Natural killer cell products and methods
The technology relates generally to the field of immunology and relates in part to compositions and methods for growing and storing modified natural killer cells, including for example, conditional chemical regulation of natural killer cell function. The technology further relates to pharmaceutical compositions and treatment of subjects using modified natural killer cells.
1. A nucleic acid, comprising a first polynucleotide and a second polynucleotide, wherein the first polynucleotide encodes a chimeric polypeptide comprising:
a) a ligand binding region; and
b) a signaling region, comprising
a MyD88 polypeptide and a CD40 cytoplasmic polypeptide region lacking the CD40 extracellular domain, or
a truncated MyD88 polypeptide lacking the TIR domain and a CD40 cytoplasmic polypeptide region lacking the CD40 extracellular domain; and
wherein the second polynucleotide encodes an IL-15 polypeptide.
2. A modified natural killer (NK) cell, comprising a first polynucleotide and a second polynucleotide, wherein the first polynucleotide encodes a chimeric polypeptide comprising:
a) a ligand binding region; and
b) a signaling region, comprising
a MyD88 polypeptide and a CD40 cytoplasmic polypeptide region lacking the CD40 extracellular domain, or
a truncated MyD88 polypeptide lacking the TIR domain and a CD40 cytoplasmic polypeptide region lacking the CD40 extracellular domain;
wherein the second polynucleotide encodes an IL-15 polypeptide.
3. The modified NK cell of claim 2 , wherein the ligand binding region comprises
a) two copies of FKBP12v36; or
b) an FKBP12 polypeptide and an FKBP-rapamycin-binding (FRB) polypeptide or FRB variant polypeptide.
4. The modified NK cell of claim 2 , which further comprises a third polynucleotide, wherein the third polynucleotide encodes
a) a chimeric antigen receptor (CAR) or a T cell receptor (TCR); further wherein the CAR or TCR targets
i. PSMA, PSCA, Muc1, CD19, RORI, Mesothelin, GD2, CD123, Muc16, CD33, CD38, CD44v6, Her2/Neu, CD20, CD30, BCMA, PRAME, NY-ESO-1, or EGFRvIII; or
ii. HER-2, PSCA, CD123, or BCMA.
5. The modified NK cell of claim 2 , which further comprises a third polynucleotide, wherein the third polynucleotide encodes a chimeric pro-apoptotic polypeptide comprising a second ligand binding region and a caspase-9 polypeptide lacking the caspase activation domain (CARD domain), and wherein the ligand binding region of the chimeric polypeptide is different than the second ligand binding domain of the chimeric pro-apoptotic polypeptide.
6. The modified NK cell of claim 2 , which further comprises a third polynucleotide, wherein the third polynucleotide encodes a chimeric pro-apoptotic polypeptide comprising a second ligand binding region and a caspase-9 polypeptide lacking the caspase activation domain (CARD domain), wherein the ligand binding region of the chimeric polypeptide comprises two copies of FKBP12v36, and wherein the second ligand binding domain of the chimeric pro-apoptotic polypeptide comprises an FRB binding polypeptide or FRB variant polypeptide, and an FKBP polypeptide.
7. The modified NK cell of claim 4 , which further comprises a fourth polynucleotide, wherein the fourth polynucleotide encodes a chimeric pro-apoptotic polypeptide comprising a second ligand binding region and a caspase-9 polypeptide lacking the caspase activation domain (CARD domain), and wherein the ligand binding region of the chimeric polypeptide
a) is different than the second ligand binding domain of the chimeric pro-apoptotic polypeptide; or
b) comprises two copies of FKBP12v36, and wherein the second ligand binding domain of the chimeric pro-apoptotic polypeptide comprises an FRB binding polypeptide or FRB variant polypeptide, and an FKBP polypeptide.
8. The modified NK cell of claim 2 , which further comprises a third polynucleotide, wherein the third polynucleotide encodes a chimeric pro-apoptotic polypeptide comprising a second ligand binding region and a caspase-9 polypeptide lacking the caspase activation domain (CARD domain), wherein the ligand binding region of the chimeric polypeptide comprises an FRB binding polypeptide or FRB variant polypeptide, and an FKBP polypeptide, and the second ligand binding domain of the chimeric pro-apoptotic polypeptide comprises two copies of FKBP12v36.
9. A modified natural killer (NK) cell, comprising a first polynucleotide and a second polynucleotide, wherein the first polynucleotide encodes a chimeric polypeptide comprising a signaling region, wherein the signaling region comprises:
a MyD88 polypeptide and a CD40 cytoplasmic polypeptide region lacking the CD40 extracellular domain, or
a truncated MyD88 polypeptide lacking the TIR domain and a CD40 cytoplasmic polypeptide region lacking the CD40 extracellular domain;
wherein the second polynucleotide encodes an IL-15 polypeptide.
