sample from a subject, said test biological sample obtained from the subject following administration of a cell therapy comprising T cells expressing a recombinant receptor, wherein the T cell in the test biological sample is genetically engineered with and/or expresses the recombinant receptor; (b) determining at least one property or attribute of a cell, from a T cell composition, that has the same clonotype or the same TCR sequence as the at least one T cell identified in (a), wherein the T cell composition comprises T cells that are, or have been derived from, T cells previously obtained from the subject prior to administering the cell therapy to the subject.

3. A method for identifying a property or attribute of a cell, the method comprising: (a) identifying the clonotype and/or a TCR sequence of all or a portion of a native TCR alpha and/or beta variable region or pair thereof of one or more T cell genetically engineered with a recombinant receptor in at least one test biological sample from a subject, wherein said clonotype is known to be, determined to be, or suspected of being present in a cell in a T cell composition, thereby identifying one or more originator T cell, wherein: the at least one test biological sample is obtained from the subject following administration of a cell therapy comprising T cells expressing the recombinant receptor; and the T cell composition comprises T cells that are or are derived from cells of a sample obtained from the subject prior to administering the cell therapy to the subject; and (b) determining at least one or property or attribute of the one or more originator T cell.

4. The method of any of claims 1-3, wherein the one or more clonotype and/or TCR sequence that is identified is present in the test biological sample at the same or increased frequency or relative frequency as in the T cell composition.

5. A method for identifying a property or attribute of a cell, the method comprising: (a) identifying one or more clonotypes and/or one or more TCR sequences of all or a portion of a native TCR alpha and/or beta variable region or pair thereof that are the same in a plurality of samples, said plurality of samples selected from one or more compositions at different stages of a cell engineering process for generating a T cell therapy and/or a biological sample from a subject following administration of the T cell therapy to the subject, said T cell therapy comprising T cells expressing the recombinant receptor, thereby identifying an originator T cell; and (b) determining at least one property or attribute of the originator T cell.

6. The method of claim 5, wherein at least one of the plurality of samples is a T cell composition at a stage of a cell engineering process, said T cell composition comprising T cells that are, or have been derived from, T cells previously obtained from the subject prior to administering the cell therapy to the subject.

7. The method of claim 5 or claim 6, wherein at least one of the plurality of samples is a test biological sample, said test biological sample obtained from the subject following administration of a cell therapy comprising T cells expressing a recombinant receptor.

8. The method of any of claims 1-4 and 7, where the test biological sample is obtained from the subject at or about or within 1 days, 3 days, 6 days, 9 days, 12 days, 15 days, 18 days, 21 days, 24 days, 27 days or 30 days, optionally at or about 12 days, 12 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days or 29 days following the administration of the cell therapy comprising the T cells expressing the recombinant receptor.

9. The method of any of claims 1-4, 7 and 8, wherein the test biological sample is obtained from a subject at a time after the subject exhibits a response to the cell therapy following the administration, said response selected from a complete response (CR), progression free survival (PFS) or a partial response (PR).

10. The method of claim 9, wherein the response is durable in the subject for at least at least 3 months, at least 6 months, at least 9 months or at least 12 months, and the test biological sample is obtained from the subject at a time when the response is still durable in the subject.

11. The method of 1-10, wherein the genetically engineered T cell in the at least one test biological sample or the plurality of samples exhibits a predetermined phenotype, function or parameter.

12. The method of claim 11, wherein the predetermined phenotype, function or attribute is an effector function associated with T cell activation state, is a cell surface phenotype or is a pharmacokinetic activity.

13. The method of claim 12, wherein the predetermined phenotype, function or attribute is a pharmacokinetic activity and the pharmacokinetic activity comprises the number or relative number of recombinant receptor-expressing T cells in the sample.

14. The method of claim 13, wherein the test biological sample is obtained from the subject at a time at or immediately after a peak T cells expressing the recombinant receptor are detectable in the blood of the subject.

15. The method of claim 14, wherein the predetermined phenotype, function or attribute is a cell surface phenotype and the cell surface phenotype is a naive phenotype or a long-lived memory phenotype.

16. The method of claim 15, wherein the cell surface phenotype comprises a phenotype surface negative for CD56 or CD45RO and/or a surface positive for CD27, CD45RA, or CCR7.

17. The method of claim 15 or claim 16, wherein the cell surface phenotype is of one or both of CD27 and CCR7.

18. The method of any of claims 1-17, wherein the at least one T cell from the at least one test biological sample or the plurality of samples is selected or isolated from a biological sample from the subject based on the predetermined phenotype, function, or attribute.

19. The method of any of claims 1-18, wherein the at least one T cell from the at least one test biological sample or the plurality of samples is positive for or expresses the recombinant receptor, optionally is surface positive for the recombinant receptor.

20. The method of claim 19, wherein the at least one T cell that is positive for or expresses the recombinant receptor is selected or isolated from a biological sample from a subject.

21. The method of any of claims 1-20 that is repeated for a plurality of subjects.

22. The method of claim 21, further comprising identifying the at least one property or parameter of originator T cells that is present in a T cell composition from a majority of subjects.

23. The method of any of claims 1-22, wherein the at least one property or parameter is identified as an attribute of a T cell composition that is predicted to increase likelihood or a desired phenotype, property or attribute of a cell therapy following administration to a subject.

24. The method of any of claims 1-23, wherein the T cell composition is an input composition that does not comprise T cells genetically engineered with the recombinant receptor.

25. The method of claim 24, wherein the input composition is obtained by isolating a population of cells comprising the T cells from a biological sample.

