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1. WO2020117526 - IMMUNOTHERAPIES USING ENHANCED iPSC DERIVED EFFECTOR CELLS

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CLAIMS

What is claimed is:

1. A cell or a population thereof, wherein

(i) the cell is (a) an induced pluripotent cell (iPSC), a clonal iPSC, or an iPS cell line cell; or (b) a derivative cell obtained from differentiating the cell of (a); and

(ii) the cell comprises:

(1) an exogenous polynucleotide encoding NKG2C; and optionally

(2) one or both of an exogenous polynucleotide encoding CD94 and an exogenous polynucleotide encoding DAP 12.

2. The cell or population thereof of claim 1, wherein the derivative cell of (i)(b) is a hematopoietic cell, and comprises longer telomeres in comparison to its native counterpart cell obtained from peripheral blood, umbilical cord blood, or any other donor tissues.

3. The cell or population thereof of claim 1, wherein the cell further comprises one or more of:

(i) a BiKE or a TriKE;

(ii) B2M null or low;

(iii) CIITA null or low;

(iv) introduced expression of HLA-G or non-cleavable HLA-G;

(v) a chimeric antigen receptor (CAR);

(vi) a partial or full peptide of a cell surface expressed exogenous cytokine or a receptor thereof;

(vii) at least one of the genotypes listed in Table 1;

(viii) deletion or reduced expression in at least one of B2M, TAPI, TAP2, tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCR a or b constant region, NKG2A, NKG2D, CD38, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, TIGIT, or any gene in the chromosome 6p21 region; and

(ix) introduced or increased expression in at least one of HLA-E, 41BBL, CD3, CD4, CD8, CD 16, CD47, CD113, CD131, CD137, CD80, PDL1, A2AR, FC receptor, an engager, and surface triggering receptor for coupling with bi- or multi- specific or universal engagers.

4. The cell or population thereof of claim 1 or 3, wherein the cell is a derivative NK or a derivative T cell, and has at least one of the following characteristics comprising:

(i) improved tumor targeting specificity,

(ii) improved persistency and/or survival,

(iii) increased resistance to native immune cells,

(iv) increased cytotoxicity,

(v) improved tumor penetration,

(vi) enhanced or acquired ADCC,

(vii) enhanced ability in migrating, and/or activating or recruiting bystander immune cells to tumor sites,

(viii) enhanced ability to reduce tumor immunosuppression, and

(ix) improved ability in rescuing tumor antigen escape,

in comparison to its native counterpart cell obtained from peripheral blood, umbilical cord blood, or any other donor tissues.

5. The cell or population thereof of claim 3, wherein (a) the BiKE or the TriKE comprises a first component for CD16, CD64, or NKG2C binding, and a second component for CD30, CD33, BCMA or EPCAM binding; (b) the TriKE comprises a first component for NKG2C binding, a second component for tumor antigen binding, and an IL15 linker between the first and the second components; wherein the tumor antigen is

(i) at least one of ADGRE2, carbonic anhydrase IX (CA1X), CCRI, CCR4,

carcinoembryonic antigen (CEA), CD3, CD5, CD7, CD8, CD10, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD44V6, CD49f, CD56, CD70, CD74, CD99, CD123, CD133,

CD 138, , CDS, CLEC12A, an antigen of a cytomegalovirus (CMV) infected cell, epithelial glycoprotein2 (EGP 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), EGFRvIII, receptor tyrosine -protein kinases erb- B2,3,4, EGFIR, EGFR-VIII, ERBB folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-a, Ganglioside G2 (GD2), Ganglioside G3 (GD3), human Epidermal Growth Factor Receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), ICAM-1, Integrin B7, Interleukin- 13 receptor subunit alpha-2 (IL-13Ra2), K- light chain, kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), LI cell adhesion molecule (Ll-CAM), LILRB2, melanoma antigen family A 1 (MAGE-A1), MICA/B, Mucin 1 (Muc-1), Mucin 16 (Muc-16), Mesothelin (MSLN), NKCSI, NKG2D ligands, c-Met, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), PRAME, prostate stem cell antigen (PSCA), PRAME prostate-specific membrane antigen (PSMA), tumor- associated glycoprotein 72 (TAG-72), TIM-3, TRBCI, TRBC2, vascular endothelial growth factor R2 (VEGF- R2), Wilms tumor protein (WT-1), and a pathogen antigen; or

(ii) CD33.

