Cells having solid tumor targeting backbone and use thereof

Abstract

To provide functionally improved effector cells that address various issues, such as the 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, efficacy against solid tumors, i.e., tumor microenvironment and related immune suppression, recruiting, trafficking and infiltration.SOLUTION: Provided are methods and compositions for obtaining functionally enhanced derivative effector cells obtained from directed differentiation of genomically engineered iPSCs. Also provided are derivative cells having stable and functional genome editing that delivers improved or enhanced therapeutic effects. Further provided are therapeutic compositions and the use thereof comprising the functionally enhanced derivative effector cells alone, or with antibodies or checkpoint inhibitors in combination therapies.SELECTED DRAWING: None

Description

Technical Field

[0001] (Related Applications) This application claims priority to U.S. Provisional Patent Application No. 63 / 329,364, filed on April 8, 2022, and U.S. Provisional Patent Application No. 63 / 380,378, filed on October 20, 2022, the disclosures of which are hereby incorporated by reference in their entireties.

[0002] (Incorporation by Reference of Sequence Listing) The sequence listing entitled 184143-641601_SL.xml, created on March 15, 2023, and having a size of 192,945 bytes, is hereby incorporated by reference in its entirety.

[0003] (Field of the Invention) The present disclosure broadly relates to the field of off-the-shelf immune cell products. More specifically, the present disclosure relates to strategies for developing multifunctional effector cells that can provide therapeutically relevant properties in vivo. The cell products developed under the present disclosure address significant limitations of patient-derived cell therapies.

Background Art

[0004] The field of adoptive cell therapy is currently focused on using patient-derived and donor-derived cells, making it particularly difficult to achieve consistent manufacturing of cancer immunotherapies and to provide therapy to all patients who could potentially benefit therefrom. There is also a need to improve the efficacy and persistence of adoptively transferred lymphocytes to promote favorable patient outcomes. Lymphocytes such as T cells and natural killer (NK) cells are powerful anti-tumor effectors that play important roles in innate and adaptive immunity. However, using these immune cells for adoptive cell therapy remains difficult, and there is an unmet need for improvement. Therefore, there is still a great opportunity to maximize the potential of T cells, NK cells, or other immune effector cells in adoptive immunotherapy.

SUMMARY OF THE INVENTION

[0005] Functionally improved effector cells are needed to address various problems such as response rate, cell depletion, loss of infused cells (survival rate and / or persistence), tumor escape due to loss of target or lineage conversion, accuracy of tumor targeting, off-target toxicity, off-tumor effects, effectiveness against solid tumors, i.e., tumor microenvironment and related immunosuppression, mobilization, transport, and infiltration.

[0006] An object of embodiments of the present invention is to provide a method and composition for generating derivative non-pluripotent cells differentiated from an iPSC (induced pluripotent stem cell) clone strain derived from a single cell, where the iPSC strain contains one or several gene modifications in its genome. In some embodiments, one or several gene modifications include one or more of DNA insertion, deletion, and substitution, and these modifications are retained and continue to function in the subsequent derived cells after differentiation, proliferation, passage, and / or transplantation.

[0007] The non-pluripotent cells derived from iPSCs of the present application include CD34 +Cells, hematopoietic endothelial cells, HSCs (hematopoietic stem and progenitor cells), hematopoietic multipotent progenitor cells, T cell precursors, NK cell precursors, T cells, NKT cells, NK cells, and B cells are included, but not limited to these. The iPSC-derived non-pluripotent cells of the present application contain one or several genetic modifications in their genomes through differentiation from iPSCs containing the same genetic modification. In some embodiments, the engineered clone iPSC differentiation strategy for obtaining genetically engineered derivative cells benefits from the fact that the likelihood of iPSC generation in the directed differentiation is not significantly adversely affected by the engineered modality of the iPSC, and the engineered modality functions as intended in the derivative cells. Furthermore, this strategy overcomes the current barriers in manipulating primary lymphocytes such as T cells or NK cells obtained from peripheral blood, namely, such cells often result in cells that lack reproducibility and uniformity and exhibit poor cell persistence with high cell death and low cell proliferation, making it difficult to manipulate such cells. Furthermore, this strategy avoids the generation of heterogeneous effector cell populations obtained in another way using a primary cell source that is initially heterogeneous.

[0008] Accordingly, in one aspect, the present invention provides a cell or a population thereof, wherein (i) the cell is (a) an immune cell, (b) an induced pluripotent cell (iPSC), or (c) a derivative effector cell obtained by differentiating iPSC, and (ii) the cell comprises a solid tumor targeting scaffold, and the solid tumor targeting scaffold comprises two or more of (a) a polynucleotide encoding a C-X-C motif chemokine receptor or a variant thereof, (b) a polynucleotide encoding a signaling redirector receptor (TGFβ-SRR) comprising a partial or complete peptide of the extracellular domain (ECD) of a transforming growth factor beta receptor (TGFβR), and (c) a polynucleotide encoding an allo-immune defense receptor (ADR). In some embodiments, the ADR is specific for 4-1BB. In some embodiments, the cell has improved transport in solid tumors, tumor microenvironment (TME) resistance, and / or alloreactivity resistance compared to the corresponding cell that does not comprise the solid tumor targeting scaffold. In various embodiments of the cell or the population thereof, the solid tumor targeting scaffold further comprises (i) CD38 knockout, (ii) a polynucleotide encoding exogenous CD16 or a variant thereof, and (iii) a polynucleotide encoding a cytokine signaling complex comprising a partial or complete peptide of an exogenous cytokine and / or its receptor expressed on the cell surface.In some embodiments of the cell or population thereof, the cell comprises (i) a chimeric antigen receptor (CAR), (ii) a deficiency of HLA-I and / or a deficiency of HLA-II, (iii) the introduction of HLA-G or non-cleavable HLA-G, or the knockout of one or both of CD58 and CD54, (iv) the disruption of at least one of B2M, CIITA, TAP1, TAP2, tapasin, NLRC5, RFXANK, RFX5, RFXAP, TCR, NKG2A, NKG2D, CD25, CD44, CD54, CD56, CD58, CD69, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT, (v) HLA-E, 4-1BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A. 2A R, antigen-specific TCR, chimeric fusion receptor (CFR), Fc receptor, antibody or its functional variant or fragment, checkpoint inhibitor, engager, and the introduction of at least one of surface trigger receptors for coupling with agonists, (vi) at least one of the genotypes listed in Table 4, and further comprises one or more of the above.

[0009] In some embodiments of the cell or population thereof, the C-X-C motif chemokine receptor comprises CXCR2 or CXCR3. In some embodiments of the cell or population thereof, the TGFβ-SRR further comprises a partial or complete peptide of the intracellular domain (ICD) of a cytokine receptor comprising IL2R, IL12R, IL18R, IL21R, or any combination thereof. In some embodiments of the cell or population thereof, (a) the cytokine receptor is IL2Rβ, thereby forming a TGFβR2-IL2Rβ redirector receptor, and the intracellular domain (ICD) of IL2Rβ comprises the amino acid sequence represented by SEQ ID NO: 11 (NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV) or (b) the cytokine receptor is IL12Rβ, thereby forming a TGFβR2-IL12Rβ redirector receptor, and the intracellular domain (ICD) of IL12Rβ comprises the amino acid sequence represented by (i) SEQ ID NO: 12 (HYFQQKVFVLLAALRPQWCSREIPDPANSTCAKKYPIAEEKTQLPLDRLLIDWPTPEDPEPLVISEVLHQVTPVFRHPPCSNWPQREKGIQGHQASEKDMMHSASSPPPPRALQAESRQLVDLYKVLESRGSDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAPLADSLEELEPQHISLSVFPSSSLHPLTFSCGDKLTLDQLKMRCDSLML) or (ii) SEQ ID NO: 13 (SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAPLADSLEELEPQ).or (c) the cytokine receptor is IL18Rβ, thereby forming a TGFβR2-IL18Rβ redirector receptor, and the intracellular domain (ICD) of IL18Rβ comprises an amino acid sequence represented by SEQ ID NO: 14 (YRVDLVLFYRHLTRRDETLTDGKTYDAFVSYLKECRPENGEEHTFAVEILPRVLEKHFGYKLCIFERDVVPGGAVVDEIHSLIEKSRRLIIVLSKSYMSNEVRYELESGLHEALVERKIKIILIEFTPVTDFTFLPQSLKLLKSHRVLKWKADKSLSYNSRFWKNLLYLMPAKTVKPGRDEPEVLPVLSES), or (d) the cytokine receptor is IL21R, thereby forming a TGFβR2-IL21R redirector receptor, and the intracellular domain (ICD) of IL21Rβ comprises an amino acid sequence represented by SEQ ID NO: 15 (SLKTHPLWRLWKKIWAVPSPERFFMPLYKGCSGDFKKWVGAPFTGSSLELGPWSPEVPSTLEVYSCHPPRSPAKRLQLTELQEPAELVESDGVPKPSFWPTAQNSGGSAYSEERDRPYGLVSIDTVTVLDAEGPCTWPCSCEDDGYPALDLDAGLEPSPGLEDPLLDAGTTVLSCGCVSAGSPGLGGPLGSLLDRLKPPLADGEDWAGGLPWGGRSPGGVSESEAGSPLAGLDMDTFDSGFVGSDCSSPVECDFTSPGDEGPPRSYLRQWVVIPPPLSSPGPQAS), or (e) the extracellular domain (ECD) of TGFβR comprises an amino acid sequence represented by SEQ ID NO: 10 (TIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQ). In some embodiments of the cell or population thereof, the cytokine receptor is,A fragment of IL2Rβ that forms a TGFβR2-trIL12Rβ redirector receptor, comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, or 97%, 98%, or 99% sequence identity to the sequence represented by SEQ ID NO: 16 (TIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNSDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAPLADSLEELEPQ), wherein the amino acid sequence represented by SEQ ID NO: 17 (VTGISLLPPLGVAISVIIIFYCYRVN) contained in SEQ ID NO: 16 may be variable.

[0010] In various embodiments of the cell or population thereof, two or more polynucleotides of the solid tumor targeting backbone are inserted into the endogenous CD38 locus to knock out CD38. In some embodiments of the cell or population thereof, a polynucleotide encoding exogenous CD16 or a variant thereof, and two or more polynucleotides of the solid tumor targeting backbone are co-expressed within a tricistronic construct. In some embodiments of the cell or population thereof, exogenous CD16 or a variant thereof may comprise at least one of: (a) high affinity non-cleavable CD16 (hnCD16), (b) F176V and S197P in the extracellular domain of CD16, (c) a complete or partial extracellular domain derived from CD64, (d) a non-natural (or non-CD16) transmembrane domain, (e) a non-natural (or non-CD16) intracellular domain, (f) a non-natural (or non-CD16) signaling domain, (g) a non-natural stimulatory domain, and (h) a transmembrane domain, signaling domain, and stimulatory domain that are not derived from CD16 and are derived from the same or different polypeptides.

[0011] In various embodiments of the cell or population thereof, the cell further comprises a cytokine signaling complex, and the cytokine signaling complex comprises: (a) a cell surface-expressed exogenous cytokine or a partial or full-length peptide of its receptor, which contains at least one of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, or their respective receptors; or (b) (i) co-expression of IL15 and IL15Rα sandwiching a self-cleaving peptide, (ii) a fusion protein of IL15 and IL15Rα, (iii) an IL15 / IL15Rα fusion protein with a shortened intracellular domain of IL15Rα, (iv) a fusion protein of IL15 and the membrane-bound Sushi domain of IL15Rα, (v) a fusion protein of IL15 and IL15Rβ, (vi) a fusion protein of IL15 and common receptor γC, wherein the common receptor γC is natural or modified, and (vii) a homodimer of IL15Rβ, at least one of which, optionally, any one of (b)(i)-(vii) can be co-expressed with the CAR in a separate construct or in a bicistronic construct; or (c) (i) a fusion protein of IL7 and IL7Rα, (ii) a fusion protein of IL7 and common receptor γC, wherein the common receptor γC is natural or modified, and (iii) a homodimer of IL7Rβ, at least one of which, optionally, any one of (c)(i)-(iii) can be co-expressed with the CAR in a separate construct or in a bicistronic expression cassette, optionally, (d) is transiently expressed.

[0012] In various embodiments of the cell or population thereof, the cell further comprises a CAR, and the CAR is (i) specific for T cells or NK cells, (ii) a bispecific antigen-binding CAR, (iii) a switchable CAR, (iv) a dimerized CAR, (v) a split CAR, (vi) a multichain CAR, (vii) an inducible CAR, (viii) co-expressed with another CAR, (ix) co-expressed with a cytokine signaling complex within a bicistronic construct, (x) co-expressed with a checkpoint inhibitor, optionally within a separate construct or within a bicistronic construct, (xi) specific for at least one tumor-associated antigen including CD19, B7H3, BCMA, CD20, CD22, CD38, CD123, CD79b, CD52, EGFR, EGP2 / EpCAM, GD2, GPRC5D, HER2, KLK2, MICA / B, MSLN, VEGF-R2, PSMA, and PDL1, and / or (xii) 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 cytomegalovirus (CMV)-infected cells, epithelial glycoprotein-2 (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-α, ganglioside G2 (GangliosideG2, GD2), ganglioside G3 (Ganglioside G3, GD3), human epidermal growth factor receptor 2 (human Epidermal Growth Factor Receptor 2, HER2), human telomerase reverse transcriptase (human telomerase reverse transcriptase, hTERT), ICAM-1, integrin B7, interleukin-13 receptor subunit alpha-2 (Interleukin-13 receptor subunit alpha-2, IL13Rα2), κ-light chain, kinase insert domain receptor (kinase insert domain receptor, KDR), Lewis A (CA19.9), Lewis Y (Lewis Y, LeY), L1 cell adhesion molecule (L1 cell adhesion molecule, L1-CAM), LILRB2, melanoma antigen family A1 (melanoma antigen family A1, MAGE-A1), MICA / B, mucin 1 (Mucin1, Muc-1), mucin 16 (Mucin16, Muc-16), mesothelin (Mesothelin, MSLN), NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, tumor fetal antigen (h5T4), PRAME, prostate stem cell antigen (prostate stem cell antigen, PSCA), PRAME prostate-specific membrane antigen (prostate-specific membrane antigen, PSMA), tumor-associated glycoprotein 72 (tumor-associated glycoprotein72, TAG-72), TIM-3, TRBCI, TRBC2, vascular endothelial growth factor R2 (vascular endothelial growth factor R2, VEGF-R2), Wilms tumor protein (Wilms tumorspecific for at least one tumor-associated antigen comprising a protein, WT-1), and a pathogen antigen, and optionally, any one of the CARs (i) to (xii) is inserted into the TCR locus and / or driven by the endogenous promoter of the TCR and / or the TCR is knocked out by the CAR insertion. In some embodiments of the cell or population thereof, the TCR locus is the constant region of TCR alpha and / or TCR beta, and optionally, the CAR is operably linked to the endogenous promoter of the TCR.

[0013] In some embodiments of the cell or population thereof, the CAR comprises (a) an extracellular domain comprising an antigen-binding domain specific for a tumor-associated antigen, (b) a transmembrane domain, and (c) an intracellular domain comprising at least one signaling domain, wherein at least one signaling domain specifically responds to the binding of the CAR to the tumor-associated antigen, thereby generating a cancer antigen-specific response.In some embodiments of the cell or population thereof, at least one signaling domain is (a) any one of 2B4 (natural killer cell receptor 2B4), 4-1BB (tumor necrosis factor receptor superfamily member 9), CD28 (T cell-specific surface glycoprotein CD28), CD3ζ (T cell surface glycoprotein CD3 zeta chain), DAP10 (hematopoietic cell signaling substance), DAP12 (TYRO protein tyrosine kinase-binding protein), DNAM1 (CD226 antigen), FcERIγ (high-affinity immunoglobulin epsilon receptor subunit gamma), IL21R (interleukin-21 receptor), IL2Rβ / IL15Rβ (interleukin-2 receptor subunit beta), IL2Rγ (cytokine receptor common subunit gamma), IL7R (interleukin-7 receptor subunit alpha), KIR2DS2 (killer cell immunoglobulin-like receptor 2DS2), NKG2D (NKG2-D type II intrinsic membrane protein), NKp30 (natural cytotoxicity triggering receptor 3), NKp44 (natural cytotoxicity triggering receptor 2), NKp46 (natural cytotoxicity triggering receptor 1), CS1 (SLAM family member 7), and CD8 (T cell surface glycoprotein CD8 alpha chain), (b) an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the cytoplasmic domain or a portion thereof of 2B4, 41BB, CD16, CD2, CD28, CD28H, CD3ζ, DAP10, DAP12, DNAM1, FcERIγ, IL21R, IL2Rβ (IL15Rβ), IL2Rγ, IL7R, KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CD3ζ1XX, CS1, or CD8, respectively, represented by SEQ ID NOs: 54-76, and / or (c) an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the cytoplasmic domain or a portion thereof of 2B4, CD28, CD3ζ, DAP10, NKG2D, CD3ζ, CD3ζ1XX, DNAM1, CS1, or a combination thereof.In some embodiments of the cell or population thereof, the intracellular domain comprises two different signaling domains, and the intracellular domain has the following forms: any one of the fusion cytoplasmic domains of CD28-CD3ζ, CD28-CD3ζ1XX, 41BB-CD3ζ, 41BB-CD3ζ1XX, 2B4-CD3ζ, and 2B4-CD3ζ1XX or a portion thereof. In some embodiments of the cell or population thereof, the transmembrane domain has at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the transmembrane region of CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8, CD8a, CD8b, CD16, CD27, CD28, CD28H, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA4, PD1, LAG3, 2B4, BTLA, DNAM1, DAP10, DAP12, FcERIγ, IL7, IL12, IL15, KIR2DL4, KIR2DS1, KIR2DS2, NKp30, NKp44, NKp46, NKG2C, NKG2D, CS1, or a T cell receptor polypeptide or a portion thereof. In some embodiments of the cell or population thereof, the transmembrane domain comprises an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the transmembrane region of 2B4, CD2, CD16, CD28, CD28H, CD3ζ, DAP10, DAP12, DNAM1, FcERIγ, KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CS1, or CD8, represented by SEQ ID NOs: 32-53, respectively. In some embodiments of the cell or population thereof, the transmembrane domain and its directly linked signaling domain are derived from the same protein or different proteins.