26. The method of any of claims 1-25, wherein the T cell composition is an output composition comprising T cells genetically engineered with the recombinant receptor.

27. The method of claim 26, wherein the output composition is the cell therapy that had been administered to the subject in (a).

28. The method of claim 26 or claim 27, wherein the output composition is produced by a process comprising: (i) incubating an input composition comprising T cells with an agent comprising a nucleic acid molecule encoding the recombinant receptor under conditions to introduce the nucleic acid encoding the recombinant receptor into cells in the composition; and (ii) stimulating the T cells, prior to, during and/or subsequent to said incubation, wherein stimulating comprises incubating the cells in the presence of a stimulating condition that induces a primary signal, signaling, stimulation, activation and/or expansion of the T cells.

29. The method of claim 28, wherein the process further comprises, prior to (i), isolating a population comprising the T cells from a biological sample.

30. The method of claim 25 or claim 29, wherein the isolating comprises, selecting the T cells from the biological sample based on surface expression of CD3 or based on surface expression of one or both of CD4 and CD8, optionally by positive or negative selection.

31. The method of claim 25, 29 or 30, wherein the isolating comprises carrying out immunoaffinity-based selection.

32. The method of any of claims 25 and 29-31, wherein the biological sample is or comprises a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cells (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a white blood cell sample, an apheresis product, or a leukapheresis product.

33. The method of any of claims 28-32, wherein the stimulating condition comprises incubation with a stimulatory reagent capable of activating one or more intracellular signaling domains of one or more components of a TCR complex and/or one or more intracellular signaling domains of one or more costimulatory molecules.

34. The method of claim 33, wherein the stimulatory reagent comprises a primary agent that specifically binds to a member of a TCR complex and a secondary agent that specifically binds to a T cell costimulatory molecule.

35. The method of claim 34, wherein the primary agent specifically binds to CD3 and/or the costimulatory molecule is selected from the group consisting of CD28, CD137 (4-1-BB), OX40, or ICOS.

36. The method of claim 34 or claim 35, wherein the primary and secondary agents comprise antibodies, optionally an anti-CD3 antibody and an anti-CD28 antibody.

37. The method of any of claims 34-36, wherein the primary and secondary agent are present on the surface of a solid support, optionally a bead.

38. The method of any of claims 28-37, wherein the stimulating of the T cells is carried out or is initiated prior to the incubating, optionally for 18-24 hours at or about 37 degrees Celsius, wherein the T cells have not been introduced with the nucleic acid encoding the recombinant receptor.

39. The method of any of claims 28-38, wherein the stimulating condition comprises a cytokine selected from among IL-2, IL-15 and IL-7.

40. The method of any of claims 28-39, wherein the stimulating of the T cells is carried out subsequent to the incubating, said T cells comprising cells introduced with the nucleic acid encoding the recombinant receptor.

41. The method of claim 40, wherein the stimulating the cells is carried out under conditions to cultivate or expand T cells introduced optionally for a period of time to achieve a threshold concentration.

42. The method of any of claims 28-40, wherein the agent comprising a nucleic acid molecule encoding the recombinant receptor is a viral vector, optionally a lentiviral vector or a gamma retroviral vector.

43. The method of any of claims 28-42, wherein: the incubating and/or stimulating is carried out in the presence of one or more test agents or conditions; or the process further comprises culturing the input composition and/or stimulated cells in the presence of one or more test agents or conditions.

44. The method of claim 43, wherein the one or more test agents or conditions comprises presence or concentration of serum; time in culture; presence or amount of a stimulating agent; the type or extent of a stimulating agent; presence or amount of amino acids; temperature; the source or cell types of the input composition; the ratio or percentage of cell types in the input composition, optionally the CD4+/CD8+ cell ratio; the presence or amount of beads; cell density; static culture; rocking culture; perfusion; the type of viral vector; the vector copy number; the presence of a transduction adjuvant; cell density of the input composition in cryopreservation; the extent of expression of the recombinant receptor; or the presence of a compound to modulate cell phenotype.

45. The method of claim 43 or claim 44, wherein the one or more test agents or conditions comprises one or more compounds from a library of test compounds.

46. The method of any of claims 1-45, wherein the test biological sample is a serum, blood or plasma sample.

47. The method of any of claims 1-46, wherein the test biological sample is or comprises a tumor sample.

48. The method of any of claims 1-47, wherein the test biological sample is obtained from the subject greater than or greater than about 7 days, 10 days, 14 days, 21 days, 28 days, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year or 2 years or more after initiation of administration of the cell therapy.

49. The method of any of claims 1-48, wherein the test biological sample is obtained from the subject greater than or greater than about 28 days after initiation of administration of the cell therapy, optionally at or about at day 29 or greater after initiation of administration of the cell therapy.

50. The method of any of claims 1-49, wherein the at least one test biological sample comprises a plurality of test biological samples, optionally at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more test biological samples.

51. The method of claim 50, wherein each of the plurality of test biological samples is obtained from the subject on different days after initiation of administration of the cell therapy, optionally on consecutive days, every other day, every third day, or once a week for a predetermined time after initiation of administration of the cell therapy.

52. The method of claim 51, wherein the identified clonotype is one whose frequency or relative frequency is retained or increased among the plurality of assessed tested biological samples over the predetermined period of time.

53. The method of any of claims 1-52, wherein the clonotype in a) and/or b) is determined by high-throughput single cell immune sequencing of nucleic acid encoding natively paired TCR chains.

54. The method of claim 53, wherein the natively paired TCR chains comprise TCR .alpha.-.beta. or TCR .gamma.-.delta. pairs.