6. The cell or population thereof of claim 3, wherein the high affinity non-cleavable CD 16 (hnCD16) or a variant thereof comprise at least one of:

(a) FI 76V and S197P in ectodomain domain of CD 16;

(b) a full or partial ectodomain originated from CD64;

(c) a non-native (or non-CD 16) transmembrane domain;

(d) a non-native (or non-CD 16) intracellular domain;

(e) a non-native (or non-CD 16) signaling domain;

(f) a non-native stimulatory domain; and

(g) transmembrane, signaling, and stimulatory domains that are not originated from CD 16, and are originated from a same or different polypeptide.

7. The cell or population thereof of claim 6, wherein

(a) the non-native transmembrane domain is derived from CD3D, CD3E, CD3G, CD3z, CD4, CD8, CD 8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, 0X40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD 16, 1L7, 1112, IL15, KIR2DL4, K1R2.DS : , NKp30, NKp44, NKp46, NKG2C, NKG2D, or T cell receptor (TCR) polypeptide;

(b) the non-native stimulatory domain is derived from CD27, CD28, 4- IBB, 0X40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP 12, CTLA-4, or NKG2D polypeptide;

(c) the non-native signaling domain is derived from EΏ3z, 2B4, DAP 10, DAP 12, DNAM1, CD137 (41BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or XKG2D polypeptide; or

(d) the non-native transmembrane domain is derived from NKG2D, the non-native stimulatory domain is derived from 2B4, and the non-native signaling domain is derived from Eϋ3z.

8.


(ix) co-expressed with a partial or full peptide of a cell surface expressed exogenous cytokine or a receptor thereof, optionally in separate constructs or in a bi-cistronic construct;

(xi) co-expressed with a checkpoint inhibitor, optionally in separate constructs or in a bi-cistronic construct;

(xii) specific to CD 19 or BCMA; and/or

(xiii) specific to any one of ADGRE2, carbonic anhydrase IX (CA1X), CCRI, CCR4, carcinoembryonic antigen (CEA), CD3, CD5, CD7, CD8, CD10, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD44V6, CD49f, CD56, CD70, CD74, CD99, CD123, CD133,

CD 138, , CDS, CLEC12A, an antigen of a cytomegalovirus (CMV) infected cell, epithelial glycoprotein2 (EGP 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), EGFRvIII, receptor tyrosine -protein kinases erb- B2,3,4, EGFIR, EGFR-VIII, ERBB folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-a, Ganglioside G2 (GD2), Ganglioside G3 (GD3), human Epidermal Growth Factor Receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), ICAM-1, Integrin B7, Interleukin- 13 receptor subunit alpha-2 (IL-13Ra2), K- light chain, kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), LI cell adhesion molecule (Ll-CAM), LILRB2, melanoma antigen family A 1 (MAGE-A1), MICA/B, Mucin 1 (Muc-1), Mucin 16 (Muc-16), Mesothelin (MSLN), NKCSI, NKG2D ligands, c-Met, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), PRAME, prostate stem cell antigen (PSCA), PRAME prostate-specific membrane antigen (PSMA), tumor- associated glycoprotein 72 (TAG-72), TIM-3, TRBCI, TRBC2, vascular endothelial growth factor R2 (VEGF- R2), Wilms tumor protein (WT-1), and a pathogen antigen;

wherein the CAR of any one of (i) to (xiii) is optionally inserted at a TCR constant region, and/or is driven by an endogenous promoter of TCR, and/or the TCR is knocked out by the CAR insertion.

