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EP-3775228-B1 - ENGINEERED IMMUNE EFFECTOR CELLS AND USE THEREOF

EP3775228B1EP 3775228 B1EP3775228 B1EP 3775228B1EP-3775228-B1

Inventors

  • VALAMEHR, BAHRAM
  • BJORDAHL, Ryan
  • GOODRIDGE, Jode
  • LEE, Tom Tong

Dates

Publication Date
20260506
Application Date
20190328

Claims (15)

  1. A construct for engineering a cell to obtain a genomically edited cell, the construct comprising: (i) a left homology arm and a right homology arm targeting CD38 at a selected position in CD38 locus (LHA/CD38, RHA/CD38) in the cell genome, operatively linked to (ii) one or more nucleic acid sequences encoding one or more exogenous proteins; wherein the genomically edited cell is an induced pluripotent cell (iPSC), a clonal iPSC, an iPS cell line cell, or an NK cell; wherein the genomically edited cell comprises a targeted integration of the one or more nucleic acid sequences encoding one or more exogenous protein at the selected position in CD38 locus, and CD38 knockout; and wherein the cell is a human cell.
  2. The construct of claim 1, wherein: (a) the one or more nucleic acid sequences encoding one or more exogenous proteins are operatively linked to, and are driven by, an endogenous promoter of CD38 upon integration; (b) the selected position in CD38 locus is in an exon of CD38; or (c) wherein the one or more exogenous proteins comprise at least one or both of: (i) a CD16 or a variant thereof; and (ii) a cell surface expressed exogenous IL15/IL15 receptor fusion protein (IL15RF) or a variant thereof; and wherein the genomically edited cell comprises the one or more nucleic acid sequences encoding CD16 and IL15RF, or variants thereof.
  3. The construct of claim 2, (i) wherein the CD16 or a variant thereof comprises at least one of: (a) F176V and S197P in ectodomain domain of CD16; (b) a full or partial ectodomain originated from CD64; (c) a non-native (or non-CD16) transmembrane domain; (d) a non-native (or non-CD16) intracellular domain; (e) a non-native (or non-CD16) signaling domain; (f) a non-native stimulatory domain; and (g) transmembrane, signaling, and stimulatory domains that are not originated from CD16, and are originated from a same or different polypeptide; or (ii) wherein the IL15RF or a variant thereof comprises at least one of: (a) co-expressed IL15 and IL15Rα by using a self-cleaving peptide; (b) a fusion protein of IL15 and IL15Rα; (c) an IL15/IL15Rα fusion protein with intracellular domain of IL15Rα truncated; (d) a fusion protein of IL15 and membrane bound Sushi domain of IL15Rα; (e) a fusion protein of IL15 and IL15Rβ; (f) a fusion protein of IL15 and common receptor γC, wherein the common receptor γC is native or modified; (g) a homodimer of IL15Rβ; and (h) a polynucleotide encoding an IL15RF comprising an amino acid sequence of at least 75%, 80%, 85%, 90%, 95% or 99% identity to SEQ ID NOs: 17, 19 or 21.
  4. The construct of claim 3, wherein the CD16 or a variant thereof comprises one or more of: (i) a non-native transmembrane domain derived from CD3D, CD3E, CD3G, CD3ζ, CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, or T cell receptor (TCR) polypeptide; (ii) a non-native stimulatory domain derived from CD27, CD28, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or NKG2D polypeptide; (iii) a non-native signaling domain derived from CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137 (41BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or NKG2D polypeptide; or (iv) a 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 CD3ζ.
  5. The construct of claim 1, wherein the genomically edited cell further comprises a chimeric antigen receptor (CAR), wherein the CAR has at least one following characteristics: (i) T cell specific or NK cell specific; (ii) bi-specific antigen binding CAR; (iii) a switchable CAR; (iv) a dimerized CAR; (v) a split CAR; (vi) a multi-chain CAR; (vii) an inducible CAR; (viii) co-expressed with another CAR; (ix) co-expressed with a partial or full peptide of a cell surface expressed exogenous cytokine or a receptor thereof; (xi) co-expressed with a checkpoint inhibitor; (xii) encoded by a nucleic acid further comprised in the construct, wherein the CAR is inserted at the selected position in CD38 locus; (xiii) inserted at a TCR locus, and/or is driven by an endogenous promoter of TCR, and/or the TCR is knocked out by the CAR insertion; (xiii) specific to CD19 or BCMA; and/or (xiv) specific to any one of ADGRE2, carbonic anhydrase IX (CAIX), CCR1, 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, CD138, 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-13Rα2), κ-light chain, kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (L1-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.