55. The method of any of claims 1-54, wherein the test biological sample comprises a plurality of T cells and the one or more clonotype is identified simultaneously or from a single reaction.

56. The method of any of claims 1-55, wherein the T cell composition comprises a plurality of T cells and the one or more clonotype is identified from a single reaction.

57. The method of any of claims 1-56, wherein the at least one property or parameter is determined by single cell gene expression profiling and/or single cell surface phenotyping.

58. The method of claim 57, wherein the at least one property or parameter is determined by single cell gene expression profiling, wherein the single cell gene expression profiling is of at least one gene product or is of the whole-transcriptome or a portion thereof.

59. The method of claim 58, wherein the at least one gene product is selected from CD4, ICOS, FOXP3, FOXP3V1, PMCH, CD80, FOXP3Y, CD86, CD70, CD40, IL-6, CD2, CD3D, GPR171, CXCL13, PD-1 (CD279), IL-2, IL-4, IL-10, CD8B, KLRK1, CCL4, RUNX3V1, RUNX3, NKG7, CD45RA, CD45RO, CD62L, CD69, CD25, CCR7, CD27, CD28, CD56, CD122, CD127, CD95, CXCR3, LFA-1, KLRG1, T-bet, CD8, IL-7R.alpha., IL-2R.beta., CD3, CD14, ROR1, granzyme B, granzyme H, CD20, CD11b, CD16, HLA-DR, PD-L1, IFN.gamma., KIRK1, caspase 2, caspase 3, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, Bcl-2, Bax, Bad, Bid, CD196 (CCR6), CTLA-4 (CD152), TIGIT (VSIG9, VSTM3), LAG-3 (CD223), 2B4 (CD244), BTLA (CD272), TIM3 (HAVCR2), VISTA (PD1-H) and CD96.

60. The method of claim 59, wherein the at least one property or parameter is determined by single cell surface phenotyping of at least one T cell surface marker.

61. The method of claim 60, wherein the at least one T cell surface marker is selected from CD4, CD8, CD45RA, CD45RO, CD62L, CD69, CD25, CCR7, CD27, CD28, CD56, CD122, CD127, T-bet, IL-7R.alpha., CD95, IL-2R.beta., CXCR3, LFA-1 or KLRG1.

62. The method of any of claims 53-61, wherein the single cell gene expression profiling or single cell surface phenotyping is coupled to or carried out in the same reaction as the single cell immune sequencing.

63. The method of any of claims 1-62, wherein the recombinant receptor is or comprises a chimeric receptor.

64. The method of claim 63, wherein the chimeric receptor is capable of binding to a target antigen that is associated with, specific to, and/or expressed on a cell or tissue of a disease, disorder or condition.

65. The method of claim 64, wherein the disease, disorder or condition is an infectious disease or disorder, an autoimmune disease, an inflammatory disease, or a tumor or a cancer.

66. The method of claim 64 or claim 65, wherein the target antigen is a tumor antigen.

67. The method of any of claims 64-66, wherein the target antigen is selected from among ROR1, B cell maturation antigen (BCMA), carbonic anhydrase 9 (CAIX), Her2/neu (receptor tyrosine kinase erbB2), L1-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen, anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), EPHa2, erb-B2, erb-B3, erb-B4, erbB dimers, EGFR vIII, folate binding protein (FBP), FCRLS, FCRHS, fetal acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kinase insert domain receptor (kdr), kappa light chain, Lewis Y, L1-cell adhesion molecule, (L1-CAM), Melanoma-associated antigen (MAGE)-Al, MAGE-A3, MAGE-A6, Preferentially expressed antigen of melanoma (PRAME), survivin, TAG72, B7-H6, IL-13 receptor alpha 2 (IL-13Ra2), CA9, GD3, HMW-MAA, CD171, G250/CAIX, HLA-AI MAGE A1, HLA-A2 NY-ESO-1, PSCA, folate receptor-a, CD44v6, CD44v7/8, avb6 integrin, 8H9, NCAM, VEGF receptors, 5T4, Fetal AchR, NKG2D ligands, CD44v6, dual antigen, a cancer-testes antigen, mesothelin, murine CMV, mucin 1 (MUC1), MUC16, PSCA, NKG2D, NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), Her2/neu, estrogen receptor, progesterone receptor, ephrinB2, CD123, c-Met, GD-2, O-acetylated GD2 (OGD2), CE7, Wilms Tumor 1 (WT-1), a cyclin, cyclin A2, CCL-1, CD138, a pathogen-specific antigen and an antigen associated with a universal tag.

68. The method of any of claims 64-66, wherein the target antigen is selected from among .alpha.v.beta.6 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), a cancer-testis antigen, cancer/testis antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), a cyclin, cyclin A2, C-C Motif Chemokine Ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), type III epidermal growth factor receptor mutation (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrinB2, ephrin receptor A2 (EPHa2), estrogen receptor, Fc receptor like 5 (FCRL5; also known as Fc receptor homolog 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), a folate binding protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gp100), glypican-3 (GPC3), G Protein Coupled Receptor 5D (GPRC5D), Her2/neu (receptor tyrosine kinase erb-B2), Her3 (erb-B3), Her4 (erb-B4), erbB dimers, Human high molecular weight-melanoma-associated antigen (HMW-MAA), hepatitis B surface antigen, Human leukocyte antigen A1 (HLA-A1), Human leukocyte antigen A2 (HLA-A2), IL-22 receptor alpha(IL-22R.alpha.), IL-13 receptor alpha 2 (IL-13R.alpha.2), kinase insert domain receptor (kdr), kappa light chain, L1 cell adhesion molecule (L1-CAM), CE7 epitope of L1-CAM, Leucine Rich Repeat Containing 8 Family Member A (LRRC8A), Lewis Y, Melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A10, mesothelin (MSLN), c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D (NKG2D) ligands, melan A (MART-1), neural cell adhesion molecule (NCAM), oncofetal antigen, Preferentially expressed antigen of melanoma (PRAME), progesterone receptor, a prostate specific antigen, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1), survivin, Trophoblast glycoprotein (TPBG also known as 5T4), tumor-associated glycoprotein 72 (TAG72), Tyrosinase related protein 1 (TRP1, also known as TYRP1 or gp75), Tyrosinase related protein 2 (TRP2, also known as dopachrome tautomerase, dopachrome delta-isomerase or DCT), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms Tumor 1 (WT-1), a pathogen-specific or pathogen-expressed antigen, or an antigen associated with a universal tag, and/or biotinylated molecules, and/or molecules expressed by HIV, HCV, HBV or other pathogens.