[0014] In various embodiments of the cell or population thereof, the tumor-associated antigen includes HER2, and the CAR comprises: (a) an extracellular domain comprising an antigen-binding domain that recognizes the HER2 (human epidermal growth factor receptor 2) antigen, wherein the antigen-binding domain comprises: (i) a heavy chain variable (VH) domain comprising a heavy chain complementary determining region 1 (H-CDR1) comprising SEQ ID NO: 103 (NYGMS), a heavy chain complementary determining region 2 (H-CDR2) comprising SEQ ID NO: 104 (TINNNGGGTYYPDSVKG), and a heavy chain complementary determining region 3 (H-CDR3) comprising SEQ ID NO: 105 (PGLLWDA), and optionally, (ii) a light chain variable (VL) domain comprising a light chain complementary determining region 1 (L-CDR1) comprising SEQ ID NO: 106 (KSSQSLLDSDGRTYLN), a light chain complementary determining region 2 (L-CDR2) comprising SEQ ID NO: 107 (LVSKLDS), and a light chain complementary determining region 3 (L-CDR3) comprising SEQ ID NO: 108 (WQGTHFPQT); (b) a transmembrane domain; and (c) an intracellular domain comprising at least one signaling domain, wherein the at least one signaling domain specifically responds to the binding of the CAR to the HER2 antigen expressed on the cancer cell, thereby generating a cancer antigen-specific response.In some embodiments of the cell or population thereof, the antigen-binding domain of the CAR comprises (a) a VH domain having at least 80% sequence identity to SEQ ID NO: 109, (b) a VL domain having at least 80% sequence identity to SEQ ID NO: 110, (c) a single chain variable fragment (scFV) comprising VH-linker-VL or VL-linker-VH, wherein the linker varies in length and sequence, and optionally, the linker has at least 80% sequence identity to SEQ ID NOs: 111-114, (d) an scFV represented by an amino acid sequence that is at least about 99%, about 98%, about 96%, about 95%, about 90%, about 85%, or about 80% identical to SEQ ID NO: 115 or SEQ ID NO: 116, each of SEQ ID NOs: 115 and 116 comprising a linker that varies in length and sequence, and / or (e) being humanized. In some embodiments of the cell or population thereof, the extracellular domain comprises one or more of (a) a signal peptide, and / or (b) a spacer / hinge. Some embodiments have a spacer / hinge that is (a) an IgG4 spacer, CD28 spacer, CD8 spacer, CH3 spacer, CH2 / CH3 spacer, or any combination thereof, (b) a short spacer of about 10 to about 80 amino acids, a medium spacer of more than 80 to about 180 amino acids, or a long spacer of more than 180 amino acids, and / or (c) an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to any of SEQ ID NOs: 96-100. In some embodiments, the spacer / hinge comprises a medium spacer, and the spacer comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 99.

[0015] In various embodiments of the cell or population thereof, the CAR comprises an amino acid sequence that is at least about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 117. In some embodiments, at least one signaling domain of the CAR specifically responds to the binding of the CAR to the HER2 antigen expressed on the cancer cell, thereby generating a cancer antigen-specific response, and the cancer cell is a breast cancer cell, an ovarian cancer cell, an endometrial cancer cell, a lung cancer cell, an esophageal cancer cell, a salivary gland cancer cell, a bladder cancer cell, a gastric cancer cell, a colorectal cancer cell, or a head and neck cancer cell.

[0016] In various embodiments, (i) the iPSC is a cloned iPSC, a single cell dissociated iPSC, an iPSC cell line cell, or an iPSC master cell bank (MCB) cell, or (ii) the derived cell is a derived CD34 +Comprising a cell, a derived hematopoietic stem progenitor cell, a derived hematopoietic pluripotent progenitor cell, a derived T cell precursor, a derived NK cell precursor, a derived T lineage cell, a derived NKT lineage cell, a derived NK lineage cell, or a derived B lineage cell, or (iii) the derived cells comprise derived effector cells having one or more functional traits not present in the corresponding primary T, NK, NKT, and / or B cells. In some embodiments, the derived cells have therapeutic properties including one or more of (i) increased cytotoxicity, (ii) improved persistence and / or survival rate, (iii) enhanced ability to migrate, activate, and / or mobilize bystander immune cells to the tumor site, (iv) improved tumor infiltration, (v) enhanced ability to reduce tumor immunosuppression, (vi) improved ability to rescue tumor antigen escape, (vii) controlled apoptosis, (viii) enhanced or acquired ADCC, and (ix) ability to avoid fratricide, as compared to their corresponding primary cells obtained from peripheral blood, cord blood, or any other donor tissue without the same gene editing. In some embodiments of the cell or population thereof, the cell is an NK lineage cell or a T lineage cell, (i) the NK lineage cell or T lineage cell has improved infiltration and / or retention at the tumor site, (ii) the NK lineage cell can mobilize and / or migrate T cells to the tumor site, or (iii) the NK lineage cell or T lineage cell can reduce tumor immunosuppression in the presence of one or more checkpoint inhibitors.

[0017] In another aspect, the present invention provides a cell or a population thereof, wherein (i) the cell is (a) an immune cell, (b) an induced pluripotent stem cell (iPSC), or (c) a derived effector cell obtained by differentiating iPSC; (ii) the cell contains a chimeric antigen receptor (CAR), and the chimeric antigen receptor (CAR) has (a) an extracellular domain containing an antigen-binding domain that recognizes a HER2 (human epidermal growth factor receptor 2) antigen, and the antigen-binding domain includes (1) a heavy-chain variable (VH) domain containing a heavy-chain complementarity-determining region 1 (H-CDR1) including SEQ ID NO: 103 (NYGMS), a heavy-chain complementarity-determining region 2 (H-CDR2) including SEQ ID NO: 104 (TINNNGGGTYYPDSVKG), and a heavy-chain complementarity-determining region 3 (H-CDR3) including SEQ ID NO: 105 (PGLLWDA), and (2) a light-chain variable (VL) domain containing a light-chain complementarity-determining region 1 (L-CDR1) including SEQ ID NO: 106 (KSSQSLLDSDGRTYLN), a light-chain complementarity-determining region 2 (L-CDR2) including SEQ ID NO: 107 (LVSKLDS), and a light-chain complementarity-determining region 3 (L-CDR3) including SEQ ID NO: 108 (WQGTHFPQT), an extracellular domain, (b) a transmembrane domain, and (c) an intracellular domain containing at least one signaling domain, and at least one signaling domain specifically responds to the binding of the CAR to the HER2 antigen expressed on cancer cells, thereby generating a cancer antigen-specific response. In some embodiments of the cell or the population thereof, the antigen-binding domain includes (a) a VH domain having at least 80% sequence identity to SEQ ID NO: 109, (b) a VL domain having at least 80% sequence identity to SEQ ID NO: 110, (c) a single-chain variable fragment (scFV) including VH-linker-VL or VL-linker-VH, the linker has different lengths and sequences, and optionally, the linker has at least 80% sequence identity to SEQ ID NOs: 111-114, (d) an scFV represented by an amino acid sequence that is at least about 99%, about 98%, about 96%, about 95%, about 90%, about 85%, or about 80% identical to SEQ ID NO: 115 or SEQ ID NO: 116, and each of SEQ ID NOs: 115 and 116 includes a linker with different lengths and sequences.and / or (e) is humanized. In some embodiments of the cell or population thereof, at least one signaling domain is (a) any one of 2B4 (natural killer cell receptor 2B4), 4-1BB (tumor necrosis factor receptor superfamily member 9), CD16 (IgG Fc region receptor III-A), CD2 (T cell surface antigen CD2), CD28 (T cell-specific surface glycoprotein CD28), CD28H (transmembrane and immunoglobulin domain-containing protein 2), CD3ζ (T cell surface glycoprotein CD3 zeta chain), DAP10 (hematopoietic cell signaling substance), DAP12 (TYRO protein tyrosine kinase-binding protein), DNAM1 (CD226 antigen), FcERIγ (high-affinity immunoglobulin epsilon receptor subunit gamma), IL21R (interleukin-21 receptor), IL2Rβ / IL15Rβ (interleukin-2 receptor subunit beta), IL2Rγ (cytokine receptor common subunit gamma), IL7R (interleukin-7 receptor subunit alpha), KIR2DS2 (killer cell immunoglobulin-like receptor 2DS2), NKG2D (NKG2-D type II integral membrane protein), NKp30 (natural cytotoxicity triggering receptor 3), NKp44 (natural cytotoxicity triggering receptor 2), NKp46 (natural cytotoxicity triggering receptor 1), CS1 (SLAM family member 7), and CD8 (T cell surface glycoprotein CD8 alpha chain); (b) an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the cytoplasmic domain or a portion thereof of 2B4, 4-1BB, CD16, CD2, CD28, CD28H, CD3ζ, CD3ζ1XX, DAP10, DAP12, DNAM1, FcERIγ, IL21R, IL2Rβ (IL15Rβ), IL2Rγ, IL7R, KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CS1, or CD8, respectively, represented by SEQ ID NOs: 54-76; and / or (c) at least about 85%, about 90%, about 95%, about 96%, about 97%,It comprises an amino acid sequence having about 98% or about 99% identity. In some embodiments of the cell or population thereof, the intracellular domain comprises two different signaling domains, and the intracellular domain has the following forms: 2B4-CD3ζ / 1XX, 2B4-DNAM1, 2B4-FcERIγ, 2B4-DAP10, CD16-DNAM1, CD16-DAP10, CD16-DAP12, CD2-CD3ζ / 1XX, CD2-DNAM1, CD2-FcERIγ, CD2-DAP10, CD28-DNAM1, CD28-FcERIγ, CD28-DAP10, CD28-DAP12, CD28-CD3ζ / 1XX, CD28H-CD3ζ / 1XX, DAP10-CD3ζ / 1XX, DAP10-DAP12, DAP12-CD3ζ / 1XX, DAP12-DAP10, DNAM1-CD3ζ / 1XX, KIR2DS2-CD3ζ / 1XX, KIR2DS2-DAP10, KIR2DS2-2B4, or NKp46-2B4, including a fusion cytoplasmic domain or a portion thereof.

[0018] In some embodiments of the cell or population thereof, the transmembrane domain is (a) CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8, CD8a, CD8b, CD16, CD27, CD28, CD28H, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA4, PD1, LAG3, 2B4, BTLA, DNAM1, DAP10, DAP12, FcERIγ, IL7, IL12, IL15, KIR2DL4, KIR2DS1, KIR2DS2, NKp30, NKp44, NKp46, NKG2C, NKG2D, CS1, or a T cell receptor polypeptide, (b) 2B4, CD2, CD16, CD28, CD28H, CD3ζ, DAP10, DAP12, DNAM1, FcERIγ, KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CS1, or CD8, or (c) 2B4, CD28, CD28H, DAP10, DNAM1, KIR2DS2, and NKG2D, and comprises an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the transmembrane region or a portion thereof. In some embodiments of the cell or population thereof, the transmembrane domain and its directly linked signaling domain are derived from the same protein or different proteins. In some embodiments of the cell or population thereof, the extracellular domain comprises one or more of (a) a signal peptide and / or (b) a spacer / hinge. Some embodiments are such that the spacer / hinge is (a) an IgG4 spacer, CD28 spacer, CD8 spacer, CH3 spacer, CH2 / CH3 spacer, or any combination thereof, (b) a short spacer of about 10 to about 80 amino acids, a medium spacer of more than 80 to about 180 amino acids, or a long spacer of more than 180 amino acids, and / or (c) an amino acid sequence having at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to any of SEQ ID NOs: 96-100.In one embodiment, the spacer / hinge includes a moderate spacer, and the spacer includes an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 99.

[0019] In some embodiments of the cell or population thereof, the CAR comprises an amino acid sequence that is at least about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 117. In some embodiments of the cell or population thereof, the cell further comprises a solid tumor targeting scaffold comprising at least one of: (a) a polynucleotide encoding a C-X-C motif chemokine receptor or a variant thereof; (b) a polynucleotide encoding a TGFβ signal redirecting receptor (TGFβ-SRR) comprising a partial or full peptide of the extracellular domain (ECD) of a transforming growth factor beta receptor (TGFβR); and (c) a polynucleotide encoding an allogeneic immune defense receptor (ADR), and optionally, the cell has improved transport in solid tumors, tumor microenvironment (TME) resistance, and / or alloreactive resistance compared to a corresponding cell that does not comprise the solid tumor targeting scaffold. In some embodiments, the ADR is specific for 4-1BB. In some embodiments of the cell or population thereof, the solid tumor targeting scaffold further comprises: (i) a CD38 knockout; (ii) a polynucleotide encoding an exogenous CD16 or a variant thereof; and (iii) a polynucleotide encoding a cytokine signaling complex comprising a partial or full peptide of an exogenous cytokine and / or its receptor expressed on the cell surface. In some embodiments of the cell or population thereof, the cell has: (i) HLA-I deficiency and / or HLA-II deficiency; (ii) introduction of HLA-G or non-cleavable HLA-G, or knockout of one or both of CD58 and CD54; (iii) disruption of at least one of B2M, CIITA, TAP1, TAP2, tapasin, NLRC5, RFXANK, RFX5, RFXAP, TCR, NKG2A, NKG2D, CD25, CD44, CD54, CD56, CD58, CD69, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT; (iv) HLA-E, 4-1BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2AIntroduction of at least one of R, antigen-specific TCR, chimeric fusion receptor (CFR), Fc receptor, antibody or its functional variant or fragment, checkpoint inhibitor, engager, and surface trigger receptor for coupling with an agonist, (v) at least one of the genotypes listed in Table 4, further comprising one or more of the above.