  6. The construct of claim 1, further comprising one or more of: (i) an exogenous promoter that drives expression of the one or more nucleic acid sequences encoding one or more exogenous proteins, wherein the exogenous promoter is constitutive, inducible, temporal-, tissue-, and/or cell type- specific; (ii) a linker sequence inserted between two nucleic acid sequences encoding exogenous proteins; (iii) an insulator at the 3' end of LHA/CD38 and an insulator at the 5' end of RHA/CD38; and (iv) a polyA signal, optionally wherein (i) the exogenous promoter is CMV, EF1α, PGK, CAG, or UBC; (ii) the linker sequence comprises a 2A sequence encoding a self-cleaving 2A peptide; and/or (iii) the linker sequence comprises an Internal Ribosome Entry Sequence (IRES).
  7. The construct of claim 1, wherein the genomically edited cell further comprises: (a) one or more following phenotypes: (i) comprising longer telomeres in comparison to its native counterpart cell obtained from peripheral blood, umbilical cord blood, or any other donor tissues; (ii) CD38 -/- CD16; (iii) CD38 -/- IL15; and/or (iv) CD38 -/- CD16 IL15, or (b) one or more of: (i) HLA-I deficiency; (ii) HLA-II deficiency; (iii) introduced expression of HLA-G or non-cleavable HLA-G; (iv) deletion or reduced expression in at least one of B2M, TAP1, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and any gene in the chromosome 6p21 region; and (v) introduced or increased expression in at least one of HLA-E, 41BBL, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A R, CAR, Fc receptor, an engager, and surface triggering receptor for coupling with bi- or multi- specific or universal engagers.
  8. The construct of claim 1, wherein the NK cell is an iPSC derived NK cell; or wherein the iPSC derived NK cell has at least one of the following characteristics comprising: (i) improved persistency and/or survival; (ii) increased resistance to native immune cells; (iii) increased cytotoxicity; (iv) improved tumor penetration; (v) enhanced or acquired ADCC; (vi) enhanced ability in migrating, and/or activating or recruiting bystander immune cells, to tumor sites; (vii) enhanced ability to reduce tumor immunosuppression; (viii) improved ability in rescuing tumor antigen escape; and (ix) reduced fratricide in the presence of anti-CD38 antibodies or CD38 CAR, in comparison to its native counterpart cell obtained from peripheral blood, umbilical cord blood, or other donor tissues, optionally, wherein the anti-CD38 antibody is daratumumab, isatuximab, or MOR202, or any of the humanized or Fc modified variants or fragments, functional equivalents and biosimilars thereof.
  9. An expression vector that comprises a construct according to any of claims 1-8.
  10. A host cell comprising a construct according to any of claims 1-8, or an expression vector of claim 9; wherein the host cell is the iPSC, the clonal iPSC, the iPS cell line cell, or the NK cell.
  11. A kit comprising (a) a construct according to any of claims 1-8, an expression vector according to claim 9, or a host cell according to claim 10; and (b) one or more medium for iPSC culturing or differentiation.
  12. A construct according to any of claims 1-8, an expression vector according to claim 9, or a host cell according to claim 10, for use in a medicament.
  13. An in vitro method of producing a genomically edited iPSC or effector cell derived therefrom using a construct according to any of claims 1-8 comprising: (i) introducing the construct to an iPSC, wherein the iPSC is a human iPSC; and (ii) incubating the iPSC for a sufficient period of time to enable targeted integration of one or more nucleic acid sequence encoding one or more exogenous protein at CD38 locus and consequently knocking out the expression of CD38 of the iPSC, thereby obtaining a genomically edited iPSC, optionally, wherein the genomically edited iPSC has improved genomic stability than iPSC undergoing integration and knocking out in separate and step-wise editing events.
  14. The method of claim 13, further comprising: (iii) directing differentiation of the genomically edited iPSC from step (ii) to obtain the effector cell comprising the same targeted integration of one or more nucleic acid sequence encoding one or more exogenous protein at CD38 locus and CD38 knockout as the genomically edited iPSC.
  15. The method of claim 13, wherein the introducing step further comprises introducing a CRISPR-cas/gNA (guide nucleic acid), a ZFN, a TALEN, a homing nuclease, or any other functional variation thereof.