69. The method of any of claims 63-68, wherein the chimeric receptor is a chimeric antigen receptor (CAR).

70. The method of any of claims 63-69, wherein the chimeric receptor comprises an extracellular domain comprising an antigen-binding domain that specifically binds a target antigen and an intracellular signaling domain comprising an ITAM.

71. The method of claim 70, wherein the antigen-binding domain is or comprises an antibody or an antibody fragment thereof, which optionally is a single chain fragment.

72. The method of claim 71, wherein the fragment comprises antibody variable regions joined by a flexible linker.

73. The method of claim 71 or claim 72, wherein the fragment comprises an scFv.

74. The method of any of claims 63-73, wherein the chimeric receptor further comprises a spacer and/or a hinge region.

75. The method of any of claims 63-74, wherein chimeric receptor comprises an intracellular signaling region.

76. The method of claim 75, wherein the intracellular signaling region comprises an intracellular signaling domain.

77. The method of claim 76, wherein the intracellular signaling domain is or comprises a primary signaling domain, a signaling domain that is capable of inducing a primary activation signal in a T cell, a signaling domain of a T cell receptor (TCR) component, and/or a signaling domain comprising an immunoreceptor tyrosine-based activation motif (ITAM).

78. The method of claim 77, wherein the intracellular signaling domain is or comprises an intracellular signaling domain of a CD3 chain, optionally a CD3-zeta (CD3.zeta.) chain, or a signaling portion thereof.

79. The method of any of claims 75-78, wherein chimeric receptor further comprises a transmembrane domain disposed between the extracellular domain and the intracellular signaling region.

80. The method of any of claims 75-79, wherein the intracellular signaling region further comprises a costimulatory signaling region.

81. The method of claim 80, wherein the costimulatory signaling region comprises an intracellular signaling domain of a T cell costimulatory molecule or a signaling portion thereof.

82. The method of claim 80 or claim 81, wherein the costimulatory signaling region comprises an intracellular signaling domain of a CD28, a 4-1BB or an ICOS or a signaling portion thereof.

83. The method of any of claims 69-82, wherein the CAR comprises an extracellular antigen-recognition domain that specifically binds to the antigen and an intracellular signaling domain comprising an ITAM, wherein optionally, the intracellular signaling domain comprises an intracellular domain of a CD3-zeta (CD3.zeta.) chain; and/or wherein the CAR further comprises a costimulatory signaling region, which optionally comprises a signaling domain of CD28 or 4-1BB.

84. The method of any of claims 1-83, wherein the T cell composition and/or cell therapy comprises CD4 and/or CD8 T cells.

85. The method of any of claims 1-84, wherein the clonotype comprises the TCR sequences of all or a portion of a native TCR alpha and/or beta variable region or pair thereof.

86. The method of any of claims 1-85, wherein the clonotype and/or TCR sequence is of a T cell genetically engineered with or expressing the recombinant receptor.

87. The method of any of claims 1-86, wherein the clonotype and/or TCR sequence is of a CD8+ T cell.
Description



CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. provisional applications No. 62/555,643, filed Sep. 7, 2017, entitled "METHODS OF IDENTIFYING CELLULAR ATTRIBUTES RELATED TO OUTCOMES ASSOCIATED WITH CELL THERAPY," the contents of which are incorporated by reference in their entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 735042013540SeqList.TXT, created Sep. 4, 2018, which is 42,585 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.

FIELD

[0003] The present disclosure relates to methods for tracking certain cells associated with a cell therapy, such as from a starting cell composition or a sample prior to administration to a subject and from a sample following administration to a subject. In some aspects, the methods include assessing one or more parameters or attributes of such cells and methods of identifying cellular attributes associated with particular desired cells. The provided methods can be used in connection with cell therapy including adoptive transfer of engineered T cells or T cell precursors.

BACKGROUND

[0004] Various methods are available for preparing and administering cells or cell compositions for therapeutic use. For example, methods are available for preparing cells, including T cells, for engineering and cell therapy, and assessment of the activity of the cells upon administration. Improved strategies are needed to assess the activity and/or survival of particular sub-populations, to improve the activity and/or survival of the cells or cell compositions, to improve the manufacturing process and/or to allow improved administration. Provided are embodiments that meet such needs.

SUMMARY

[0005] Provided herein are methods for identifying a property or attribute of a cell including (a) identifying the clonotype and/or a TCR sequence of all of a portion of a native TCR alpha and/or beta variable region or pair thereof, of at least one T cell from at least one test biological sample from a subject, said test biological sample obtained from the subject following administration of a cell therapy containing T cells expressing a recombinant receptor, wherein the T cell in the test biological sample is genetically engineered with and/or expresses the recombinant receptor; (b) identifying, from a T cell composition, a cell that has the same clonotype or the same TCR sequence as the at least one T cell identified in (a), thereby identifying an originator T cell, wherein the T cell composition contains T cells that are, or have been derived from, T cells previously obtained from the subject prior to administering the cell therapy to the subject; and (c) determining at least one property or attribute of the originator T cell.