[0020] In some embodiments of the cell or population thereof, the C-X-C motif chemokine receptor comprises CXCR2 or CXCR3. In some embodiments of the cell or population thereof, the TGFβ-SRR further comprises a partial or complete peptide of the intracellular domain (ICD) of a cytokine receptor comprising IL2R, IL12R, IL18R, IL21R, or any combination thereof. In some embodiments of the cell or population thereof, (a) the cytokine receptor is IL2Rβ, thereby forming a TGFβR2-IL2Rβ redirector receptor, and the intracellular domain (ICD) of IL2Rβ comprises the amino acid sequence represented by SEQ ID NO: 11 (NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV) or (b) the cytokine receptor is IL12Rβ, thereby forming a TGFβR2-IL12Rβ redirector receptor, and the intracellular domain of IL12Rβ comprises the amino acid sequence represented by SEQ ID NO: 12 (HYFQQKVFVLLAALRPQWCSREIPDPANSTCAKKYPIAEEKTQLPLDRLLIDWPTPEDPEPLVISEVLHQVTPVFRHPPCSNWPQREKGIQGHQASEKDMMHSASSPPPPRALQAESRQLVDLYKVLESRGSDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAPLADSLEELEPQHISLSVFPSSSLHPLTFSCGDKLTLDQLKMRCDSLML) or SEQ ID NO: 13 (SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAPLADSLEELEPQ) or (c) the cytokine receptor is IL18Rβ, therebyForms a TGFβR2-IL18Rβ redirector receptor, and the intracellular domain (ICD) of IL18Rβ comprises the amino acid sequence represented by SEQ ID NO: 14 (YRVDLVLFYRHLTRRDETLTDGKTYDAFVSYLKECRPENGEEHTFAVEILPRVLEKHFGYKLCIFERDVVPGGAVVDEIHSLIEKSRRLIIVLSKSYMSNEVRYELESGLHEALVERKIKIILIEFTPVTDFTFLPQSLKLLKSHRVLKWKADKSLSYNSRFWKNLLYLMPAKTVKPGRDEPEVLPVLSES), or (d) the cytokine receptor is IL21R, whereby a TGFβR2-IL21R redirector receptor is formed, and the intracellular domain (ICD) of IL21Rβ comprises the amino acid sequence represented by SEQ ID NO: 15 (SLKTHPLWRLWKKIWAVPSPERFFMPLYKGCSGDFKKWVGAPFTGSSLELGPWSPEVPSTLEVYSCHPPRSPAKRLQLTELQEPAELVESDGVPKPSFWPTAQNSGGSAYSEERDRPYGLVSIDTVTVLDAEGPCTWPCSCEDDGYPALDLDAGLEPSPGLEDPLLDAGTTVLSCGCVSAGSPGLGGPLGSLLDRLKPPLADGEDWAGGLPWGGRSPGGVSESEAGSPLAGLDMDTFDSGFVGSDCSSPVECDFTSPGDEGPPRSYLRQWVVIPPPLSSPGPQAS), or (e) the extracellular domain (ECD) of TGFβR comprises the amino acid sequence represented by SEQ ID NO: 10 (TIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQ). In some embodiments of the cell or population thereof, the cytokine receptor is a fragment of IL2Rβ, wherebyForm a TGFβR2-trIL12Rβ redirector receptor comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, or 97%, 98%, or 99% sequence identity to the sequence represented by SEQ ID NO: 16 (TIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNSDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAPLADSLEELEPQ), and the amino acid sequence represented by SEQ ID NO: 17 (VTGISLLPPLGVAISVIIIFYCYRVN) contained in SEQ ID NO: 16 may be variable.

[0021] In some embodiments of the cell or population thereof, one or more polynucleotides of the solid tumor targeting backbone are inserted into the endogenous CD38 locus to knock out CD38. In some embodiments, a polynucleotide encoding exogenous CD16 or a variant thereof, and two or more polynucleotides of the solid tumor targeting backbone are co-expressed within a tricistronic construct. In some embodiments, the exogenous CD16 or a variant thereof may comprise at least one of (a) high-affinity non-cleavable CD16 (hnCD16), (b) F176V and S197P in the extracellular domain of CD16, (c) a complete or partial extracellular domain derived from CD64, (d) a non-native (or non-CD16) transmembrane domain, (e) a non-native (or non-CD16) intracellular domain, (f) a non-native (or non-CD16) signaling domain, (g) a non-native stimulatory domain, and (h) a transmembrane domain, a signaling domain, and a stimulatory domain that are not derived from CD16 and are derived from the same or different polypeptides.In some embodiments of the cell or population thereof, the cell further comprises a cytokine signaling complex, and the cytokine signaling complex comprises: (a) a cell surface-expressed exogenous cytokine or a partial or complete peptide of its receptor, which comprises at least one of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, or their respective receptors, or (b) (i) co-expression of IL15 and IL15Rα sandwiching a self-cleaving peptide, (ii) a fusion protein of IL15 and IL15Rα, (iii) an IL15 / IL15Rα fusion protein with a shortened intracellular domain of IL15Rα, (iv) a fusion protein of IL15 and the membrane-bound Sushi domain of IL15Rα, (v) a fusion protein of IL15 and IL15Rβ, (vi) a fusion protein of IL15 and common receptor γC, wherein the common receptor γC is natural or modified, and (vii) a homodimer of IL15Rβ, wherein at least one of (b)(i)-(vii) can be co-expressed with the CAR in a separate construct or in a bicistronic construct, or (c) (i) a fusion protein of IL7 and IL7Rα, (ii) a fusion protein of IL7 and common receptor γC, wherein the common receptor γC is natural or modified, and (iii) a homodimer of IL7Rβ, wherein at least one of (c)(i)-(iii) is optionally co-expressed with the CAR in a separate construct or in a bicistronic expression cassette, and optionally, (d) is transiently expressed. In some embodiments, (i) the CAR is co-expressed with the cytokine signaling complex in a bicistronic construct and / or (ii) the CAR is inserted into the TCR locus and optionally operably linked to the endogenous promoter of the TCR. In some embodiments, (i) the TCR locus is the constant region of TCR alpha and / or TCR beta and / or (ii) the TCR is knocked out by the CAR insertion.

[0022] In some embodiments, at least one signaling domain of the CAR specifically responds to the binding of the CAR to the HER2 antigen expressed on the cancer cell, thereby generating a cancer antigen-specific response, and the cancer cell is a breast cancer cell, an ovarian cancer cell, an endometrial cancer cell, a lung cancer cell, an esophageal cancer cell, a salivary gland cancer cell, a bladder cancer cell, a gastric cancer cell, a colorectal cancer cell, or a head and neck cancer cell.

[0023] In various embodiments, (i) the iPSC is a cloned iPSC, a single-cell dissociated iPSC, an iPSC cell line cell, or an iPSC master cell bank (MCB) cell, or (ii) the derived cell comprises a derived CD34 + cell, a derived hematopoietic stem progenitor cell, a derived hematopoietic pluripotent progenitor cell, a derived T cell precursor, a derived NK cell precursor, a derived T lineage cell, a derived NKT lineage cell, a derived NK lineage cell, or a derived B lineage cell, or (iii) the derived cell comprises a derived effector cell having one or more functional traits not present in the corresponding primary T, NK, NKT, and / or B cells. In some embodiments, the derived cell has therapeutic properties including one or more of (i) increased cytotoxicity, (ii) improved persistence and / or survival rate, (iii) enhanced ability to migrate, activate, or mobilize bystander immune cells to the tumor site, (iv) improved tumor infiltration, (v) enhanced ability to reduce tumor immunosuppression, (vi) improved ability to rescue tumor antigen escape, (vii) controlled apoptosis, (viii) enhanced or acquired ADCC, and (ix) ability to avoid fratricide, compared to its corresponding primary cell obtained from peripheral blood, cord blood, or any other donor tissue that does not have the same gene editing. In some embodiments of the cell or population thereof, the cell is an NK lineage cell or a T lineage cell, and (i) the NK lineage cell or T lineage cell has improved infiltration and / or retention at the tumor site, (ii) the NK lineage cell can mobilize and / or migrate T cells to the tumor site, or (iii) the NK lineage cell or T lineage cell can reduce tumor immunosuppression in the presence of one or more checkpoint inhibitors.

[0024] In another aspect, the present invention provides a composition comprising the cells or a population thereof provided herein. In various embodiments of the composition, the composition further comprises one or more therapeutic agents. In some embodiments of the composition, the one or more therapeutic agents include a peptide, a cytokine, a checkpoint inhibitor, a mitogen, a growth factor, a small molecule RNA, dsRNA (double-stranded RNA), a mononuclear cell, a feeder cell, a feeder cell component or a supplement factor 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). In some embodiments of the composition where the therapeutic agent is a checkpoint inhibitor, the checkpoint inhibitor is (a) PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A 2AOne or more antagonist checkpoint molecules including R, BATE, BTLA, CD39, CD47, CD73, CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxp1, 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, lirilumab, monalizumab, nivolumab, pembrolizumab, and derivatives or functional equivalents thereof; or (c) at least one of atezolizumab, nivolumab, and pembrolizumab. In some embodiments of the composition wherein the therapeutic agent is an antibody, the antibody includes (a) an anti-CD20 antibody, an anti-HER2 antibody, an anti-CD52 antibody, an anti-EGFR antibody, an anti-CD123 antibody, an anti-GD2 antibody, an anti-PDL1 antibody, or an anti-CD38 antibody; or (b) rituximab, bertuzumab, ofatumumab, ublituximab, ocaratuzumab, obinutuzumab, trastuzumab, pertuzumab, alemtuzumab, cetuximab, dinutuximab, avelumab, daclizumab, basiliximab, M-A251, 2A3, BC69, 24204, 22722, 24212, MAB23591, FN50, 298614, AF2359, CY1G4, DF1513, vibatuzumab, RG7356, G44-26, 7G3, CSL362, elotuzumab, daratumumab, isatuximab, MOR202, and humanized or Fc-modified variants or fragments thereof and functional equivalents and biosimilars thereof, one or more of them.In embodiments of the composition in which the therapeutic agent is an engager, the engager may comprise (i) a bi-specific T cell engager (BiTE), (ii) a bispecific killer cell engager (BiKE), or (iii) a tri-specific killer cell engager (TriKE), or the engager comprises (a) a first binding domain that recognizes the extracellular portion of CD3, CD28, CD5, CD16, CD64, CD32, CD33, CD89, NKG2C, NKG2D, or any functional variant thereof of a cell or bystander immune effector cell, and (b) a second binding domain specific for an antigen comprising any one of B7H3, CD10, CD19, CD20, CD22, CD24, CD30, CD33, CD34, CD38, CD44, CD52, CD79a, CD79b, CD123, CD138, CD179b, CEA, CLEC12A, CS-1, DLL3, EGFR, EGFRvIII, EpCAM, FLT-3, FOLR1, FOLR3, GD2, gpA33, HER2, HM1.24, LGR5, MSLN, MCSP, MICA / B, Muc1, Muc16, PDL1, PSMA, PAMA, P-cadherin, ROR1, or VEGF-R2.

[0025] In another aspect, the present invention is a therapeutic use of the composition provided herein by introducing the composition into a subject in need of adoptive cell therapy, wherein the subject has an autoimmune disorder, a hematological malignancy, a solid tumor, cancer, or a viral infection. In another aspect, the present invention provides a master cell bank (MCB) comprising the clonal iPSCs provided herein.

[0026] In another aspect, the present invention provides a method for producing the derived cells provided herein, wherein the derived cells are immune effector cells, and the method comprises: (i) obtaining genetically engineered iPSCs, wherein the iPSCs comprise a solid tumor targeting backbone comprising two or more of: (a) a polynucleotide encoding a C-X-C motif chemokine receptor or a variant thereof; (b) a polynucleotide encoding a TGFβ signal redirecting receptor (TGFβ-SRR) comprising a partial or full peptide of the extracellular domain (ECD) of a transforming growth factor beta receptor (TGFβR); and (c) a polynucleotide encoding an allogeneic immune defense receptor (ADR); (ii) differentiating the genetically engineered iPSCs into derived CD34 + cells; and (iii) differentiating the derived CD34 + cells into immune effector cells, wherein the immune effector cells retain the solid tumor targeting backbone. In some embodiments, the ADR is specific for 4-1BB. In some embodiments of the production method, obtaining genetically engineered iPSCs comprising a solid tumor targeting backbone comprises: (a) integrating two or more polynucleotides for co-expression at the endogenous CD38 locus and knocking out CD38, wherein the two or more polynucleotides for co-expression are within a cistronic construct and the polynucleotides encode at least two of: (i) a C-X-C motif chemokine receptor; (ii) a TGFβ-SSR; and (iii) an allogeneic immune defense receptor (ADR). In some embodiments of the production method, (i) the cistronic construct further comprises a polynucleotide encoding an exogenous CD16 or a variant thereof, (ii) the C-X-C motif chemokine receptor comprises CXCR2 or CXCR3, (iii) the TGFβ-SRR comprises a TGFβR2-IL2Rβ, TGFβR2-IL12Rβ, TGFβR2-IL18Rβ, or TGFβR2-trIL12Rβ redirecting receptor, or (iv) the ADR is specific for 4-1BB or CD38.

[0027] In various embodiments of the manufacturing method, the method further comprises genetically engineering iPSCs comprising a solid tumor targeting scaffold by integrating a polynucleotide encoding a chimeric antigen receptor (CAR) into the TCR locus, optionally, (i) the CAR is operably linked to the endogenous promoter of the TCR and / or (ii) the TCR is knocked out by the CAR insertion. In some embodiments, the CAR is co-expressed with a cytokine signaling complex within a bicistronic construct, or the TCR locus is the constant region of TCR alpha and / or TCR beta. In some embodiments, the cytokine signaling complex comprises at least one of (i) a fusion protein of IL7 and IL7Rα, (ii) a fusion protein of IL15 and IL15Rα, and (iii) an IL15 / IL15Rα fusion protein with a shortened intracellular domain of IL15Rα.

[0028] In some embodiments of the manufacturing method, the CAR is (i) specific to a tumor-associated antigen, (ii) specific to a solid tumor-associated antigen, (iii) specific to a pan-tumor antigen, or (iv) specific to one of B7H3, BCMA, CD19, CD38, CD79b, EGP2 / EpCAM, GPRC5D, HER2, KLK2, MICA / B, and MR1. In some embodiments, a CAR specific to the HER2 antigen expressed on cancer cells comprises a heavy chain variable (VH) domain comprising (i) a heavy chain complementarity determining region 1 (H-CDR1) comprising SEQ ID NO: 103 (NYGMS), a heavy chain complementarity determining region 2 (H-CDR2) comprising SEQ ID NO: 104 (TINNNGGGTYYPDSVKG), and a heavy chain complementarity determining region 3 (H-CDR3) comprising SEQ ID NO: 105 (PGLLWDA), and optionally (ii) a light chain variable (VL) domain comprising a light chain complementarity determining region 1 (L-CDR1) comprising SEQ ID NO: 106 (KSSQSLLDSDGRTYLN), a light chain complementarity determining region 2 (L-CDR2) comprising SEQ ID NO: 107 (LVSKLDS), and a light chain complementarity determining region 3 (L-CDR3) comprising SEQ ID NO: 108 (WQGTHFPQT), the antigen-binding domain. In some embodiments of the manufacturing method, the antigen-binding domain comprises (a) a VH domain having at least 80% sequence identity to SEQ ID NO: 109, (b) a VL domain having at least 80% sequence identity to SEQ ID NO: 110, (c) a single-chain variable fragment (scFV) comprising VH-linker-VL or VL-linker-VH, the linker differing in length and sequence, and optionally, the linker having at least 80% sequence identity to SEQ ID NOs: 111-114, (d) an scFV represented by an amino acid sequence that is at least about 99%, about 98%, about 96%, about 95%, about 90%, about 85%, or about 80% identical to SEQ ID NO: 115 or SEQ ID NO: 116, each of SEQ ID NOs: 115 and 116 comprising a linker differing in length and sequence, and / or (e) being humanized.

[0029] In some embodiments of the manufacturing method, the method comprises: (a) introduction of HLA-I deficiency and / or HLA-II deficiency; (b) deletion or disruption of one or more of B2M, CIITA, TAP1, TAP2, Tapasin, NLRC5, RFXANK, RFX5, RFXAP, TCR, NKG2A, NKG2D, CD25, CD44, CD54, CD56, CD58, CD69, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT; and / or (c) introduction of at least one of HLA-G, HLA-E, 4-1BBL, CD3, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A

[0030] further comprises genetically engineering iPSCs comprising a tumor solid targeting scaffold by one or more of: introduction of at least one of R, antigen-specific TCR, chimeric fusion receptor (CFR), Fc receptor, antibody or a functional variant or fragment thereof, checkpoint inhibitor, engager, and surface trigger receptor for coupling with an agonist. In some embodiments, the genomic manipulation comprises targeted editing. In some embodiments, the targeted editing is performed by CRISPR, ZFN, TALEN, homing nuclease, homologous recombination, or any other functional variation of these methods.In another aspect, the present invention provides a method of treating a subject in need of adoptive cell therapy, the method comprising injecting effector cells into the subject, wherein the effector cells comprise the derived cells or a population thereof provided herein. In some embodiments of the treatment method, the effector cells comprise a chimeric antigen receptor (CAR) specific for the HER2 antigen expressed on cancer cells, and the CAR comprises: (i) a heavy-chain variable (VH) domain comprising a heavy-chain complementarity-determining region 1 (H-CDR1) comprising SEQ ID NO: 103 (NYGMS), a heavy-chain complementarity-determining region 2 (H-CDR2) comprising SEQ ID NO: 104 (TINNNGGGTYYPDSVKG), and a heavy-chain complementarity-determining region 3 (H-CDR3) comprising SEQ ID NO: 105 (PGLLWDA); and optionally, (ii) a light-chain variable (VL) domain comprising a light-chain complementarity-determining region 1 (L-CDR1) comprising SEQ ID NO: 106 (KSSQSLLDSDGRTYLN), a light-chain complementarity-determining region 2 (L-CDR2) comprising SEQ ID NO: 107 (LVSKLDS), and a light-chain complementarity-determining region 3 (L-CDR3) comprising SEQ ID NO: 108 (WQGTHFPQT). The CAR is at the TRAC locus and CAR expression is driven by an endogenous TCR promoter. The subject in need of adoptive cell therapy has breast cancer, ovarian cancer, endometrial cancer, lung cancer, esophageal cancer, salivary gland cancer, bladder cancer, gastric cancer, colorectal cancer, or head and neck cancer.