9. The cell or population thereof of claim 3, wherein the cell further comprises a partial or full peptide of a cell surface expressed exogenous cytokine or a receptor thereof, and wherein the exogenous cytokine or a receptor thereof

(a) comprises at least one of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and respective receptor thereof; or

(b) comprises at least one of:

(i) co-expression of IL15 and IL15Ra by suing a self-cleaving peptide;

(ii) a fiision protein of IL15 and IL15Ra;

(iii) an IL15/IL15Ra fusion protein with intracellular domain of IL15Ra truncated;

(iv) a fusion protein of IL15 and membrane bound Sushi domain of IL15Ra;

(v) a fusion protein of IL15 and I L 15 R b;

(vi) a fusion protein of IL15 and common receptor yC, wherein the common receptor yC is native or modified; and

(vii) a homodimer of IL15RP; wherein any one of (i)-(vii) can be co-expressed with a CAR in separate constructs or in a bi-cistronic construct;

and optionally,

(c) is transiently expressed.

10. The cell or population thereof of claim 3, wherein the cell is a derivative NK or a derivative T cell, wherein

(i) the derivative NK cell is capable of recruiting, and/or migrating T cells to tumor sites;

(ii) the derivative NK or the derivative T cell is capable of reducing tumor

immunosuppression in the presence of one or more checkpoint inhibitors.

11. The cell or population thereof of claim 8 or 10, wherein the checkpoint inhibitors are antagonists to one or more checkpoint molecules comprising PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A2aR, BATE, BTLA, CD39, CD47, CD73, CD94, CD96, CD 160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA/B, NR4A2, MAFB, OCT-2, Rara (retinoic acid receptor alpha), TLR3, VISTA, NKG2A/HLA-E, or inhibitory KIR.

12. The cell or population thereof of claim 10, wherein the checkpoint inhibitors comprise:

(a) one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102,

IPH43, IPH33, lirimumab, monalizumab, nivolumab, pembrolizumab, and their derivatives or functional equivalents; or

(b) at least one of atezolizumab, nivolumab, and pembrolizumab.

13. The cell or population thereof of claim 1, wherein the derivative cell comprises derivative CD34 cell, derivative hematopoietic stem and progenitor cell, derivative

hematopoietic multipotent progenitor cell, derivative T cell progenitor, derivative NK cell progenitor, derivative T cell, derivative NKT cell, derivative NK cell, or derivative B cell.

14. The cell or population thereof of claim 1, wherein the cell comprises:

(i) one or more exogenous polynucleotides integrated in a selected locus; or

(ii) more than two exogenous polynucleotides integrated in different selected loci.

15. The cell or population thereof of claim 14, wherein the selected locus comprises at least one of AAVS1, CCR5, ROSA26, collagen, HTRP, Hl l, GAPDH, RUNX1, B2M, TAPI, TAP2, tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCR a or b constant region, NKG2A, NKG2D, CD38, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, TIGIT, or any gene in the chromosome 6p21 region.

16. The cell or population thereof of claim 15, wherein the selected locus is a constant region of TCR a.

17. A composition compri sing the cell or population thereof of any one of the claims 1-16.

18. A composition for therapeutic use comprising the derivative cell of any one of the claims 1-16, and one or more therapeutic agents.

19. The composition of claim 18, wherein the therapeutic agents comprise a peptide, a cytokine, a checkpoint inhibitor, a mitogen, a growth factor, a small RNA, a dsRNA (double stranded RNA), mononuclear blood cells, feeder cells, feeder cell components or replacement factors thereof, a vector comprising one or more polynucleic acids of interest, an antibody, a chemotherapeutic agent or a radioactive moiety, or an immunomodulatory drug (IMiD).

20. The composition of claim 19, wherein the checkpoint inhibitor comprises

(a) one or more antagonists checkpoint molecules comprising PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4- IBB, 4-1BBL, A2aR, BATE, BTLA, CD39, CD47, CD73, CD94, CD96, CD 160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA/B, NR4A2, MAFB, OCT-2, Rara (retinoic acid receptor alpha), TLR3, VISTA, NKG2A/HLA-E, or inhibitory KIR;

(b) one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102,

IPH43, IPH33, lirimumab, monalizumab, nivolumab, pembrolizumab, and their derivatives or functional equivalents;

(c) at least one of atezolizumab, nivolumab, and pembrolizumab.