[0006] Provided herein are methods for identifying a property or attribute of a cell including (a) identifying the clonotype and/or a TCR sequence of all of a portion of a native TCR alpha and/or beta variable region or pair thereof, of at least one T cell from at least one test biological sample from a subject, said test biological sample obtained from the subject following administration of a cell therapy containing T cells expressing a recombinant receptor, wherein the T cell in the test biological sample is genetically engineered with and/or expresses the recombinant receptor; (b) determining at least one property or attribute of a cell, from a T cell composition, that has the same clonotype or the same TCR sequence as the at least one T cell identified in (a), wherein the T cell composition contains T cells that are, or have been derived from, T cells previously obtained from the subject prior to administering the cell therapy to the subject.

[0007] In some embodiments, the genetically engineered T cell in the test biological sample exhibits a predetermined phenotype, function or parameter. In some embodiments, the predetermined phenotype, function or attribute is an effector function associated with T cell activation state, is a cell surface phenotype or is a pharmacokinetic activity. In some cases, the predetermined phenotype, function or attribute is a pharmacokinetic activity and the pharmacokinetic activity includes determining the number or relative number of recombinant receptor-expressing T cells in the sample. In some aspects, the predetermined phenotype, function or attribute is a cell surface phenotype and the cell surface phenotype is a naive phenotype or a long-lived memory phenotype.

[0008] Provided herein is a method for identifying a property or attribute of a cell including identifying the clonotype and/or a TCR sequence of all or a portion of a native TCR alpha and/or beta variable region or pair thereof of one or more T cell genetically engineered with a recombinant receptor in at least one test biological sample from a subject, wherein said clonotype is known to be, determined to be, or suspected of being present in a cell in a T cell composition, thereby identifying one or more originator T cell, wherein: the at least one test biological sample is obtained from the subject following administration of a cell therapy containing T cells expressing the recombinant receptor; and the T cell composition contains T cells that are or are derived from cells of a sample obtained from the subject prior to administering the cell therapy to the subject; and determining at least one or property or attribute of the one or more originator T cell. In some embodiments, the one or more clonotype and/or TCR sequence that is identified is present in the test biological sample at the same or increased frequency or relative frequency as in the T cell composition.

[0009] Provided herein is a method for identifying a property or attribute of a cell including identifying one or more clonotypes and/or one or more TCR sequences of all or a portion of a native TCR alpha and/or beta variable region or pair thereof that are the same in a plurality of samples, said plurality of samples selected from one or more compositions at different stages of a cell engineering process for generating a T cell therapy and/or a biological sample from a subject following administration of the T cell therapy to the subject, said T cell therapy containing T cells expressing the recombinant receptor, thereby identifying an originator T cell; and determining at least one property or attribute of the originator T cell.

[0010] Provided herein are methods for assessing clonal diversity of a sample containing T cells including identifying one or more clonotypes and/or one or more TCR sequences of all or a portion of a native TCR alpha and/or beta variable region or pair thereof in one of a plurality of samples containing T cells, at different stages of a cell engineering process for generating a T cell therapy and/or following administration of the T cell therapy to a subject, said T cell therapy containing T cells expressing the recombinant receptor and determining the clonal diversity in each of the plurality of samples. In some embodiments, the method further includes determining at least one property or attribute of the one or more cells in the plurality of samples. In some embodiments, the method further includes comparing the clonal diversity of each of the plurality of samples.

[0011] In some embodiments, the comparing includes determining the increase or decrease in clonal diversity in the plurality of samples from the same subject. In some cases, the clonal diversity is determined based on the relative frequency of the one or more clonotypes and/or one or more TCR sequences. In some embodiments, the determining the clonal diversity is represented as clonality, Shannon-adjusted clonality or top 25 clonality of each of the plurality of samples. In some embodiments, the determining the clonal diversity is represented as Shannon-adjusted clonality in each of the plurality of samples.

[0012] In some embodiments, at least one of the plurality of samples is a T cell composition at a stage of a cell engineering process, said T cell composition containing T cells that are, or have been derived from, T cells previously obtained from the subject prior to administering the cell therapy to the subject. In some of any such embodiments, at least one of the plurality of samples is a test biological sample, said test biological sample obtained from the subject following administration of a cell therapy containing T cells expressing a recombinant receptor. In some embodiments, the method further includes determining a phenotype, function or parameter of the one or more cells in the plurality of sample, prior to the identifying. In some embodiments, the genetically engineered T cell in the test biological sample exhibits a predetermined phenotype, function or parameter. In some examples, the predetermined phenotype, function or attribute is an effector function associated with T cell activation state, is a cell surface phenotype or is a pharmacokinetic property. In some cases, the predetermined phenotype, function or attribute is a pharmacokinetic property and the pharmacokinetic property includes the number or relative number of recombinant receptor-expressing T cells in the sample. In some embodiments, the predetermined phenotype, function or attribute is a cell surface phenotype and the cell surface phenotype is a naive phenotype or a long-lived memory phenotype. In some embodiments, the cell surface phenotype is determined based on surface expression of one or both of CD27 and CCR7.