[0031] In some embodiments of the treatment method, the effector cells further comprise a solid tumor targeting scaffold, and the effector cells comprise (i) at the CD38 locus, two or more of (a) a polynucleotide encoding CXCR2, (b) a polynucleotide encoding a TGFβR2-IL18Rβ redirector receptor or a TGFβR2-trIL12Rβ redirector receptor, and (c) a polynucleotide encoding an allogeneic immune defense receptor (ADR), (ii) at the CD38 locus, a polynucleotide encoding exogenous CD16 or a variant thereof, (iii) at the TRAC locus, a polynucleotide encoding a fusion protein of IL7 and IL7Rα, and (iv) CD38 knockout and TCR knockout. In some embodiments, the ADR is specific for 4-1BB. In some embodiments of the treatment method, the method further comprises administering to the subject one or more therapeutic agents, and the one or more therapeutic agents are (i) a cytokine, antibody, engager, checkpoint inhibitor, chemotherapeutic agent or radioactive moiety, or immunomodulatory drug (IMiD), (ii) an anti-CD38 antibody comprising daratumumab, isatuximab, or MOR202, (iii) an engager comprising a BiTE (bispecific T cell engager) or TriKE (trispecific killer cell engager), (iv) a checkpoint inhibitor comprising atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lirimumab, monalizumab, nivolumab, or pembrolizumab, and / or (v) a chemotherapeutic agent comprising cyclophosphamide and fludarabine (Cy / Flu). In some embodiments of the treatment method, the effector cells comprise CD38 knockout and TCR knockout and optionally an ADR specific for 4-1BB, the method comprises administering an anti-CD38 antibody to the subject, and the method requires minimal or no lymphodepletion comprising administering Cy / Flu to the subject. In some embodiments of the treatment method, the effector cells are allogeneic, and injecting the effector cells into the subject is performed in an out-patient setting.

[0032] In another aspect, the present invention provides a method of improving adoptive cell therapy in the treatment of a subject having a solid tumor, the method comprising administering a population of derived cells provided herein, optionally, wherein the derived cells have improved transport, tumor microenvironment (TME) resistance, and / or alloreactive resistance in the solid tumor compared to the corresponding cells that do not contain the solid tumor targeting backbone.

[0033] In another aspect, the present invention provides a method of improving anti-HER2 monoclonal antibody (mAb) therapy, the method comprising introducing into a subject in need thereof a composition comprising effector cells comprising a polynucleotide encoding CasMab250-CAR, a polynucleotide encoding CXCR2, a polynucleotide encoding TGFβ-SRR, and a polynucleotide encoding exogenous CD16 or a variant thereof, and introducing an anti-HER2 mAb into the subject. In various embodiments, the anti-HER2 mAb is trastuzumab (Herceptin™).

[0034] In one aspect, the present invention provides a method for selecting NK cells comprising a transgene of interest. Some embodiments include: (i) obtaining engineered NK cells comprising a construct that co-expresses at least one of: a transgene of interest and a cell surface-expressed exogenous cytokine or a partial or full peptide of its receptor, wherein the exogenous cytokine or its receptor comprises IL15 or at least one of its respective receptors; or any of the following (1)-(7): (1) co-expression of IL15 and IL15Rα sandwiching a self-cleaving peptide; (2) a fusion protein of IL15 and IL15Rα; (3) an IL15 / IL15Rα fusion protein with a truncated intracellular domain of IL15Rα; (4) a fusion protein of IL15 and the membrane-bound Sushi domain of IL15Rα; (5) a fusion protein of IL15 and IL15Rβ; (6) a fusion protein of IL15 and the common receptor γC, wherein the common receptor γC is either native or modified; and (7) a homodimer of IL15Rβ; (ii) culturing the cells without supplying exogenous IL15 cytokine to the engineered NK cells; and (iii) collecting NK cells that proliferate without exogenous IL15 cytokine, thereby selecting engineered NK cells comprising the transgene of interest.

[0035] Various objects and advantages of the compositions and methods provided herein will become apparent from the following description when taken in conjunction with the accompanying drawings, which illustrate specific embodiments of the invention by way of example and illustration.

Brief Description of the Drawings

[0036]

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[0037] Genome modification of iPSCs (induced pluripotent stem cells) can include one or more of insertion, deletion, or substitution of polynucleotides. Exogenous gene expression in genome-engineered iPSCs often encounters problems such as gene silencing or decreased gene expression after long-term clonal expansion of the original genome-engineered iPSCs, after cell differentiation, and in dedifferentiated cell types derived from genome-engineered iPSCs. On the other hand, directly manipulating primary immune cells such as T cells or NK cells is difficult and poses an obstacle to the preparation and delivery of engineered immune cells for adoptive cell therapy. In various embodiments, the present invention provides an efficient and reliable targeted approach for stably integrating one or more exogenous genes including suicide genes and other functional modalities, which confers improved therapeutic properties related to engraftment, trafficking, homing, migration, cytotoxicity, viability, maintenance, proliferation, lifespan, self-renewal, persistence, and / or survival rate to iPSC-derived cells including, but not limited to, hematopoietic stem and progenitor cells (HSCs), T cell progenitor cells, NK cell progenitor cells, T lineage cells, NKT lineage cells, and NK lineage cells.

[0038] Definitions Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by those skilled in the art. Further, unless the context requires otherwise, singular terms shall include the plural, and plural terms shall include the singular.

[0039] It is to be understood that the present invention is not limited to the specific methodologies, protocols, and reagents, etc. described herein and may, therefore, vary. The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention, which is defined only by the claims.

[0040] As used herein, the articles “a,” “an,” and “the” are used herein to refer to one or more than one (i.e., at least one) of the grammatical objects of the article. By way of example, “an element” means one element or more than one element.

[0041] The use of the alternative (e.g., “or”) should be understood to mean either one of the alternatives, both, or any combination thereof.

[0042] The term “and / or” should be understood to mean either one or both of the alternatives.

[0043] As used herein, the terms "about" or "approximately" refer to an amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length that varies by an amount of 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% as compared to the referenced amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length. In one embodiment, the terms "about" or "approximately" refer to a range of amounts, levels, values, numbers, frequencies, percentages, dimensions, sizes, quantities, weights, or lengths that are ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the referenced amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length.

[0044] As used herein, the terms "substantially" or "essentially" refer to an amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length that is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more as compared to the referenced amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length. In one embodiment, the terms "substantially the same" or "essentially the same" refer to a range of amounts, levels, values, numbers, frequencies, percentages, dimensions, sizes, quantities, weights, or lengths that are substantially identical to the referenced amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length.

[0045] As used herein, the terms "substantially free of" and "essentially free of" are used interchangeably and, when used to describe a composition such as a cell population or a culture medium, refer to a composition that is, for example, free of 95%, 96%, 97%, 98%, 99% or more of a particular substance or source thereof, or undetectable by conventional means. The terms "free of" or "essentially free of" a particular component or substance in a composition also mean that such component or substance is (1) not present in the composition at any concentration, or (2) present in the composition at a low concentration that is functionally inert. A similar meaning may be applied to the term "absent", which refers to the absence of a particular substance or source thereof in a composition.

[0046] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" are to be interpreted inclusively as indicating the stated steps, or elements, or groups of steps or elements but not as precluding any other steps, or elements, or groups of steps or elements. In certain embodiments, the terms "include", "have", "contain" and "comprise" are used synonymously.

[0047] "Consisting of" means including, and limited to, all those things following the phrase "consisting of". Thus, the phrase "consisting of" indicates that the listed elements are necessary or essential and that no other elements may be present.

[0048] The term "consisting essentially of" means including any elements recited after the phrase, and being limited to other elements that do not interfere with or contribute to the activities or operations specified in the disclosure of the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are necessary or essential, but that other elements are optional and may or may not be present depending on whether they affect the activities or operations of the recited elements.

[0049] Throughout this specification, references to "one embodiment", "an embodiment", "a particular embodiment", "related embodiments", "specific embodiments", "additional embodiments", or "further embodiments", or combinations thereof, mean that the particular features, structures, or characteristics described in connection with the embodiment are included in at least one embodiment of the invention. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Further, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0050] The term "ex vivo" generally refers to activities performed outside of a living body, such as experiments or measurements performed within or on biological tissue in an artificial environment outside of the body, preferably with minimal variation of natural conditions. In certain embodiments, an "ex vivo" procedure includes living cells or tissues that are removed from a living body and cultured in an experimental apparatus, typically under aseptic conditions, for a period of several hours or up to about 24 hours, but in some cases up to 48 hours or more depending on the circumstances. In certain embodiments, such tissues or cells may be collected and frozen and later thawed for ex vivo processing. Tissue culture experiments or procedures that last longer than a few days using living cells or tissues are typically considered to be "in vitro", but in certain embodiments, the term may be used interchangeably with ex vivo.

[0051] The term "in vivo" generally refers to activities occurring within a living body.

[0052] As used herein, the terms "reprogramming," "dedifferentiation," "increase in cellular potency," or "increase in developmental potential" refer to methods of increasing the ability of a cell or dedifferentiating a cell to a less differentiated state. For example, a cell with increased cellular potency has more developmental plasticity (i.e., can differentiate into more cell types) compared to the same cell in an un-reprogrammed state. In other words, a reprogrammed cell is a cell in a less differentiated state than the same cell in an un-reprogrammed state.

[0053] As used herein, the term "differentiation" is the process by which unspecialized ("uncommitted") or less specialized cells acquire the characteristics of specialized cells, such as blood cells or muscle cells. Differentiated or induced-differentiated cells are cells that assume a more specialized ("committed") position within a cell lineage. The term "committed," when applied to the process of differentiation, refers to cells that have progressed along a differentiation pathway to a position where, under normal circumstances, they will continue to differentiate into a specific cell type or subset of cell types and, under normal circumstances, cannot differentiate into different cell types or revert to a less differentiated cell type. As used herein, the term "pluripotency" refers to the ability of a cell (i.e., the embryo itself) to form all cell lineages of a living organism or somatic cells. For example, embryonic stem cells are a type of pluripotent stem cell that can form cells from each of the three germ layers, the ectoderm, mesoderm, and endoderm. Pluripotency is a range of developmental potential that extends from less primitive and less pluripotent cells (e.g., epiblast stem cells or EpiSCs) that cannot give rise to a complete organism to more primitive and more pluripotent cells (e.g., embryonic stem cells) that can give rise to a complete organism.

[0054] As used herein, the term "induced pluripotent stem cell" or "iPSC" refers to stem cells that are produced in vitro from induced or modified differentiated adult, neonatal, or fetal cells, i.e., reprogrammed to cells that can differentiate into all tissues of the three germ layers or dermal layer: mesoderm, endoderm, and ectoderm. In some embodiments, the reprogramming process uses reprogramming factors and / or small molecule chemical driving methods. The iPSCs produced do not refer to naturally occurring cells.

[0055] As used herein, the term "embryonic stem cell" refers to naturally occurring pluripotent stem cells of the inner cell mass of the blastocyst. Embryonic stem cells are pluripotent and give rise to all derivatives of the three primary germ layers, i.e., ectoderm, endoderm, and mesoderm, during development. They do not contribute to the extraembryonic membranes or placenta (i.e., are not totipotent).

[0056] As used herein, the term "pluripotent stem cell" refers to a cell having the developmental potential to differentiate into cells of one or more germ layers (i.e., ectoderm, mesoderm, and endoderm), but not all three. Thus, pluripotent cells can also be referred to as "partially differentiated cells". Pluripotent cells are known in the art and examples of pluripotent cells include adult stem cells such as, for example, hematopoietic stem cells and neural stem cells. "Pluripotency" indicates that a cell can form many types of cells in a given lineage, but not cells of other lineages. For example, pluripotent hematopoietic cells can form many different types of blood cells (red, white, platelets, etc.), but cannot form neurons. Thus, the term "multipotency" refers to the state of a cell having a lower degree of developmental potential than totipotency and pluripotency.

[0057] Pluripotency can be determined in part by evaluating the pluripotency characteristics of cells. The characteristics of pluripotency include, but are not limited to, (i) pluripotent stem cell morphology, (ii) the potential for unlimited self-renewal, (iii) the expression of pluripotent stem cell markers including, but not limited to, SSEA1 (mouse only), SSEA3 / 4, SSEA5, TRA1-60 / 81, TRA1-85, TRA2-54, GCTM-2, TG343, TG30, CD9, CD29, CD133 / prominin, CD140a, CD56, CD73, CD90, CD105, OCT4, NANOG, SOX2, CD30 and / or CD50, (iv) the ability to differentiate into all three somatic lineages (ectoderm, mesoderm, and endoderm), (v) teratoma formation consisting of the three somatic lineages, and (vi) the formation of embryoid bodies consisting of cells from the three somatic lineages, but not limited to these.

[0058] Two types of pluripotency have been described to date, a "primed" or "quasi-stable" state of pluripotency similar to the epiblast stem cells (EpiSC) of the late blastocyst, and a "naïve" or "ground" state similar to the inner cell mass of the early / pluripotent pre-implantation blastocyst. While both pluripotent states exhibit the characteristics as described above, the naïve or ground state further exhibits (i) pre-inactivation or reactivation of the X chromosome in female cells, (ii) improved clonality and survival rate in single cell culture, (iii) an overall decrease in DNA methylation, (iv) a reduction in the deposition of the H3K27me3 repressive chromatin mark on the promoters of developmental regulatory genes, and (v) a decrease in the expression of differentiation markers compared to primed state pluripotent cells. The standard methodology of cell reprogramming, in which exogenous pluripotency genes are introduced into somatic cells, expressed, and then either silenced or removed from the resulting pluripotent cells, generally appears to have the characteristics of the primed state of pluripotency. Under standard pluripotent cell culture conditions, such cells remain in the primed state and the characteristics of the ground state are not observed unless the expression of the exogenously introduced genes is maintained.

[0059] As used herein, the term "pluripotent stem cell morphology" refers to the classical morphological traits of embryonic stem cells. The morphology of normal embryonic stem cells is characterized by a high nucleus-to-cytoplasm ratio, prominent nucleoli, and a typical intercellular spacing, and is round and small in shape.

[0060] As used herein, the term "subject" refers to any animal, preferably a human patient, a domestic animal, or other farm animal.

[0061] "Pluripotency factor" or "reprogramming factor" refers to an agent that can increase the developmental potential of a cell, either alone or in combination with other agents. Pluripotency factors include, but are not limited to, polynucleotides, polypeptides, and small molecules that can increase the developmental potential of a cell. Exemplary pluripotency factors include, for example, transcription factors and small molecule reprogramming agents.

[0062] "Culture" or "cell culture" refers to the maintenance, growth, and / or differentiation of cells in an in vitro environment. "Cell culture medium", "culture medium" (singular in each case is "medium"), "supplemental component", and "medium supplement component" refer to the nutrient compositions for culturing cell cultures.

[0063] "Culturing" or "maintaining" refers to, for example, maintaining (sustaining), propagating (growing), and / or differentiating tissues or cells outside the body in a sterile plastic (or coated plastic) cell culture dish or flask. "Culturing" or "maintaining" can utilize a medium as a source of nutrients, hormones, and / or other factors useful for the growth and / or maintenance of cells.

[0064] As used herein, the term "mesoderm" refers to one of the three germ layers that appears during early embryonic development and gives rise to various specialized cell types including blood cells of the circulatory system, muscle, heart, dermis, skeleton, and other supportive and connective tissues.

[0065] As used herein, the terms "definitive hemogenic endothelium" (HE) or "pluripotent stem cell-derived definitive hemogenic endothelium" (iHE) refer to a subset of endothelial cells that give rise to hematopoietic stem and progenitor cells in a process called endothelial-to-hematopoietic transition. Hematopoietic cell development in the embryo proceeds sequentially from the lateral plate mesoderm through angioblasts to definitive hemogenic endothelial cells and hematopoietic precursors.

[0066] The terms "hematopoietic stem and progenitor cells", "hematopoietic stem cells", "hematopoietic progenitor cells", or "hematopoietic progenitor cells" refer to cells that are committed to the hematopoietic lineage but are capable of further hematopoietic differentiation and include pluripotent hematopoietic stem cells (blood cells), myeloid precursors, megakaryocyte precursors, erythroid precursors, and lymphocyte precursors. Hematopoietic stem and progenitor cells (progenitor cell, HSC) are pluripotent stem cells that give rise to all blood cell types including the myeloid lineage (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells), and the lymphoid lineage (T cells, B cells, NK cells). As used herein, the term "secondary hematopoietic stem cells" refers to CD34 + hematopoietic cells that can give rise to both mature myeloid and lymphoid cell types including T lineage cells, NK lineage cells, and B lineage cells. Hematopoietic cells also include various subsets of primitive hematopoietic cells that give rise to primitive erythrocytes, megakaryocytes, and macrophages.