21. The composition of claim 19, wherein the antibody comprises:

(a) anti-CD20, anti-HER2, anti-CD52, anti-EGFR, anti-CD123, anti-GD2, anti-PDLl, and/or anti-CD38 antibody;

(b) one or more of retuximab, veltuzumab, ofatumumab, ublituximab, ocaratuzumab, obinutuzumab, trastuzumab, pertuzumab, alemtuzumab, certuximab, dinutuximab, avelumab, daratumumab, isatuximab, MOR202, 7G3, CSL362, elotuzumab, and their humanized or Fc modified variants or fragments and their functional equivalents and biosimilars; or

(c) daratumumab.

22. Therapeutic use of the therapeutic composition of any one of the claims 17-21 by introducing the composition to a subject suitable for adoptive cell therapy, wherein the subject has an autoimmune disorder; a hematological malignancy; a solid tumor; cancer, or a virus infection.

23. A method of manufacturing the derivative cell of any one of the claims 1-16 comprising differentiating an iPSC, wherein the iPSC comprises: an exogenous NKG2C; and optionally one or both of an exogenous CD94, and an exogenous DAP 12; and optionally one or more of:

(i) a BiKE or a TriKE;

(ii) B2M null or low;

(iii) CIITA null or low;

(iv) introduced expression of HLA-G or non-cleavable HLA-G;

(v) a chimeric antigen receptor (CAR);

(vi) a partial or full peptide of a cell surface expressed exogenous cytokine or a receptor thereof;

(vii) at least one of the genotypes listed in Table 1;

(viii) deletion or reduced expression in at least one of B2M, TAPI, TAP2, tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCR a or b constant region, NKG2A, NKG2D, CD38, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, TIGIT, or any gene in the chromosome 6p21 region; and

(ix) introduced or increased expression in at least one of HLA-E, 41BBL, CD3, CD4, CD8, CD 16, CD47, CD113, CD131, CD137, CD80, PDL1, A2AR, TCR, Fc receptor, an engager, or surface triggering receptor for coupling with bi- or multi- specific or universal engagers.

24. The method of manufacturing the derivative cells of claim 23, further comprising genomically engineering iPSC to obtain a clonal iPSC having at least one genotype listed in Table 1.

25. The method of manufacturing the derivative cell of claim 23, wherein the genomic engineering comprises targeted editing.

26. The method of manufacturing the derivative cell of claim 25, wherein the targeted editing comprising deletion, insertion, or in/del, and wherein the targeted editing is carried out by CRISPR, ZFN, TALEN, homing nuclease, homology recombination, or any other functional variation of these methods.

27. CRISPR mediated editing of clonal iPSCs, wherein the edited clonal iPSCs comprise:

(a) an exogenous NKG2C; and optionally one or both of an exogenous CD94, and an exogenous DAP 12; and optionally one or more of:

(i) a BiKE or a TriKE;

(ii) a high affinity non-cleavable CD 16 (hnCD16) or a variant thereof; and

(iii) a chimeric antigen receptor (CAR); or

(b) at least one of the genotypes listed in Table 1 ;

wherein the CAR is optionally inserted at TCR constant region, and/or is driven by an endogenous promoter of TCR, and/or the TCR is knocked out by the CAR insertion.

28. A method of preventing or reducing tumor antigen escape and/or tumor relapse, comprising administering to a subject under the treatment effector cells comprising:

(a) (i) an exogenous NKG2C; and optionally one or both of an exogenous CD94, and an exogenous DAP 12; and optionally,

(ii) one or more of: a BiKE or a TriKE; a high affinity non-cleavable CD 16

(hnCD16) or a variant thereof; a chimeric antigen receptor (CAR); or

(b) at least one of the genotypes listed in Table 1 ; and

an antigen specific monoclonal antibody, or any of the humanized or Fc modified variants or fragments, functional equivalents and biosimilars thereof, wherein the antigen targeted by the antibody is different from tumor antigen recognized by the CAR, the BiKE or the TriKE.