10. The modified NK cell of claim 2 , wherein the signaling region comprises a truncated MyD88 polypeptide lacking the TIR domain and a CD40 cytoplasmic polypeptide region lacking the CD40 extracellular domain.
11. The modified NK cell of claim 2 , wherein the truncated MyD88 polypeptide lacking the TIR domain comprises the amino acid sequence of
a) SEQ ID NO: 119 or an amino acid sequence that is 90% identical to SEQ ID NO: 119; or
b) SEQ ID NO: 2 or an amino acid sequence that is 90% identical to SEQ ID NO: 2.
12. The modified NK cell of claim 9 , wherein the truncated MyD88 polypeptide lacking the TIR domain comprises the amino acid sequence of
a) SEQ ID NO: 119 or an amino acid sequence that is 90% identical to SEQ ID NO: 119; or
b) SEQ ID NO: 2 or an amino acid sequence that is 90% identical to SEQ ID NO: 2.
13. The modified NK cell of claim 2 , wherein
a) the modified NK cells
i. are or have been cryostored; or
ii. have been stored at a temperature of −150° C. or below; or
iv have not been contacted with exogenous IL-15; or
b) the ligand which binds to the ligand binding domain of the chimeric polypeptide is
i. rimiducid, AP20187 or AP1510; or
ii. rapamycin or a rapalog.
14. A method for stimulating an immune response comprising administering modified NK cells of claim 2 to a subject;
wherein
(a) the subject has a disease or condition associated with an elevated level of expression of a target antigen expressed by a target cell; or
(b) a tumor has been detected in the subject.
15. A method for stimulating an immune response comprising administering (i) modified NK cells claim 2 to a subject and (ii) a ligand that binds to the ligand binding region of the chimeric polypeptide; wherein the ligand is administered after the modified NK cells are administered to the subject.
16. The method of claim 15 , wherein the subject has a disease or condition associated with an elevated level of expression of a target antigen expressed by a target cell;
further wherein the ligand is administered to the subject in amount effective to reduce the number or concentration of the target antigen or target cells in the subject.
17. The method of claim 14 , wherein
(a) the subject has cancer;
(b) the subject has been diagnosed as having a hyperproliferative disease;
(c) the subject has been diagnosed with sickle cell anemia or metachromatic eukodystrophy;
(d) the subject has been diagnosed with a condition selected from the group consisting of a primary immune deficiency condition, hemophagocytosis lymphohistiocytosis (HAH) or another hemophagocytic condition, an inherited marrow failure condition, a hemoglobinopathy, a metabolic condition, and an osteoclast condition;
(e) the subject has been diagnosed with a disease or condition selected from the group consisting of Severe Combined Immune Deficiency (SCID), Combined Immune Deficiency (CID), Congenital T-cell Defect/Deficiency, Common Variable Immune Deficiency (CVID), Chronic Granulomatous Disease, IPEX (Immune deficiency, polyendocrinopathy, enteropathy, X-linked) or IPEX-like, Wiskott-Aldrich Syndrome, CD40 Ligand Deficiency, Leukocyte Adhesion Deficiency, DOCA 8 Deficiency, IL-10 Deficiency/IL-IO Receptor Deficiency, GATA 2 deficiency, X-linked lymphoproliferative disease (XAP), Cartilage Hair Hypoplasia, Shwachman Diamond Syndrome, Diamond Blackfan Anemia, Dyskeratosis Congenita, Fanconi Anemia, Congenital Neutropenia, Sickle Cell Disease, Thalassemia, Mucopolysaccharidosis, Sphingolipidoses, and Osteopetrosis;
(f) the subject has been diagnosed with leukemia; or
(g) the subject has been diagnosed with an infection of viral etiology selected from the group consisting HIV, influenza, Herpes, viral hepatitis, Epstein Bar, polio, viral encephalitis, measles, chicken pox, Cytomegalovirus (CMV), adenovirus (ADV), HHV-6 (human herpesvirus 6, I), and Papilloma virus, or has been diagnosed with an infection of bacterial etiology selected from the group consisting of pneumonia, tuberculosis, and syphilis, or has been diagnosed with an infection of parasitic etiology selected from the group consisting of malaria, trypanosomiasis, leishmaniasis, trichomoniasis, and amoebiasis.
18. A method for reducing the number of modified NK cells in the event of a negative symptom or condition, comprising administering to a subject who has been previously been administered modified NK cells of claim 5 a ligand that binds to the second ligand binding region of the chimeric pro-apoptotic polypeptide in an amount effective to reduce the number or concentration of the modified NK cells in the subject;
wherein the amount is effective to kill
(a) at least 30% of the cells that express the chimeric pro-apoptotic polypeptide;
(b) at least 60% of the cells that express the chimeric pro-apoptotic polypeptide; or
(c) at least 90% of the cells that express the chimeric pro-apoptotic polypeptide further wherein the negative symptom or condition is graft-versus-host disease.