[0067] As used herein, the terms "T lymphocyte" and "T cell" are used interchangeably and refer to a major type of white blood cell that has completed maturation in the thymus and has various roles in the immune system including the identification of specific foreign antigens in the body and the activation and inactivation of other immune cells in an MHC class I-restricted manner. T cells can be any T cell, e.g., cultured T cells, e.g., primary T cells, or T cell lines, e.g., Jurkat, SupT1, etc., or T cells obtained from a mammal. T cells are CD3 +It can be a T cell. T cells are CD4 + / CD8 + double-positive T cells, CD4 + helper T cells (e.g., Th1 and Th2 cells), CD8 + T cells (e.g., cytotoxic T cells), peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor infiltrating lymphocytes (TILs), memory T cells, naive T cells, regulatory T cells, gamma delta T cells (γδT cells), etc., including but not limited to, can be any type of T cell and can be at any stage of development. Additional types of helper T cells include cells such as Th3 (Treg), Th17, Th9, or Tfh cells. Additional types of memory T cells include cells such as central memory T cells (Tcm cells), effector memory T cells (Tem cells and TEMRA cells). The term "T cell" can also refer to genetically engineered T cells such as T cells modified to express a T cell receptor (TCR) or a chimeric antigen receptor (CAR). T cells or T cell-like effector cells can also be differentiated from stem cells or progenitor cells ("derived T cells" or "derived T cell-like effector cells", or collectively "derived T lineage cells"). Derived T cell-like effector cells can have a T cell lineage in some respects, but at the same time have one or more functional characteristics not present in primary T cells. In this application, T cells, T cell-like effector cells, derived T cells, derived T cell-like effector cells, or derived T lineage cells are collectively referred to as "T lineage cells".

[0068] "CD4 +"CD4 cells" refer to a subset of T cells that express CD4 on their surface and are associated with the cellular immune response. They are characterized by their secretion profile after stimulation, which may include the secretion of cytokines such as IFN-gamma, TNF-alpha, IL2, IL4, and IL10. The "CD4" molecule is a 55kD glycoprotein originally defined as a differentiation antigen of T lymphocytes, but is also found on other cells including monocytes / macrophages. The CD4 antigen is a member of the immunoglobulin supergene family and is involved as an associated recognition element in MHC (major histocompatibility complex) class II-restricted immune responses. In T lymphocytes, it defines the helper / inducer subset.

[0069] "CD8 + cells" refer to a subset of T cells that express CD8 on their surface, are MHC class I-restricted, and function as cytotoxic T cells. The "CD8" molecule is a differentiation antigen found on thymocytes and cytotoxic and suppressor T lymphocytes. The CD8 antigen is a member of the immunoglobulin supergene family and is an associated recognition element in major histocompatibility complex class I-restricted interactions.

[0070] As used herein, the terms "NK cell" or "natural killer cell" refer to a subset of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the absence of the T cell receptor (CD3). NK cells can be any NK cell, such as a cultured NK cell (e.g., a primary NK cell), or an NK cell derived from a cultured or expanded NK cell, or a cell line NK cell (e.g., NK-92), or an NK cell obtained from a mammal having a healthy or diseased state. As used herein, the terms "adaptive NK cell" and "memory NK cell" are interchangeable and phenotypically are CD3 - and CD56 + and express at least one of NKG2C and CD57, and optionally CD16, but lack the expression of one or more of PLZF, SYK, FceRγ, and EAT-2, and refer to a subset of NK cells. In some embodiments, CD56 +Isolated subpopulations of NK cells include the expression of CD16, NKG2C, CD57, NKG2D, NCR ligands, NKp30, NKp40, NKp46, activating and inhibitory KIR, NKG2A, and / or DNAM-1. CD56 + can be weakly positive (dim) or strongly positive (bright) expression. NK cells or NK cell-like effector cells can be differentiated from stem cells or progenitor cells (“derived NK cells” or “derived NK cell-like effector cells”, or collectively “derived NK lineage cells”). Derived NK cell-like effector cells may have the NK cell lineage in some respects, but at the same time have one or more functional traits that are not present in primary NK cells. In this application, NK cells, NK cell-like effector cells, derived NK cells, derived NK cell-like effector cells, or derived NK lineage cells are collectively referred to as “NK lineage cells”.

[0071] As used herein, the terms “NKT cells” or “natural killer T cells” or “NKT lineage cells” refer to CD1d-restricted T cells that express a T cell receptor (TCR). Different from conventional T cells that detect peptide antigens presented by conventional major histocompatibility (MHC) molecules, NKT cells recognize lipid antigens presented by CD1d, a non-classical MHC molecule. Two types of NKT cells are recognized. Invariant or type I NKT cells express a canonical α chain (Vα24-Jα18 in humans) associated with a very limited TCR repertoire - limited range of β chains (Vβ11 in humans). A second population of NKT cells, called non-classical or non-invariant type II NKT cells, presents a more heterogeneous use of TCRαβ. Type I NKT cells are considered suitable for immunotherapy. Adaptive or invariant (type I) NKT cells can be identified by the expression of one or more of the markers TCR Va24-Ja18, Vb11, CD1d, CD3, CD4, CD8, aGalCer, CD161, and CD56.

[0072] The term "effector cell" generally applies to specific cells in the immune system that perform specific activities in response to stimulation and / or activation, or cells that bring about a specific function upon activation. As used herein, the term "effector cell" includes immune cells, "differentiated immune cells", and primary or differentiated cells that have been edited and / or regulated to perform specific activities in response to stimulation and / or activation, and in some contexts is interchangeable with them. Non-limiting examples of effector cells include primary or iPSC-derived T cells, NK cells, NKT cells, B cells, macrophages, and neutrophils.

[0073] As used herein, terms such as "isolated" refer to a cell or population of cells that has been separated from its original environment, i.e., the environment of the isolated cells does not substantially contain at least one component found in the environment in which the "non-isolated" reference cells are present. This term includes, for example, cells isolated from a tissue or biopsy sample and removed from some or all of the components as found in their natural environment. This term also includes cells that have been removed from at least one, some, or all of the components because the cells are found in a non-natural environment, such as a cell culture or cell suspension. Thus, an "isolated cell" is partially or completely separated from at least one component that includes other substances, cells, or cell populations, whether found naturally or when growing, storing, or persisting in a non-natural environment. Specific examples of isolated cells include partially pure cell compositions, substantially pure cell compositions, and cells cultured in a medium not found in nature. Isolated cells can be obtained by separating the desired cell or population thereof from other substances or cells in the environment, or by removing one or more other cell populations or subpopulations from the environment.

[0074] As used herein, terms such as "purify" refer to increasing purity. For example, the purity can be increased to at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%.

[0075] As used herein, the term "encoding" refers to the unique property of a specific sequence of nucleotides in a polynucleotide such as a gene, cDNA, or mRNA that functions as a template for the synthesis of other polymers and macromolecules in the defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or the defined sequence of amino acids and the biological processes resulting therefrom and their resulting biological properties. Thus, when the transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system, that gene encodes the protein. Both the nucleotide sequence that is identical to the mRNA sequence and which is usually set forth in the sequence listing, the coding strand, and the non-coding strand that is used as a template for transcription of the gene or cDNA can be said to "encode" the protein or other product of that gene or cDNA.

[0076] "Construct" refers to a macromolecule or molecular complex that includes a polynucleotide that is delivered to a host cell either in vitro or in vivo. As used herein, "vector" refers to any nucleic acid construct that can induce the delivery or transfer of foreign genetic material into a target cell and can replicate and / or express in the target cell. Thus, the term "vector" includes the construct being delivered. A vector can be a linear or circular molecule. A vector may or may not be integrated. The main types of vectors include, but are not limited to, plasmids, episomal vectors, viral vectors, cosmids, and artificial chromosomes. Viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, Sendai viral vectors, and the like.

[0077] When used occasionally throughout this application, the expression "TRAC_[construct]" (where "[construct]" is a variable expression construct having components and their arrangement that are specified in a given context) means that the expression construct is inserted into the TRAC locus to knock out the TCR, and the components of the expression construct are expressed or co-expressed under the control of the endogenous TCR promoter.

[0078] When used occasionally throughout this application, the expression "CD38_[construct]" (where "[construct]" is a variable expression construct having components and their arrangement that are specified in a given context) means that the expression construct is inserted into the CD38 locus to knock out CD38, and the components of the expression construct are expressed or co-expressed, whether under the control of the endogenous CD38 promoter in the construct or under an exogenous promoter.

[0079] "Integration" means that one or more nucleotides of a construct are stably inserted into the cell genome, i.e., covalently bound to a nucleic acid sequence within the chromosomal DNA of the cell. "Targeted integration" means that the nucleotides of the construct are inserted into the chromosomal or mitochondrial DNA of the cell at a pre-selected site or "integration site". As used herein, the term "integration" further refers to a process that includes the insertion of one or more exogenous sequences or nucleotides of a construct, with or without deletion of endogenous sequences or nucleotides at the integration site. If there is a deletion at the insertion site, "integration" may further include replacement of the nucleotides deleted by the endogenous sequence or one or more inserted nucleotides.

[0080] As used herein, the term "exogenous" is intended to mean that the reference molecule or activity is introduced into the host cell or is non-native to the host cell. A molecule can be introduced, for example, by introducing the coding nucleic acid into the host's genetic material, such as by integrating it into the host's chromosome or introducing it as extrachromosomal genetic material, such as a plasmid. Thus, the term when used in reference to the expression of a coding nucleic acid refers to introducing the coding nucleic acid into the cell in an expressible form. The term "endogenous" refers to a reference molecule or activity that is present in the host cell. Similarly, this term, when used in reference to the expression of a coding nucleic acid, refers to the expression of a coding nucleic acid that is contained within the cell and not exogenously introduced.

[0081] As used herein, a "gene of interest" or "polynucleotide sequence of interest" is a DNA sequence that, when placed under the control of appropriate regulatory sequences, is transcribed into RNA and optionally translated into a polypeptide in vivo. The gene of interest or polynucleotide can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and synthetic DNA sequences. For example, the gene of interest can encode an miRNA, shRNA, natural polypeptide (i.e., a polypeptide found in nature) or a fragment thereof, variant polypeptide (i.e., a mutant of a natural polypeptide having less than 100% sequence identity to the natural polypeptide) or a fragment thereof, engineered polypeptide or peptide fragment, therapeutic peptide or polypeptide, imaging marker, selectable marker, etc.

[0082] As used herein, the term "polynucleotide" refers to a nucleotide in polymeric form of any length, which can be either deoxyribonucleotide or ribonucleotide, or any analog thereof. The sequence of a polynucleotide is composed of four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), and when the polynucleotide is RNA, thymine is uracil (U). Polynucleotides include genes or gene fragments (e.g., probes, primers, ESTs, or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. "Polynucleotide" also refers to both double-stranded and single-stranded molecules.

[0083] As used herein, the terms "peptide", "polypeptide", and "protein" are used interchangeably and refer to a molecule having amino acid residues covalently linked by peptide bonds. A polypeptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids in a polypeptide. As used herein, these terms refer to both short chains, which are generally also referred to as peptides, oligopeptides, and oligomers in the art, and long chains, which are generally referred to as polypeptides or proteins in the art. "Polypeptide" includes, for example, among others, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins. Polypeptides include natural polypeptides, recombinant polypeptides, synthetic polypeptides, or combinations thereof.

[0084] As used herein, the term "subunit" refers to each individual polypeptide chain of a protein complex, and each individual polypeptide chain can form a stable folded structure by itself. Many protein molecules are composed of two or more subunits, where the amino acid sequences can be identical, similar, or completely different for each subunit. For example, the CD3 complex is composed of CD3α, CD3ε, CD3δ, CD3γ, and CD3ζ subunits, which form CD3ε / CD3γ, CD3ε / CD3δ, and CD3ζ / CD3ζ dimers. Within a single subunit, contiguous portions of the polypeptide chain often fold into compact, locally semi-independent units called "domains". Many protein domains can further contain independent "structural subunits", also called subdomains, that contribute to the common function of the domain. Thus, as used herein, the term "subdomain" refers to a protein domain within a larger domain, such as a binding domain within the extracellular domain of a cell surface receptor, or a stimulatory domain or signaling domain within the intracellular domain of a cell surface receptor.

[0085] "Operably-linked" or "operatively linked" is interchangeable with "operably connected" or "operatively connected" and refers to the association of nucleic acid sequences (or amino acids in a polypeptide having multiple domains) on a single nucleic acid fragment such that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence or functional RNA (i.e., the coding sequence or functional RNA is under the transcriptional control of the promoter) if it can affect the expression of that coding sequence or functional RNA. The coding sequence can be operably linked to regulatory sequences in the sense or antisense orientation. As a further example, a receptor binding domain can be operably connected to an intracellular signaling domain such that binding of the receptor to a ligand converts a signal in response to that binding.

[0086] "Fusion protein" or "chimeric protein," as used herein, is a protein produced through genetic manipulation to join two or more partial or complete polynucleotides encoding sequences encoding separate proteins, and expression of these joined polynucleotides results in a single peptide or multiple polypeptides having functional properties derived from each of the original proteins or fragments thereof. A linker (or spacer) peptide can be added between two adjacent polypeptides of different sources in a fusion protein.

[0087] As used herein, the term "genetic imprint" refers to genetic or epigenetic information that contributes to the preferential therapeutic properties of the source cell or iPSC and can be retained in iPSCs derived from the source cell and / or hematopoietic lineage cells derived from iPSCs. As used herein, a "source cell" is a non-pluripotent cell that can be used to generate iPSCs through reprogramming, and iPSCs derived from the source cell can further differentiate into specific cell types including any hematopoietic lineage cells. iPSCs derived from the source cell, and cells differentiated therefrom, may collectively be referred to as "derived" cells or "derivatives" depending on the context. For example, derivative effector cells, or derivative NK cells or derivative T cells used throughout this application are cells differentiated from iPSCs when compared to their primary counterparts obtained from natural / native sources such as peripheral blood, cord blood, or other donor tissues. As used herein, the genetic imprint conferring preferential therapeutic properties is incorporated into iPSCs by reprogramming selected source cells that are specific to the donor, disease, or treatment response, or by introducing gene editing modalities into iPSCs using genome editing. In the context of source cells obtained from specifically selected donors, diseases, or treatment situations, the genetic imprint contributing to preferential therapeutic properties may include a heritable phenotype, i.e., a context-specific genetic or epigenetic modification representing preferential therapeutic properties, passed on to derivative cells of the selected source cell, regardless of whether underlying molecular events have been identified.Source cells that are specific for a donor, disease, or treatment response may contain genetic imprints that can be retained in iPSCs and their derived hematopoietic cells. Such genetic imprints include, for example, pre - arranged single - specific TCRs from virus - specific T cells or invariant natural killer T (iNKT) cells, traceable and desirable gene polymorphisms, such as homozygosity for a point mutation encoding a high - affinity CD16 receptor in a selected donor, and predetermined HLA requirements, i.e., selected HLA - compatible donor cells that exhibit haplotypes in an increased population, but are not limited to these. As used herein, preferential therapeutic properties include improved engraftment, transport, homing, viability, self - renewal, persistence, regulation and modification of the immune response, survival rate, and cytotoxicity of the derived cells. Preferential therapeutic properties may also be related to antigen - targeted receptor expression, HLA presentation or its absence, resistance to the tumor microenvironment, induction of bystander immune cells and immune modification, improved on - target specificity with reduced off - tumor effects, and / or resistance to treatments such as chemotherapy. When iPSCs incorporating genetic imprints conferring preferential therapeutic properties are differentiated to obtain derived cells having one or more therapeutic properties, such derived cells are also referred to as "synthetic cells". Generally, synthetic cells, when compared to their closest corresponding primary cells, have one or more non - natural cell functions, whether the synthetic cells are differentiated from engineered pluripotent cells or are obtained by manipulating primary cells from natural / native sources such as peripheral blood, cord blood, or other donor tissues. For example, synthetic effector cells, or synthetic NK cells or synthetic T cells, as used throughout this application, are cells differentiated from genomically modified iPSCs compared to their primary counterparts obtained from natural / native sources such as peripheral blood, cord blood, or other donor tissues. In some embodiments, synthetic cells have one or more non - natural cell functions when compared to their closest corresponding primary cells.