Description

RELATED APPLICATION This application claims priority to U.S. Provisional Application Serial No. 62/649,781, filed March 29, 2018, U.S. Provisional Application Serial No. 62/774,052, filed November 30, 2018, and International Application No. PCT/US 18/67289, filed December 21, 2018. REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY This application references a Computer Readable Form (CRF) of a Sequence Listing in ASCII text format submitted with this application, entitled 13601-204-228_SEQ_LISTING.txt, was created on March 28, 2018, and is 45,867 bytes in size. FIELD OF THE INVENTION The present disclosure is broadly concerned with the field of off-the-shelf immunocellular products. More particularly, the present disclosure is concerned with methods and constructs for developing multifunctional effector cells capable of delivering therapeutically relevant properties in vivo. The cell products developed under the present disclosure address critical limitations of patient-sourced cell therapies. BACKGROUND OF THE INVENTION The field of adoptive cell therapy is currently focused on using patient- and donor- sourced cells, which makes it particularly difficult to achieve consistent manufacturing of cancer immunotherapies and to deliver therapies to all patients who may benefit. There is also the need to improve the efficacy and persistence of adoptively transferred lymphocytes to promote favorable patient outcome. Lymphocytes such as T cells and natural killer (NK) cells are potent anti-tumor effectors that play an important role in innate and adaptive immunity. However, the use of these immune cells for adoptive cell therapies remain to be challenging and have unmet needs for improvement. Therefore, there are significant opportunities remain to harness the full potential of T and NK cells, or other lymphocytes in adoptive immunotherapy. SUMMARY OF THE INVENTION There is a need for functionally improved effector cells that address issues ranging from response rate, cell exhaustion, loss of transfused cells (survival and/or persistence), tumor escape through target loss or lineage switch, tumor targeting precision, off-target toxicity, off-tumor effect, to efficacy against solid tumors, i.e., tumor microenvironment and related immune suppression, recruiting, trafficking and infiltration. It is an object of the present invention to provide methods and compositions to generate derivative non-pluripotent cells differentiated from a single cell derived iPSC (induced pluripotent stem cell) clonal line, which iPSC line comprises one or several genetic modifications in its genome. Said one or several genetic modifications include DNA insertion, deletion, and substitution, and which modifications are retained and remain functional in subsequently derived cells after differentiation, expansion, passaging and/or transplantation. The iPSC derived non-pluripotent cells of the present invention include, but not limited to, CD34 cells, hemogenic endothelium cells, HSCs (hematopoietic stem and progenitor cells), hematopoietic multipotent progenitor cells, T cell progenitors, NK cell progenitors, T cells, NKT cells, NK cells, and B cells. The iPSC derived non-pluripotent cells of the present invention comprise one or several genetic modifications in their genome through differentiation from an iPSC comprising the same genetic modifications. The engineered clonal iPSC differentiation strategy for obtaining genetically engineered derivative cells requires that the developmental potential of the iPSC in a directed differentiation is not adversely impacted by the engineered modality in the iPSC, and also that the engineered modality functions as intended in the derivative cell. Further, this strategy overcomes the present barrier in engineering primary lymphocytes, such as T cells or NK cells obtained from peripheral blood, as such cells are difficult to engineer, with engineering of such cells often lacking reproducibility and uniformity, resulting in cells exhibiting poor cell persistence with high cell death and low cell expansion. Moreover, this strategy avoids production of a heterogenous effector cell population otherwise obtained using primary cell sources which are heterogenous to start with. The present invention provides a construct for engineering a cell to obtain a genomically edited cell, the construct comprising: (i) a left homology arm and a right homology arm targeting CD38 at a selected position in CD38 locus (LHA/CD38, RHA/CD38) in the cell genome, operatively linked to (ii) one or more nucleic acid sequences encoding one or more exogenous proteins; wherein the genomically edited cell is an induced pluripotent cell (iPSC), a clonal iPSC, or an iPS cell line cell, or an NK cell; and wherein the genomically edited cell comprises a targeted integration of the one or more nucleic acid sequences encoding one or more exogenous protein at the selected position in CD38 locus, and CD38 knockout. In one embodiment of t