[0013] In some embodiments, the test biological sample is obtained from the subject at or about or within 1 days, 3 days, 6 days, 9 days, 12 days, 15 days, 18 days, 21 days, 24 days, 27 days or 30 days, optionally at or about 12 days, 12 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days or 29 days following the administration of the cell therapy containing the T cells expressing the recombinant receptor. In some embodiments, the test biological sample is obtained from a subject at a time after the subject exhibits a response to the cell therapy following the administration, said response selected from a complete response (CR), progression free survival (PFS) or a partial response (PR). In some cases, the response is durable in the subject for at least at least 3 months, at least 6 months, at least 9 months or at least 12 months, and the test biological sample is obtained from the subject at a time when the response is still durable in the subject. In some embodiments, the genetically engineered T cell in the test biological sample exhibits a predetermined phenotype, function or parameter.

[0014] In some embodiments, the predetermined phenotype, function or attribute is an effector function associated with T cell activation state, is a cell surface phenotype or is a pharmacokinetic activity. In some embodiments, the predetermined phenotype, function or attribute is a pharmacokinetic activity and the pharmacokinetic activity includes the number or relative number of recombinant receptor-expressing T cells in the sample. In some embodiments, the test biological sample is obtained from the subject at a time at or immediately after a peak T cells expressing the recombinant receptor are detectable in the blood of the subject. In some embodiments, the predetermined phenotype, function or attribute is a cell surface phenotype and the cell surface phenotype is a naive phenotype or a long-lived memory phenotype. In some examples, the cell surface phenotype includes a phenotype surface negative for CD56 or CD45RO and/or a surface positive for CD27, CD45RA, or CCR7. In some cases, the cell surface phenotype is of one or both of CD27 and CCR7.

[0015] In some embodiments, the at least one T cell from the at least one test biological sample or the plurality of samples is selected or isolated from a biological sample from the subjects based on the predetermined phenotype, function, or attribute. In some embodiments, the at least one T cell from the at least one test biological sample or the plurality of samples is positive for or expresses the recombinant receptor, optionally is surface positive for the recombinant receptor. In some cases, the at least one T cell that is positive for or expresses the recombinant receptor is selected or isolated from a biological sample from a subject.

[0016] In some of any such embodiments, the method is repeated for a plurality of subjects.

[0017] In some embodiments, the method includes identifying the at least one property or parameter of originator T cells or T cells in the sample that is present in a T cell composition from a majority of subjects.

[0018] In some embodiments, the at least one property or parameter is identified as an attribute of a T cell composition that is predicted to increase likelihood or a desired property, phenotype, attribute or outcome of a cell therapy following administration to a subject.

[0019] In some of any such embodiments, the T cell composition is an input composition that does not contain T cells genetically engineered with the recombinant receptor. In some aspects, the input composition is obtained by isolating a population of cells containing the T cells from a biological sample.

[0020] In some of any such embodiments, the T cell composition is an output composition containing T cells genetically engineered with the recombinant receptor. In some cases, the output composition is the cell therapy administered to the subject. In some embodiments, the output composition is produced by a process including: (i) incubating an input composition containing T cells with an agent containing a nucleic acid molecule encoding the recombinant receptor under conditions to introduce the nucleic acid encoding the recombinant receptor into cells in the population; and (ii) stimulating the cells, prior to, during and/or subsequent to said incubation, wherein stimulating includes incubating the cells in the presence of a stimulating condition that induces a primary signal, signaling, stimulation, activation and/or expansion of the cells. In some embodiments, the process further includes, prior to (i), isolating the population of cells from a biological sample.

[0021] In some of any such embodiments, the isolating includes selecting cells (e.g. T cells) from the biological sample based on surface expression of CD3 or based on surface expression of one or both of CD4 and CD8, optionally by positive or negative selection. In some cases, the isolating includes carrying out immunoaffinity-based selection.

[0022] In some embodiments, the biological sample is or contains a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cells (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a white blood cell sample, an apheresis product, or a leukapheresis product.

[0023] In some embodiments, the stimulating condition includes incubation with a stimulatory reagent capable of activating one or more intracellular signaling domains of one or more components of a TCR complex and/or one or more intracellular signaling domains of one or more costimulatory molecules. In some aspects, the stimulatory reagent contains a primary agent that specifically binds to a member of a TCR complex and a secondary agent that specifically binds to a T cell costimulatory molecule. In some cases, the primary agent specifically binds to CD3 and/or the costimulatory molecule is selected from the group consisting of CD28, CD137 (4-1-BB), OX40, or ICOS. In some embodiments, the primary and secondary agents contain antibodies, optionally an anti-CD3 antibody and an anti-CD28 antibody. In some embodiments, the primary and secondary agent are present on the surface of a solid support, optionally a bead.

[0024] In some of any such embodiments, the stimulating the cells is carried out or is initiated prior to the incubating, optionally for 18-24 hours at or about 37 degrees Celsius, wherein the T cells have not been introduced with the nucleic acid encoding the recombinant receptor. In some cases, the stimulating condition includes a cytokine selected from among IL-2, IL-15 and IL-7. In some embodiments, the stimulating cells is carried out subsequent to the incubating, optionally for a period of time to achieve a threshold concentration. In some embodiments, the T cells including cells introduced with the nucleic acid encoding the recombinant receptor. In some embodiments, the stimulating the cells is carried out under conditions to cultivate or expand T cells introduced.

[0025] In some embodiments, the agent containing a nucleic acid molecule encoding the recombinant receptor is a viral vector, optionally a lentiviral vector or a gamma retroviral vector.