[0088] As used herein, the term "enhanced therapeutic properties" refers to the therapeutic properties of a cell that are enhanced as compared to typical immune cells of the same general cell type. For example, NK cells having "enhanced therapeutic properties" have enhanced, improved, and / or increased therapeutic properties as compared to typical unmodified and / or naturally occurring NK cells. The therapeutic properties of immune cells can include, but are not limited to, cell engraftment, trafficking, homing, viability, self-renewal, persistence, regulation and modification of the immune response, survival rate, and cytotoxicity. The therapeutic properties of immune cells are also manifested by antigen-targeting receptor expression, HLA presentation or lack thereof, resistance to the tumor microenvironment, induction of bystander immune cells and immune modification, improved on-target specificity with reduced off-tumor effects, and / or resistance to treatments such as chemotherapy.

[0089] As used herein, the term "engager" refers to a molecule, such as a fusion polypeptide, that can form a linkage between an immune cell (e.g., a T cell, NK cell, NKT cell, B cell, macrophage, neutrophil) and a tumor cell and can activate the immune cell. Examples of engagers include, but are not limited to, bispecific T cell engagers (BiTEs), bispecific killer cell engagers (BiKEs), trispecific killer cell engagers (TriKEs), or multispecific killer cell engagers, or universal engagers that can be compatible with multiple immune cell types.

[0090] As used herein, the term "surface trigger receptor" refers to a receptor that can induce or initiate an immune response, such as a cytotoxic response. The surface trigger receptor can be engineered and expressed in effector cells, such as T cells, NK cells, NKT cells, B cells, macrophages, or neutrophils. In some embodiments, the surface trigger receptor facilitates the binding of a bispecific or multispecific antibody between an effector cell and a specific target cell (e.g., a tumor cell), independent of the natural receptor and cell type of the effector cell. Using this approach, iPSCs containing a universal surface trigger receptor can be generated and differentiated into populations of various effector cell types that express the universal surface trigger receptor. "Universal" means that the surface trigger receptor can be expressed and activated in any effector cell regardless of cell type, and all effector cells expressing the universal receptor can bind or link to an engager recognized by the surface trigger receptor, regardless of the tumor-binding specificity of the engager. In some embodiments, an engager having the same tumor targeting specificity is used to bind to the universal surface trigger receptor. In some embodiments, an engager having different tumor targeting specificities is used to bind to the universal surface trigger receptor. Thus, one or more effector cell types may be used to kill one specific type of tumor cell or two or more types of tumors. The surface trigger receptor generally includes a co-stimulatory domain for activation of the effector cell and an anti-epitope specific for an epitope of the engager. The bispecific engager is specific for the anti-epitope of the surface trigger receptor at one end and specific for a tumor antigen at the other end.

[0091] As used herein, the term "safety switch protein" refers to an engineered protein designed to prevent potential toxicity or other adverse effects of cell therapy. In some examples, the expression of the safety switch protein is conditionally controlled to address concerns about the safety of transplanted engineered cells that have permanently integrated the gene encoding the safety switch protein into the genome. This conditional regulation can vary and may include post-translational activation via small molecules and control by tissue-specific and / or transient transcriptional regulation. Safety switch proteins may mediate induction of apoptosis, inhibition of protein synthesis, DNA replication, growth arrest, transcriptional and post-transcriptional gene regulation, and / or antibody-mediated depletion. In some examples, the safety switch protein is activated by an exogenous molecule, such as a prodrug, and upon activation, induces apoptosis and / or cell death of the therapeutic cells. Examples of safety switch proteins include, but are not limited to, suicide genes such as caspase 9 (or caspase 3 or 7), thymidine kinase, cytosine deaminase, B cell CD20, modified EGFR, and any combination thereof. In this strategy, the prodrug administered upon the occurrence of an adverse event is activated by the suicide gene product to kill the transduced cells.

[0092] As used herein, the term "pharmaceutically active protein or peptide" refers to a protein or peptide capable of achieving a biological and / or pharmaceutical effect on an organism. A pharmaceutically active protein has curative or palliative properties against a disease and can be administered to improve, relieve, alleviate, reverse or lessen the severity of the disease. A pharmaceutically active protein also has preventive properties and is used to prevent the onset of a disease or, if it has occurred, to reduce the severity of such a disease or pathological condition. "Pharmaceutically active protein" includes the whole protein or peptide or a pharmaceutically active fragment thereof. This term also includes pharmaceutically active analogs of the protein or peptide or analogs of fragments of the protein or peptide. The term "pharmaceutically active protein" also refers to a plurality of proteins or peptides that act cooperatively or synergistically to bring about a therapeutic effect. Examples of pharmaceutically active proteins or peptides include, but are not limited to, receptors, binding proteins, transcription and translation factors, tumor growth inhibitory proteins, antibodies or fragments thereof, growth factors, and / or cytokines.

[0093] As used herein, the term "signaling molecule" refers to any molecule that modifies, participates in, inhibits, activates, reduces, or increases cell signaling. "Cell signaling" refers to the transmission of molecular signals in the form of chemical modifications by the recruitment of protein complexes along a pathway that ultimately causes a biochemical event within a cell. Examples of signaling pathways are known in the art and include, but are not limited to, G protein-coupled receptor signaling, tyrosine kinase receptor signaling, integrin signaling, Toll-like receptor signaling, ligand-gated ion channel signaling, ERK / MAPK signaling pathway, Wnt signaling pathway, cAMP-dependent pathway, and IP3 / DAG signaling pathway.

[0094] As used herein, the term "targeting modality" refers to a molecule, e.g., a polypeptide, that is genetically incorporated into a cell and promotes the specificity of an antigen and / or epitope, including but not limited to: i) antigen specificity when associated with a native chimeric antigen receptor (CAR) or T cell receptor (TCR); ii) engager specificity when associated with a monoclonal antibody or bispecific engager; iii) targeting of transformed cells; iv) targeting of cancer stem cells; and v) other targeting strategies in the absence of a specific antigen or surface molecule.

[0095] As used herein, the term "specific" or "specificity" can be used to refer to the ability of a molecule, e.g., a receptor or engager, to selectively bind to a target molecule, as opposed to non-specific or non-selective binding.

[0096] As used herein, the term "adoptive cell therapy" refers to a cell-based immunotherapy involving the infusion of autologous or allogeneic lymphocytes, regardless of whether the immune cells are isolated from a human donor, are effector cells obtained from in vitro differentiation of pluripotent cells, whether they are genetically modified, or whether they are primary donor cells that have been passaged, expanded, or immortalized ex vivo after isolation from a donor.

[0097] As used herein, "radiation" refers to the emission or transmission of energy in the form of waves or particles. Exemplary forms of radiation include, but are not limited to, electromagnetic radiation (e.g., radio waves, microwaves, infrared rays, visible light, ultraviolet rays, X-rays, and gamma rays), particle radiation (e.g., alpha rays, beta rays, proton radiation, and neutron radiation), and acoustic radiation (e.g., ultrasonic waves, sound waves, and seismic waves). In various embodiments, the amount of radiation is measured in Grays (Gy), which is defined as the absorption of 1 joule of radiation energy per kilogram of matter. In radiation therapy, the amount of radiation applied varies depending on the type and stage of the cancer being treated. In curative cases, typical doses for solid epithelial tumors range from 60 to 80 Gy, while lymphomas are typically treated with 20 to 40 Gy. Prophylactic (adjuvant) doses are typically about 45 to 60 Gy divided into fractions of 1.8 to 2 Gy (e.g., in the case of breast cancer, head and neck cancer). In various embodiments, radiation can be used as a sensitizing agent as disclosed herein.

[0098] As used herein, “radiation therapy” or “radiotherapy” are used interchangeably to refer to a type of cancer treatment involving the use of radiation to damage cells by destroying the genetic material that controls how cells grow and divide. Both healthy and cancer cells are damaged by radiation therapy, but the goal of radiation therapy is to spare normal, healthy cells as much as possible. The term “radiation therapy” often refers to external beam radiation therapy, where high-energy beams (e.g., X-rays, gamma rays, photons, protons, neutrons, ions, and any other form of energy applicable to such treatments) are generated by a machine external to the subject being treated and directed at precise points on the subject's body. However, the term “radiation therapy” also includes brachytherapy, where seeds, ribbons, or capsules containing or connected to a radiation source are placed inside or near a tumor or cancer cells within the subject's body. Low-dose rate implants, high-dose rate implants, and permanent implants are included in brachytherapy. The term “radiation therapy” also includes systemic radiation therapy, where a radiopharmaceutical (e.g., a radioactive pharmaceutical or radionuclide containing a radioactive peptide) is administered orally or intravenously to the subject and accumulates in the region of the body where the tumor or cancer cells are located. Similar to antibody-drug conjugates where an antibody that binds to a tumor antigen is linked to a toxic drug, radiopharmaceuticals incorporate radioactive compounds linked to a targeting molecule (such as an antibody) that specifically binds to a tumor antigen. Examples of radioactive compounds useful in radiopharmaceuticals include, but are not limited to, calcium-47, carbon-11, carbon-14, chromium-51, cobalt-57, cobalt-58, erbium-169, fluorine-18, gallium-67, gallium-68, hydrogen-3, indium-111, iodine-123, iodine-125, iodine-131, indium-59, krypton-81m, lutetium-177, nitrogen-13, oxygen-15, phosphorus-32, radium-223, rubidium-82, samarium-153, selenium-75, sodium-22, sodium-24, strontium-89, technetium-99m, thallium-201, xenon-133, and yttrium-90.In various embodiments, radiation therapy can be used as a sensitizer as disclosed herein.

[0099] As used herein, "lymphocyte depletion" and "lymphodepletion" are typically used interchangeably to refer to the destruction of lymphocytes and T cells prior to immunotherapy. The purpose of lymphodepletion prior to the administration of adoptive cell therapy is to promote the homeostatic expansion of effector cells and to eliminate other competing elements of the immune system that compete for regulatory immune cells and homeostatic cytokines. Thus, lymphodepletion is typically achieved by administering one or more chemotherapeutic agents to a subject prior to the first dosing of adoptive cell therapy. In various embodiments, lymphodepletion is performed several hours to several days prior to the first dosing of adoptive cell therapy. Exemplary chemotherapeutic agents useful for lymphodepletion include, but are not limited to, cyclophosphamide (CY), fludarabine (FLU), and those described below. However, sufficient lymphocyte depletion with anti-CD38 mAb can provide an alternative conditioning process for this iNK cell therapy without or with minimal need for a CY / FLU-based lymphodepletion procedure, as further described herein.

[0100] As used herein, "homing" or "transporting" refers to the active navigation (migration) of cells to a target site (e.g., a cell, a tissue (e.g., a tumor), or an organ). A "homing molecule" refers to a molecule that directs a cell to a target site. In some embodiments, the homing molecule functions to recognize and / or initiate an interaction of the cell with the target site. In some embodiments, the homing molecule is a chemokine receptor. As used herein, a "chemokine receptor" refers to a cell surface molecule that binds a chemokine. A chemokine receptor can include a naturally occurring chemokine receptor or a recombinant chemokine receptor or variants thereof. Exemplary chemokine receptors include, but are not limited to, CXC chemokine receptors (e.g., CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, or CXCR7), CC chemokine receptors (e.g., CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, or CCR11), CX3C chemokine receptors (e.g., CX3CR1), XC chemokine receptors (e.g., XCR1), or variants thereof.

[0101] As used herein, a "therapeutically sufficient amount" includes, within the scope of its meaning, a non-toxic but sufficient and / or effective amount of the particular therapeutic agent and / or pharmaceutical composition to which it refers and provides the desired therapeutic effect. The exact amount required will vary for each subject depending on factors such as the overall health of the patient, the patient's age, and the stage and severity of the condition being treated. In certain embodiments, a "therapeutically sufficient amount" is sufficient and / or effective to alleviate, reduce, and / or improve at least one symptom associated with the disease or condition of the subject being treated.

[0102] Differentiation of pluripotent stem cells requires changes in the culture system, such as stimulants in the culture medium and changes in the physical state of the cells. The most common strategy is to utilize the formation of embryoid bodies (EBs) as a common and important intermediate for initiating lineage-specific differentiation. An "embryoid body" is a three-dimensional cluster that has been shown to mimic embryonic development, generating multiple lineages within the three-dimensional region. Through a differentiation process that typically takes several hours to several days, simple EBs (e.g., aggregated pluripotent stem cells induced to differentiate) continue to mature and grow into cystic EBs, which typically takes several days to several weeks, at which point they are further processed to continue differentiating. EB formation is initiated by bringing pluripotent stem cells into proximity with each other in a three-dimensional multi-layer cluster of cells. Typically, this is achieved by one of several methods, including sedimenting pluripotent cells in droplets, sedimenting cells in "U" bottom well plates, or by mechanical agitation. Since aggregates maintained in pluripotent culture maintenance medium do not form proper EBs, aggregates of pluripotent stem cells require further cues for differentiation in order to promote EB growth. Therefore, aggregates of pluripotent stem cells need to be transferred to a differentiation medium that provides cues for induction into the selected lineage. EB-based culture of pluripotent stem cells typically generates a moderately proliferating population of differentiated cells within the EB cell cluster (i.e., the germ layers of ectoderm, mesoderm, and endoderm). EBs have been shown to promote cell differentiation, but generate heterogeneous cells in various differentiation states due to inconsistent exposure of the three-dimensional structure of the cells to cues for differentiation in the environment. In addition, EBs are cumbersome to create and maintain. Furthermore, cell differentiation by EB formation is accompanied by moderate cell proliferation, which also leads to a decrease in differentiation efficiency.

[0103] In contrast, "aggregate formation", unlike "EB formation", can be used to proliferate a population of pluripotent stem cell-derived cells. For example, during the proliferation of pluripotent stem cells based on aggregates, the culture medium is selected to maintain proliferation and pluripotency. Cell proliferation generally increases the size of the aggregates that form larger aggregates, which can be dissociated mechanically or enzymatically into smaller aggregates to maintain cell proliferation in culture and increase the number of cells. Unlike EB culture, cells cultured within aggregates in the maintenance culture medium maintain pluripotency markers. Pluripotent stem cell aggregates require additional cues for differentiation to induce differentiation.

[0104] As used herein, "monolayer differentiation" is a term that refers to a differentiation method different from the differentiation of cells into three-dimensional multilayer clusters, i.e., "EB formation". Monolayer differentiation, among other advantages disclosed herein, particularly avoids the need for EB formation to initiate differentiation. Since monolayer culture does not mimic embryogenesis as in the case of EB formation, differentiation into a specific lineage is considered minimal compared to the differentiation of all three germ layers in EB formation.

[0105] As used herein, "dissociated cells" or "single dissociated cells" refer to cells that are substantially separated or purified from other cells or from a surface (e.g., the surface of a culture plate). For example, cells can be dissociated from an animal or tissue by mechanical or enzymatic methods. Alternatively, cells that aggregate in vitro can be dissociated from each other enzymatically or mechanically, for example, by dissociation into a suspension of clusters, single cells, or a mixture of single cells and clusters. In yet another alternative embodiment, adherent cells can be dissociated from a culture plate or other surface. Thus, dissociation includes disrupting cell interactions with the extracellular matrix (ECM) and substrate (such as the culture surface), or disrupting the ECM between cells.

[0106] As used herein, "Master Cell Bank" or "MCB" refers to a clonal master engineered iPSC line, which is a clonal population of iPSCs that have been engineered, characterized, tested, qualified, expanded, and shown to reliably serve as starting cell material for the production of cell-based therapeutics by directed differentiation in a manufacturing environment. In various embodiments, the MCB is maintained, stored, and / or cryopreserved in multiple containers to prevent genetic mutations and / or the potential for contamination by reducing and / or eliminating the total number of times the iPSC line is passaged, thawed, or handled during the manufacturing process.

[0107] As used herein, "feeder cell" or "feeder" refers to a type of cell that is co-cultured with a second type of cell to provide an environment in which the second type of cell can grow, proliferate, or differentiate, and the feeder cell provides stimulation, growth factors, nutrients, and supports the second cell type. The feeder cells may be derived from a different species than the cells they support. For example, certain types of human cells, including stem cells, can be supported by primary cultures of mouse embryonic fibroblasts or immortalized mouse embryonic fibroblasts. In another example, peripheral blood-derived cells or transformed leukemia cells support the growth and maturation of natural killer cells. Feeder cells can typically be inactivated by treatment with mitotic inhibitors such as irradiation or mitomycin to prevent them from growing more than the cells they support when co-cultured with other cells. Feeder cells can include endothelial cells, stromal cells (e.g., epithelial cells or fibroblasts), and leukemia cells. Without limiting the foregoing, one particular type of feeder cell can be a human feeder such as human dermal fibroblasts. Another type of feeder cell can be mouse embryonic fibroblasts (MEF). Generally, various feeder cells can be used in part to maintain pluripotency, direct differentiation into specific lineages, enhance proliferation capacity, and promote maturation into specialized cell types such as effector cells.