[0026] In some of any such embodiments, the incubating and/or stimulating is carried out in the presence of one or more test agents or conditions; or the process further includes culturing the input composition and/or stimulated cells in the presence of one or more test agents or conditions. In some embodiments, the one or more test agents or conditions includes presence or concentration of serum; time in culture; presence or amount of a stimulating agent; the type or extent of a stimulating agent; presence or amount of amino acids; temperature; the source or cell types of the input composition; the ratio or percentage of cell types in the input composition, optionally the CD4+/CD8+ cell ratio; the presence or amount of beads; cell density; static culture; rocking culture; perfusion; the type of viral vector; the vector copy number; the presence of a transduction adjuvant; cell density of the input composition in cryopreservation; the extent of expression of the recombinant receptor; or the presence of a compound to modulate cell phenotype. In some embodiments, the one or more test agents or conditions includes one or more compounds from a library of test compounds.

[0027] In some of any such embodiments, the test biological sample is a serum, blood or plasma sample. In some embodiments, the test biological sample is or contains a tumor sample. In some embodiments, the test biological sample is obtained from the subject greater than or greater than about 7 days, 10 days, 14 days, 21 days, 28 days, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year or 2 years or more after initiation of administration of the cell therapy. In some embodiments, the test biological sample is obtained from the subject greater than or greater than about 28 days after initiation of administration of the cell therapy, optionally at or about at day 29 or greater after initiation of administration of the cell therapy.

[0028] In some of any such embodiments, the at least one test biological sample contains a plurality of test biological samples, optionally at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more test biological samples. In some aspects, each of the plurality of test biological samples is obtained from the subject on different days after initiation of administration of the cell therapy, optionally on consecutive days, every other day, every third day, or once a week for a predetermined time after initiation of administration of the cell therapy.

[0029] In some embodiments, the identified clonotype is one whose frequency or relative frequency is retained or increased among the plurality of assessed tested biological samples over the predetermined period of time. In some embodiments, the clonotype and/or TCR sequence is determined by high-throughput or next-generation nucleic acid sequencing. In some embodiments, the clonotype and/or TCR sequence is determined by high-throughput or next-generation RNA sequencing (RNAseq). In some embodiments, the clonotype and/or TCR sequence is determined by high-throughput or next-generation nucleic acid sequencing of one or more regions of the TCR.alpha., TCR.beta., TCR.gamma. and TCR.delta. expressed in the T cell.

[0030] In some cases, the clonotype and/or TCR sequence is determined by high-throughput single cell immune sequencing of nucleic acid encoding natively paired TCR chains. In some aspects, the natively paired TCR chains contain TCR .alpha.-.beta. or TCR .gamma.-.delta. pairs.

[0031] In some of any such embodiments, the test biological sample contains a plurality of T cells and the one or more clonotype is identified simultaneously or from a single reaction.

[0032] In some of any such embodiments, the T cell composition contains a plurality of T cells and the one or more clonotype is identified from a single reaction.

[0033] In some embodiments, the at least one property or parameter is determined by single cell gene expression profiling and/or single cell surface phenotyping. In some cases, the at least one property or parameter is determined by single cell gene expression profiling, wherein the single cell gene expression profiling is of at least one gene product or is of the whole-transcriptome or a portion thereof. In some embodiments, the at least one gene product is selected from CD4, ICOS, FOXP3, FOXP3V1, PMCH, CD80, FOXP3Y, CD86, CD70, CD40, IL-6, CD2, CD3D, GPR171, CXCL13, PD-1 (CD279), IL-2, IL-4, IL-10, CD8B, KLRK1, CCL4, RUNX3V1, RUNX3, NKG7, CD45RA, CD45RO, CD62L, CD69, CD25, CCR7, CD27, CD28, CD56, CD122, CD127, CD95, CXCR3, LFA-1, KLRG1, T-bet, CD8, IL-7R.alpha., IL-2R.beta., CD3, CD14, ROR1, granzyme B, granzyme H, CD20, CD11b, CD16, HLA-DR, PD-L1, IFN.gamma., KIRK1, caspase 2, caspase 3, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, Bcl-2, Bax, Bad, Bid, CD196 (CCR6), CTLA-4 (CD152), TIGIT (VSIG9, VSTM3), LAG-3 (CD223), 2B4 (CD244), BTLA (CD272), TIM3 (HAVCR2), VISTA (PD1-H) and CD96.

[0034] In some embodiments, the at least one property or parameter is determined by single cell surface phenotyping of at least one T cell surface marker. In some cases, the at least one T cell surface marker is selected from CD4, CD8, CD45RA, CD45RO, CD62L, CD69, CD25, CCR7, CD27, CD28, CD56, CD122, CD127, T-bet, IL-7R.alpha., CD95, IL-2R.beta., CXCR3, LFA-1 or KLRG1.

[0035] In some embodiments, the single cell gene expression profiling or single cell surface phenotyping is coupled to or carried out in the same reaction as the single cell immune sequencing. In some embodiments, identification of the clonotype and/or TCR sequence includes barcoded nucleic acid sequencing.

[0036] In some of any such embodiments the recombinant receptor is or contains a chimeric receptor. In some cases, the chimeric receptor is capable of binding to a target antigen that is associated with, specific to, and/or expressed on a cell or tissue of a disease, disorder or condition. In some aspects, the disease, disorder or condition is an infectious disease or disorder, an autoimmune disease, an inflammatory disease, or a tumor or a cancer.