[0108] As used herein, a "feeder-free" (FF) environment refers to an environment such as a culture condition, cell culture, or culture medium that is essentially free of feeders or stromal cells and / or has not been pretreated by culturing feeder cells. A "pretreated" medium refers to a medium taken after feeder cells have been cultured in the medium for a period such as at least one day. The pretreated medium contains many mediator substances, including growth factors and cytokines secreted from feeder cells cultured in the medium. In some embodiments, the feeder-free environment contains neither feeder cells nor stromal cells and has not been pretreated by culturing feeder cells.

[0109] "Functional," as used in connection with genome editing or modification of iPSCs and derived non-pluripotent cells differentiated therefrom, or genome editing or modification of non-pluripotent cells and derived iPSCs reprogrammed therefrom, means (1) at the gene level, success of transgenic or controlled gene expression such as inducible or transient expression at a desired cell developmental stage achieved by knock-in, knockout, knockdown gene expression, direct genome editing or modification, or "passage" via differentiation or reprogramming from a starting cell first genome-engineered, or (2) at the cell level - (i) modification of gene expression obtained in the cell through direct genome editing; (ii) modification of gene expression maintained in the cell through "passage" via differentiation or reprogramming from a starting cell first genome-engineered; (iii) downstream gene regulation in the cell as a result of modification of gene expression that appears only at an earlier developmental stage of the cell or only in the starting cell that gives rise to the cell via differentiation or reprogramming, or (iv) success of removal, addition, or modification of cell functions / characteristics by enhanced or newly achieved cell functions or attributes shown in mature cell products initially derived from genome editing or modification performed on iPSCs, precursors, or dedifferentiated cells.

[0110] "HLA deficiency", including HLA class I deficiency, HLA class II deficiency, or both, refers to cells in which the surface expression level of the complete MHC complex containing HLA class I protein heterodimers and / or HLA class II heterodimers is insufficient, or no longer maintained, or reduced, and the reduced or decreased level is lower than the level naturally detectable by other cells or synthetic methods.

[0111] As used herein, "modified HLA-deficient iPSC" refers to HLA-deficient iPSCs that are further modified by introducing genes that express proteins related to, but not limited to, improved differentiation ability, antigen targeting, antigen presentation, antibody recognition, persistence, immune evasion, resistance to suppression, proliferation, costimulation, cytokine stimulation, cytokine production (autocrine or paracrine), chemotaxis, and cytotoxicity, such as non-classical HLA class I proteins (e.g., HLA-E and HLA-G), chimeric antigen receptors (CARs), T cell receptors (TCRs), CD16Fc receptors, BCL11b, NOTCH, RUNX1, IL15, 4-1BB, DAP10, DAP12, CD24, CD3ζ, 4-1BBL, CD47, CD113, and PDL1. Cells with "modified HLA deficiency" include cells other than iPSCs.

[0112] The term "ligand" refers to a substance that forms a complex with a target molecule and generates a signal by binding to a site on the target. The ligand may be a natural or artificial substance that can specifically bind to the target. The ligand may be in the form of a protein, peptide, antibody, antibody complex, conjugate, nucleic acid, lipid, polysaccharide, monosaccharide, small molecule, nanoparticle, ion, neurotransmitter, or any other molecular entity that can specifically bind to the target. The target to which the ligand binds can be a protein, nucleic acid, antigen, receptor, protein complex, or cell. A ligand that binds to a target and changes its function, inducing a signal transduction response, is called an "agonistic" or "agonist" ligand. A ligand that binds to a target and blocks or reduces the signal transduction response is "antagonistic" or an "antagonist".

[0113] The term "antibody" is used in the broadest sense herein and generally refers to an immune response generating molecule containing at least one binding site that specifically binds to a target, where the target can be an antigen or a receptor that can interact with a particular antibody. For example, NK cells are activated by the binding of an antibody or its Fc region to its Fc-gamma receptor (FcγR), thereby inducing antibody-dependent cellular cytotoxicity (ADCC)-mediated effector cell activation. The specific fragment or portion of the antigen or receptor to which the antibody binds, or generally the target, is known as an epitope or antigenic determinant. The term "antibody" includes, but is not limited to, antibody mimetics that mimic the structure and / or function of an antibody or a specific fragment or portion thereof, including natural antibodies and their variants, fragments of natural antibodies and their variants, peptibodies and their variants, and single-chain antibodies and their fragments. Antibodies can be murine antibodies, human antibodies, humanized antibodies, camel IgG, single variable new antigen receptors (VNARs), shark heavy chain antibodies (Ig-NARs), chimeric antibodies, recombinant antibodies, single domain antibodies (dAbs), anti-idiotypic antibodies, bispecific, multispecific, or multimeric antibodies, or fragments thereof. Anti-idiotypic antibodies are specific for binding to the idiotype of another antibody, and the idiotype is the antigenic determinant of the antibody. Bispecific antibodies can be BiTEs (bispecific T cell engagers) or BiKEs (bispecific killer cell engagers), and multispecific antibodies can be TriKEs (trispecific killer cell engagers).Non-limiting examples of antibody fragments include Fab, Fab’, F(ab’)2, F(ab’)3, Fv, Fabc, pFc, Fd, single-chain variable fragment (scFv), tandem scFv (scFv)2, single-chain Fab (scFab), disulfide stabilized Fv (dsFv), minibody, diabody, triabody, tetrabody, single-domain antigen binding fragment (sdAb), camel heavy-chain IgG and Nanobody® fragment, heavy-chain-only antibody (VHH), and other antibody fragments that maintain the binding specificity of the whole antibody.

[0114] "Fc receptor" is abbreviated as FcR and is classified based on the type of antibody it recognizes. For example, those that bind to the most common class of antibody IgG are called Fc-gamma receptors (FcγR), those that bind to IgA are called Fc-alpha receptors (FcαR), and those that bind to IgE are called Fc-epsilon receptors (FcεR). The classes of FcR are also distinguished by the cells that express them (macrophages, granulocytes, natural killer cells, T and B cells) and the signal transduction properties of each receptor. Fc-gamma receptors (FcγR) include several members with different molecular structures and thus different antibody affinities, such as FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), and FcγRIIIB (CD16b).

[0115] "Chimeric receptor" is a general term used to describe engineered artificial or hybrid receptor protein molecules that are created to contain two or more portions of amino acid sequences derived from at least two different proteins. Chimeric receptor proteins are engineered to confer on cells the ability to initiate signal transduction and carry out downstream functions upon binding of an agonist ligand to the receptor. Exemplary "chimeric receptors" include, but are not limited to, chimeric antigen receptors (CARs), chimeric fusion receptors (CFRs), chimeric Fc receptors (CFcRs), and fusions of two or more receptors.

[0116] The term "chimeric Fc receptor", abbreviated as CFcR, is used to describe an engineered Fc receptor in which the native transmembrane domain and / or intracellular signaling domain has been modified or replaced with a non-native transmembrane domain and / or intracellular signaling domain. In some embodiments of the chimeric Fc receptor, in addition to one or both of the transmembrane and signaling domains being non-native, one or more stimulatory domains are introduced into the intracellular portion of the engineered Fc receptor to enhance receptor-induced cell activation, proliferation, and function. Unlike chimeric antigen receptors (CARs) that include an antigen-binding domain to a target antigen, chimeric Fc receptors bind to an Fc fragment, or the Fc region of an antibody, or the Fc region contained in an engager or binding molecule, and can either bring target cells into proximity or activate cell functions by binding to the molecule without bringing the cells into proximity. For example, Fcγ receptors can be engineered to include a selected transmembrane domain, stimulatory domain, and / or signaling domain in the intracellular region that generates the CFcR in response to IgG binding at the extracellular domain. In one example, CFcR is produced by engineering CD16, an Fcγ receptor, by replacing its transmembrane domain and / or intracellular domain. To further improve the binding affinity of the CD16-based CFcR, the extracellular domain of CD64 or a high-affinity variant of CD16 (e.g., F176V) can be incorporated. In some embodiments of CFcR that include the high-affinity CD16 extracellular domain, the proteolytic cleavage site containing serine at position 197 has been removed or the extracellular domain of the receptor has been replaced to be non-cleavable, i.e., not shed, thereby yielding an hnCD16-based CFcR.

[0117] It has been confirmed that CD16, an FcγR receptor, has two isoforms, Fc receptor FcγRIIIa (CD16a) and FcγRIIIb (CD16b). CD16a is a transmembrane protein expressed by NK cells that binds to monomeric IgG attached to target cells to activate NK cells and promote antibody-dependent cell cytotoxicity (ADCC). As used herein, "high-affinity CD16", "non-cleavable CD16", or "high-affinity non-cleavable CD16" (abbreviated as hnCD16) refers to natural or non-natural variants of CD16. Wild-type CD16 has low affinity and, when NK cells are activated, is subjected to extracellular domain shedding, a proteolytic cleavage process that regulates the cell surface density of various cell surface molecules on leukocytes. F176V and F158V are exemplary CD16 polymorphic variants with high affinity. CD16 variants in which the cleavage site (positions 195-198) in the membrane-proximal region (positions 189-212) is altered or eliminated do not shed. The cleavage site and the membrane-proximal region are described in detail in International Publication No. WO 2015 / 148926, the entire disclosure of which is incorporated herein by reference. The S197P variant of CD16 is a non-cleavable version of CD16. CD16 variants containing both F158V and S197P have high affinity and are non-cleavable. Another exemplary high-affinity non-cleavable CD16 (hnCD16) variant is an engineered CD16 that contains an extracellular domain derived from one or more of the three exons of the CD64 extracellular domain.

[0118] In some embodiments, generally in the context of treating tumors and with respect to the solid tumor microenvironment, cells are provided herein that include a set of engineered components that collectively complement (and in some cases synergize with) one another to enhance the activity of effector cells. The selected set of engineered components is referred to herein as a "scaffold" for its compatibility with any tumor antigen-binding molecule expressed in effector cells, including but not limited to CARs, antibodies, bispecific antibodies, and TCRs. However, the term "scaffold" does not require any particular physical relationship between the individual components of the set or any particular intracellular location for them, although a particular association and / or arrangement (e.g., the order in a co-expression construct of two or more of the individual components) can be optimized for higher expression levels or ease of processing, among other considerations in a manufacturing setting. For example, the scaffold can include the incorporation of two expression cassettes, each at a different location in the cell's genome. In some embodiments, the scaffold includes multiple genomic modifications such as the insertion of one or more polynucleotides and / or modifications to knock out one or more genes. The modifications can be made simultaneously or sequentially. Non-limiting examples of effector cell functions that can be enhanced by modification of the scaffold include autonomously improving cell growth, proliferation, expansion, and / or effector function without contacting the additionally supplied soluble cytokine in vitro or in vivo, and enhancing the homing, trafficking, exhaustion, or reduction of alloreactive host immune cells, as well as retention at the tumor site, and the tumor cells can be sensitized to synergize with the functional properties provided to the effector cells. The solid tumor-targeting scaffold of the present disclosure provides, for example, a master cell bank that provides a source of starting cells that can be modified by the simple addition of a tumor antigen-binding molecule for the indication intended to be treated, and is then used as a source for differentiating enhanced effector cells having therapeutic properties for one or more intended tumor indications, and can be particularly beneficial in the context of iPSCs that include the scaffold.

[0119] I. Cells and Compositions Useful for Adoptive Cell Therapies with Enhanced Properties Provided herein is a strategy for systematically manipulating the regulatory circuitry of clonal iPSCs while enhancing the therapeutic properties of derivative cells differentiated from iPSCs without affecting the differentiation potential and cellular developmental biology of iPSCs and their derivative cells. The iPSC-derived cells are functionally improved, and a combination of select modalities is suitable for adoptive cell therapy after being introduced into the cells at the iPSC level through genome engineering. Previously, it was unclear whether modified iPSCs, including one or more gene edits provided, retained the ability to enter cellular development while maintaining modified activity and / or properties, and / or the ability to mature into functional differentiated cells. Unexpected failures during directed cell differentiation from iPSCs can be attributed to aspects including, but not limited to, the specific gene expression or lack thereof at the developmental stage, the requirements for HLA complex presentation, protein shedding of introduced surface expression modalities, and the need to reconfigure the differentiation protocol to allow for changes in cell phenotype and / or function. This application demonstrates that one or more selected genome modifications provided herein do not adversely affect iPSC differentiation potential, and that functional effector cells derived from the engineered iPSCs have enhanced and / or acquired therapeutic properties resulting from individual or combined genome modifications that are retained in the effector cells after iPSC differentiation. Furthermore, all genome modifications and combinations thereof that may be described in the context of iPSCs and iPSC-derived effector cells are applicable to primary-derived cells, including primary immune cells such as T cells, NK cells, or immunomodulatory cells, whether cultured or expanded, and the modifications result in engineered immune cells useful for adoptive cell therapy.

[0120] Furthermore, although CAR-T cells have been shown to be effective and potent in treating some hematological malignancies, engineered T cell therapies have had limited success in dealing with solid tumors. Unlike liquid tumors where uniformly expressed antigens are accessible and can be effectively targeted, tumor access, lack of tumor-specific antigen targets, and antigen heterogeneity are significant barriers to the success of CAR-T cell development in solid tumors. In addition, the inherent genetic engineering variability seen in patient- and donor-derived immune cells limits the widespread application of CAR-T cell therapy. This application provides genomic engineering modalities in the form of solid tumor-targeting scaffolds, as well as other gene modalities, to improve on-target specificity with reduced off-tumor effects, to avoid allogeneic rejection, and to overcome challenges that increase in solid tumors, particularly in the context of an off-the-shelf adoptive cell therapy setting using effector cells derived from engineered iPSCs.

[0121] 1. Overexpression of C-X-C motif chemokine receptor Chemokines are a family of homogeneous serum proteins of approximately 7 to approximately 16 kDa, originally characterized by their ability to induce leukocyte migration. Most chemokines have four characteristic cysteines (Cys) and are classified into C-X-C (or alpha, CXC), C-C (or beta), C (or gamma), and CX3C (or delta) chemokine classes according to the motif shown by the first two cysteines. The C-X-C (or alpha, CXC) subfamily is further classified into two groups: ELR-CXC chemokines and non-ELR-CXC chemokines according to the presence of an ELR motif (Glu-Leu-Arg) preceding the first cysteine.

[0122] CXC chemokine receptor 2 (CXCR2), also known as CD128, interleukin 8 receptor beta (IL8Rβ), or L8 receptor type B, is a chemokine receptor mainly expressed by neutrophils, mast cells, monocytes, and macrophages. CD56 dim NK cells are known to express CXCR2, but their expression can be downregulated upon NK cell activation. T cells typically do not express CXCR2. iPSCs and iPSC-derived T cells do not express CXCR2 unless they are transduced with an exogenous polynucleotide encoding CXCR2 as disclosed in this application. Chemokine IL8 (also known as CXCL8) is secreted by mononuclear macrophages, neutrophils, eosinophils, T lymphocytes, epithelial cells, and fibroblasts and functions as a chemotactic factor by inducing neutrophils to the site of infection. CXCL8 is also secreted by tumor cells and promotes tumor migration, invasion, angiogenesis, and metastasis. CXCL8 is one of the ligands for multiple CXC chemokine receptors including CXCR1 and CXCR2. Additional chemokines known to bind to CXCR2 include, but are not limited to, CXCL1, GROβ (CXCL2), CXCL3, CXCL5, CXCL6, and CXCL7.

[0123] CXC chemokine receptor 3 (CXCR3), also known as G Protein-coupled Receptor 9 (GPR9) and CD183, is a G protein-coupled receptor that binds to chemokines CXCL9, CXCL10, and CXCL11. CXCR3 is mainly expressed in activated T-helper type 1 (Th1) lymphocytes, but is also present in natural killer cells, macrophages, dendritic cells, and subsets of B lymphocytes. The interaction between CXCR3 and its ligands is involved in inducing receptor-bearing cells to specific parts of the body, particularly sites of inflammation, immune disorders, and immunodeficiency.