[0037] In some of any such embodiments, the target antigen is a tumor antigen. In some embodiments, the target antigen is selected from among ROR1, B cell maturation antigen (BCMA), carbonic anhydrase 9 (CAIX), Her2/neu (receptor tyrosine kinase erbB2), L1-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen, anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), EPHa2, erb-B2, erb-B3, erb-B4, erbB dimers, EGFR vIII, folate binding protein (FBP), FCRL5, FCRH5, fetal acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kinase insert domain receptor (kdr), kappa light chain, Lewis Y, L1-cell adhesion molecule, (L1-CAM), Melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, Preferentially expressed antigen of melanoma (PRAME), survivin, TAG72, B7-H6, IL-13 receptor alpha 2 (IL-13Ra2), CA9, GD3, HMW-MAA, CD171, G250/CAIX, HLA-AI MAGE A1, HLA-A2 NY-ESO-1, PSCA, folate receptor-a, CD44v6, CD44v7/8, avb6 integrin, 8H9, NCAM, VEGF receptors, 5T4, Foetal AchR, NKG2D ligands, CD44v6, dual antigen, a cancer-testes antigen, mesothelin, murine CMV, mucin 1 (MUC1), MUC16, PSCA, NKG2D, NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), Her2/neu, estrogen receptor, progesterone receptor, ephrinB2, CD123, c-Met, GD-2, O-acetylated GD2 (OGD2), CE7, Wilms Tumor 1 (WT-1), a cyclin, cyclin A2, CCL-1, CD138, a pathogen-specific antigen and an antigen associated with a universal tag.

[0038] In some embodiments, the target antigen is selected from among .alpha.v.beta.6 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), a cancer-testis antigen, cancer/testis antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), a cyclin, cyclin A2, C-C Motif Chemokine Ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), type III epidermal growth factor receptor mutation (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrinB2, ephrin receptor A2 (EPHa2), estrogen receptor, Fc receptor like 5 (FCRL5; also known as Fc receptor homolog 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), a folate binding protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gp100), glypican-3 (GPC3), G Protein Coupled Receptor 5D (GPRC5D), Her2/neu (receptor tyrosine kinase erb-B2), Her3 (erb-B3), Her4 (erb-B4), erbB dimers, Human high molecular weight-melanoma-associated antigen (HMW-MAA), hepatitis B surface antigen, Human leukocyte antigen A1 (HLA-A1), Human leukocyte antigen A2 (HLA-A2), IL-22 receptor alpha(IL-22R.alpha.), IL-13 receptor alpha 2 (IL-13R.alpha.2), kinase insert domain receptor (kdr), kappa light chain, L1 cell adhesion molecule (L1-CAM), CE7 epitope of L1-CAM, Leucine Rich Repeat Containing 8 Family Member A (LRRC8A), Lewis Y, Melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A10, mesothelin (MSLN), c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D (NKG2D) ligands, melan A (MART-1), neural cell adhesion molecule (NCAM), oncofetal antigen, Preferentially expressed antigen of melanoma (PRAME), progesterone receptor, a prostate specific antigen, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1), survivin, Trophoblast glycoprotein (TPBG also known as 5T4), tumor-associated glycoprotein 72 (TAG72), Tyrosinase related protein 1 (TRP1, also known as TYRP1 or gp75), Tyrosinase related protein 2 (TRP2, also known as dopachrome tautomerase, dopachrome delta-isomerase or DCT), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms Tumor 1 (WT-1), a pathogen-specific or pathogen-expressed antigen, or an antigen associated with a universal tag, and/or biotinylated molecules, and/or molecules expressed by HIV, HCV, HBV or other pathogens. Antigens targeted by the receptors in some embodiments include antigens associated with a B cell malignancy, such as any of a number of known B cell marker. In some embodiments, the antigen is or includes CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Igkappa, Iglambda, CD79a, CD79b or CD30.

[0039] In some embodiments, the chimeric receptor is a chimeric antigen receptor (CAR). In some cases, the chimeric receptor contains an extracellular domain containing an antigen-binding domain. In some embodiments, the antigen-binding domain is or contains an antibody or an antibody fragment thereof, which optionally is a single chain fragment. In some aspects, the fragment contains antibody variable regions joined by a flexible linker. In some embodiments, the fragment contains an scFv.

[0040] In some of any such embodiments, the chimeric receptor further contains a spacer and/or a hinge region. In some cases, the chimeric receptor contains an intracellular signaling region. In some embodiments, the intracellular signaling region contains an intracellular signaling domain. In some embodiments, the intracellular signaling domain is or contains a primary signaling domain, a signaling domain that is capable of inducing a primary activation signal in a T cell, a signaling domain of a T cell receptor (TCR) component, and/or a signaling domain containing an immunoreceptor tyrosine-based activation motif (ITAM). In some cases, the intracellular signaling domain is or contains an intracellular signaling domain of a CD3 chain, optionally a CD3-zeta (CD3.zeta.) chain, or a signaling portion thereof.

[0041] In some of any such embodiments, chimeric receptor further contains a transmembrane domain disposed between the extracellular domain and the intracellular signaling region. In some aspects, the intracellular signaling region further contains a costimulatory signaling region. In some embodiments, the costimulatory signaling region contains an intracellular signaling domain of a T cell costimulatory molecule or a signaling portion thereof. In some cases, the costimulatory signaling region contains an intracellular signaling domain of a CD28, a 4-1BB or an ICOS or a signaling portion thereof. In some embodiments, the costimulatory signaling region is between the transmembrane domain and the intracellular signaling region.

[0042] In some of any such embodiments, the T cell composition and/or cell therapy contains CD4 and/or CD8 T cells.

[0043] In some of any such embodiments, the clonotype contains the TCR sequences of all or a portion of a native TCR alpha and/or beta variable region or pair thereof. In some embodiments, the clonotype and/or TCR sequence is of a T cell genetically engineered with or expressing the recombinant receptor. In some embodiments, the clonotype and/or TCR sequence is of a CD8+ T cell.


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