[0124] In various embodiments, the present application provides effector cells or iPSCs that are genetically engineered to include a solid tumor targeting backbone that includes a C-X-C motif chemokine receptor, among other editing and other gene modalities contemplated and described herein. In various embodiments, the C-X-C motif chemokine receptor includes CXCR2 or CXCR3, or variants thereof. A non-limiting example of the amino acid sequence of human CXCR2 is that registered as UniProtKB number P25025. In one embodiment, CXCR2 includes an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 1. In some embodiments, CXCR2 includes an amino acid sequence that is at least 90% identical to SEQ ID NO: 1. In some embodiments, CXCR2 includes an amino acid sequence that is at least 95% identical to SEQ ID NO: 1. In some embodiments, CXCR2 includes the amino acid sequence of SEQ ID NO: 1. In some embodiments, variants of CXCR2 include CXCR2 isoforms represented by SEQ ID NO: 2, 3, 4, 5, or 6. In some embodiments, variants of CXCR2 include an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 99% identical to any one of SEQ ID NO: 2, 3, 4, 5, and 6. In some embodiments, variants of CXCR2 include an amino acid sequence that is at least 90% identical to any one of SEQ ID NO: 2, 3, 4, 5, and 6. In some embodiments, variants of CXCR2 include an amino acid sequence that is at least 95% identical to any one of SEQ ID NO: 2, 3, 4, 5, and 6. In some embodiments, variants of CXCR2 include the amino acid sequence of SEQ ID NO: 2. In some embodiments, variants of CXCR2 include the amino acid sequence of SEQ ID NO: 3. In some embodiments, variants of CXCR2 include the amino acid sequence of SEQ ID NO: 4. In some embodiments, variants of CXCR2 include the amino acid sequence of SEQ ID NO: 5. In some embodiments, variants of CXCR2 include the amino acid sequence of SEQ ID NO: 6.As used throughout this specification and this application, the percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions x 100), taking into account the number of gaps that need to be introduced for the optimal alignment of the two sequences and the length of each gap. The comparison of sequences and the determination of the percent identity between two sequences can be performed using mathematical algorithms recognized in the art.

[0125] SEQ ID NO:1 MEDFNMESDSFEDFWKGEDLSNYSYSSTLPPFLLDAAPCEPESLEINKYFVVIIYALVFLLSLLGNSLVMLVILYSRVGRSVTDVYLLNLALADLLFALTLPIWAASKVNGWIFGTFLCKVVSLLKEVNFYSGILLLACISVDRYLAIVHATRTLTQKRYLVKFICLSIWGLSLLLALPVLLFRRTVYSSNVSPACYEDMGNNTANWRMLLRILPQSFGFIVPLLIMLFCYGFTLRTLFKAHMGQKHRAMRVIFAVVLIFLLCWLPYNLVLLADTLMRTQVIQETCERRNHIDRALDATEILGILHSCLNPLIYAFIGQKFRHGLLKILAIHGLISKDSLPKDSRPSFVGSSSGHTSTTL (CXCR2 of 360 amino acids; UniProtKB accession number P25025)

[0126] SEQ ID NO:2 MEDFNMESDSFEDFW (CXCR2 isoform 1 of 15 amino acids (residues 1 - 15 of CXCR2); UniProtKB accession number Q6LCZ7)

[0127] SEQ ID NO:3 MEDFNMESDSFEDFWKGEDLSNYSYSSTLPPFLLDAAPCEPESLEINKYFVVIIYALVFL LSLLGNSLVMLVILYSRVGRSVTDVYLLNLALADLLFALTLPIWAASKVNGWIFGTFLCK VVSLLKEVNFYSGILLLACISVDRYLAIVHATRTLTQKRYLVKFICLSIWGLSLLLALPV LLFRRTVYSSNVSPACYEDM (200 - amino acid CXCR2 isoform 2 (residues 1 - 200 of CXCR2); UniProtKB accession number C9JW47)

[0128] SEQ ID NO: 4 MEDFNMESDSFEDFWKGEDLSNYSYSSTLPPFLLDAAPCEPESLEINKYFVVIIYALVFL LSLLGNSLVMLVILYSRVGRSVTDVYLLNLALADLLFALTLPIWAASKVNGWIFGTFLCK VVSLLKEVNFYSGIL (135 - amino acid CXCR2 isoform 3 (residues 1 - 135 of CXCR2); UniProtKB accession number C9JG19)

[0129] SEQ ID NO: 5 MEDFNMESDSFEDFWKGEDLSNYSYSSTLPPFLLDAAPCEPESLEINKYFVVIIYALVFL LSLLGNSLVMLVILYSRVGRSVTDVYLLNLALADLLFALTLPIWAASKVNGWIFGTFLCK VVSLLKEVNFYSGILLLACISVDRYLAIVHATRTLTQKRYLVKFICLSIWGL (172 - amino acid CXCR2 isoform 4 (residues 1 - 172 of CXCR2); UniProtKB accession number C9J1J7)

[0130] SEQ ID NO: 6 MEDFNMESDSFEDFWKGEDLSNYSYSSTLPPFLLDAAPCEPESLEINKYFVVIIYALVFL LSLLGNSLVMLVILYSRVGRSVTDVYLLNLALADLLFALTLPIWAASKVNGWIFGTFLCK VVSLLKEVNFYSGILLLA (138 amino acid CXCR2 isoform 5 (residues 1 - 138 of CXCR2); UniProtKB number C9J2F9)

[0131] A non - limiting example of the amino acid sequence of human CXCR3 is the one registered as UniProtKB number P49682. In one embodiment, CXCR3 comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 7. In some embodiments, CXCR3 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 7. In some embodiments, CXCR3 comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 7. In some embodiments, CXCR3 comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, variants of CXCR3 comprise the CXCR3 isoforms represented by SEQ ID NO: 8 or 9. In some embodiments, variants of CXCR3 comprise an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 8 or 9. In some embodiments, variants of CXCR3 comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 8 or 9. In some embodiments, variants of CXCR3 comprise an amino acid sequence that is at least 95% identical to SEQ ID NO: 8 or 9. In some embodiments, variants of ICXCR3 comprise the amino acid sequence of SEQ ID NO: 8. In some embodiments, variants of CXCR3 comprise the amino acid sequence of SEQ ID NO: 9.

[0132] SEQ ID NO: 7 MVLEVSDHQVLNDAEVAALLENFSSSYDYGENESDSCCTSPPCPQDFSLNFDRAFLPALYSLLFLLGLLGNGAVAAVLLSRRTALSSTDTFLLHLAVADTLLVLTLPLWAVDAAVQWVFGSGLCKVAGALFNINFYAGALLLACISFDRYLNIVHATQLYRRGPPARVTLTCLAVWGLCLLFALPDFIFLSAHHDERLNATHCQYNFPQVGRTALRVLQLVAGFLLPLLVMAYCYAHILAVLLVSRGQRRLRAMRLVVVVVVAFALCWTPYHLVVLVDILMDLGALARNCGRESRVDVAKSVTSGLGYMHCCLNPLLYAFVGVKFRERMWMLLLRLGCPNQRGLQRQPSSSRRDSSWSETSEASYSGL (CXCR3 of 368 amino acids; UniProtKB accession number P49682)

[0133] SEQ ID NO: 8 MELRKYGPGRLAGTVIGGAAQSKSQTKSDSITKEFLPGLYTAPSSPFPPSQVSDHQVLNDAEVAALLENFSSSYDYGENESDSCCTSPPCPQDFSLNFDRAFLPALYSLLFLLGLLGNGAVAAVLLSRRTALSSTDTFLLHLAVADTLLVLTLPLWAVDAAVQWVFGSGLCKVAGALFNINFYAGALLLACISFDRYLNIVHATQLYRRGPPARVTLTCLAVWGLCLLFALPDFIFLSAHHDERLNATHCQYNFPQVGRTALRVLQLVAGFLLPLLVMAYCYAHILAVLLVSRGQRRLRAMRLVVVVVVAFALCWTPYHLVVLVDILMDLGALARNCGRESRVDVAKSVTSGLGYMHCCLNPLLYAFVGVKFRERMWMLLLRLGCPNQRGLQRQPSSSRRDSSWSETSEASYSGL (CXCR3 isoform 2 of 415 amino acids; UniProtKB accession number P49682-2)

[0134] SEQ ID NO: 9 MVLEVSDHQVLNDAEVAALLENFSSSYDYGENESDSCCTSPPCPQDFSLNFDRAFLPALYSLLFLLGLLGNGAVAAVLLSRRTALSSTDTFLLHLAVADTLLVLTLPLWAVDAAVQWVFGSGLCKVAGALFNINFYAGALLLACISFDRYLNIVHATQLYRRGPPARVTLTCLAVWGLCLLFALPDFIFLSAHHDERLNATHCQYNFPQGSSSGSGCGCCSCAWAAPTREGSRGSHRLPAGIHPGLRPQRPPTRACEAGIRAPLSPI (CXCR3 isoform 3 of 267 amino acids; UniProtKB accession number P49682-3)

[0135] In various embodiments, a polynucleotide encoding a C-X-C motif chemokine receptor or a variant thereof is inserted into a selected locus of a primary-derived effector cell or iPSC to induce functional effector cells containing the same gene editing via directed differentiation. In some embodiments, the selected locus for C-X-C motif chemokine receptor insertion includes a safe harbor locus, a locus that is intended to be disrupted or knocked out, and a locus that provides an endogenous promoter for spatial and / or temporal control of exogenous gene expression. In some embodiments, the selected locus for C-X-C motif chemokine receptor insertion includes AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR, NKG2A, NKG2D, CD38, CD25, CD69, CD44, CD58, CD54, CD56, CD71, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT. In one embodiment, the selected locus for C-X-C motif chemokine receptor insertion is the TCR locus. In one embodiment, the selected locus for C-X-C motif chemokine receptor insertion is the CD38 locus.

[0136] In some embodiments, the C-X-C motif chemokine receptor is co-expressed with one or more exogenous polynucleotides encoding a polypeptide of interest via separate expression constructs or a single bicistronic or tricistronic expression cassette. In some embodiments, a single bicistronic or tricistronic expression cassette comprising the C-X-C motif chemokine receptor and one or more exogenous polynucleotides encoding a polypeptide of interest comprises a 2A sequence, such that the C-X-C motif chemokine receptor and the additional polynucleotide are in a single open reading frame (ORF). The bicistronic design allows for the coordinated expression of multiple polynucleotides under the same control mechanism that can be selected, for example, to incorporate an inducible promoter for the expression of a single ORF, for both timing and quantity. Self-cleaving peptides are found in members of the Picornaviridae family, including those of the Aphthovirus genus such as foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV), Thosea asigna virus (TaV), and porcine tescho virus-1 (PTV-I) (Donnelly, ML, et al, J. Gen. Virol, 82, 1027-101(2001); Ryan, MD, et al., J. Gen. Virol., 72, 2727-2732(2001)), as well as those of the Cardiovirus genus such as Theiler's murine encephalomyelitis virus and encephalomyocarditis virus. The 2A peptides derived from FMDV, ERAV, PTV-I, and TaV are sometimes referred to as "F2A", "E2A", "P2A", and "T2A", respectively.In some embodiments, the exogenous polynucleotide that can be co-expressed with a C-X-C motif chemokine receptor encodes one or more polypeptides including a CAR, CD16 or a variant thereof, a cytokine, a cytokine receptor, a cytokine signaling complex, a chimeric fusion receptor, a chimeric Fc receptor, an engager, a checkpoint inhibitor, an Fc receptor, or an antibody or a functional variant or fragment thereof. In one embodiment, the exogenous polynucleotide co-expressed with a C-X-C motif chemokine receptor in a bicistronic cassette does not encode a CAR. In one embodiment, at least one exogenous polynucleotide co-expressed with a C-X-C motif chemokine receptor in a bicistronic cassette encodes exogenous CD16. In some embodiments, the primary-derived or derived effector cells comprising a C-X-C motif chemokine receptor or a variant thereof are T lineage cells. In some embodiments, the primary-derived or derived effector cells comprising a C-X-C motif chemokine receptor or a variant thereof are NK lineage cells.

[0137] Further provided herein is a master cell bank comprising sorted single cells and expanded cloned engineered iPSCs having at least one modification or phenotype as provided herein, including but not limited to a C-X-C motif chemokine receptor or a variant thereof, the cell bank providing a renewable source for producing ready-to-use engineered homogeneous cell therapy products, including but not limited to induced NK cells and T cells, which can be mass-produced at a significant scale in a manner that is clearly defined, homogeneous in composition, and cost-effective, for further engineering of cloned engineered iPSCs.

[0138] 2. Exogenously introduced TGFβ redirector receptor Transforming growth factor-beta (TGFβ) is a pleiotropic immunosuppressive cytokine that has a complex role in tumorigenesis, including epithelial-mesenchymal transition, angiogenesis, tumor cell motility and metastasis, cancer associated fibroblast (CAF) proliferation, and immunosuppression. TGFβ exists in its latent form in the tumor microenvironment and is known to suppress T cell effector functions, in part through Smad-mediated downregulation of the target genes granzyme, perforin, and interferon. Furthermore, detection of the TGFβ gene expression signature correlates with T cell exclusion from tumors and resistance to immunotherapy. One aspect of the present application provides a multi-element solid tumor targeting scaffold design incorporating a synthetic transforming growth factor-beta receptor (TGFβR) signaling redirector receptor, which, among other edits contemplated and described herein, comprises allogeneic effector cells derived from genetically engineered iPSCs for generally better efficacy in tumors and particularly in solid tumors. Generally, a "signaling (or signal) redirector receptor" or "SRR" redirects signaling of the extracellular domain from a first receptor (e.g., a TGFβ receptor) through the intracellular domain of a different receptor (e.g., a cytokine receptor) by joining the extracellular domain of the first receptor to the intracellular domain of a different receptor. In the context of TGFβR, the signaling redirector receptor may be referred to throughout the present application as the "TGFβR redirector" or "TGFβR redirector receptor" or "TGFβ signal redirector receptor" or "TGFβ-SRR".

[0139] In some embodiments, iPSCs and derivative cells therefrom comprise a polynucleotide encoding a TGFβ-SRR, which comprises a partial or complete peptide of the extracellular domain (ECD) of TGFβR. In some embodiments, the TGFβ-SRR comprises (i) the extracellular domain of the transforming growth factor beta receptor (TGFβR) or a fragment thereof, and (ii) the intracellular domain (ICD) of a cytokine receptor comprising IL2R, IL12R, IL18R, IL21R, or any combination thereof, or a fragment thereof.

[0140] In some embodiments, the TGFβ-SRR comprising the above ECD and ICD further comprises a transmembrane domain (TM). In various embodiments, the transmembrane (TM) domain of the TGFβ-SRR can be (i) derived from the same molecule providing the intracellular domain, (ii) derived from the same molecule providing the extracellular domain, or (iii) modified or replaced with the transmembrane domain of any other membrane-bound protein. In some embodiments, the cytokine receptor providing the intracellular domain or a fragment thereof of the TGFβ-SRR comprises at least one of IL2R, IL4R, IL6R, IL7R, IL9R, IL10R, IL11R, IL12R, IL15R, IL18R, and IL21R.

[0141] In one embodiment, the extracellular domain (ECD) of TGFβR comprises an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 10. In some embodiments, the extracellular domain (ECD) of TGFβR comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 10. In some embodiments, the extracellular domain (ECD) of TGFβR comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 10. In some embodiments, the extracellular domain (ECD) of TGFβR comprises the amino acid sequence of SEQ ID NO: 10. In one embodiment, the intracellular domain (ICD) of IL2Rβ comprises an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 11. In some embodiments, the intracellular domain (ICD) of IL2Rβ comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 11. In some embodiments, the intracellular domain (ICD) of IL2Rβ comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 11. In some embodiments, the intracellular domain (ICD) of IL2Rβ comprises the amino acid sequence of SEQ ID NO: 11. In one embodiment, the intracellular domain (ICD) of IL12Rβ comprises an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 12. In some embodiments, the intracellular domain (ICD) of IL12Rβ comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 12. In some embodiments, the intracellular domain (ICD) of IL12Rβ comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 12. In some embodiments, the intracellular domain (ICD) of IL12Rβ comprises the amino acid sequence of SEQ ID NO: 12. In one embodiment, a fragment of the intracellular domain of IL12Rβ comprises an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 13.In some embodiments, a fragment of the intracellular domain of IL12Rβ comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 13. In some embodiments, a fragment of the intracellular domain of IL12Rβ comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 13. In some embodiments, a fragment of the intracellular domain of IL12Rβ comprises the amino acid sequence of SEQ ID NO: 13. In one embodiment, the intracellular domain (ICD) of IL18Rβ comprises an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 14. In some embodiments, the intracellular domain (ICD) of IL18Rβ comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 14. In some embodiments, the intracellular domain (ICD) of IL18Rβ comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 14. In some embodiments, the intracellular domain (ICD) of IL18Rβ comprises the amino acid sequence of SEQ ID NO: 14. In one embodiment, the intracellular domain (ICD) of IL21Rβ comprises an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 15. In some embodiments, the intracellular domain (ICD) of IL21Rβ comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 15. In some embodiments, the intracellular domain (ICD) of IL21Rβ comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 15. In some embodiments, the intracellular domain (ICD) of IL21Rβ comprises the amino acid sequence of SEQ ID NO: 15.

[0142] SEQ ID NO: 10 TIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQ (ECD of TGFβR)

[0143] Sequence number 11 NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV (ICD of IL2Rβ)

[0144] Sequence number 12 HYFQQKVFVLLAALRPQWCSREIPDPANSTCAKKYPIAEEKTQLPLDRLLIDWPTPEDPEPLVISEVLHQVTPVFRHPPCSNWPQREKGIQGHQASEKDMMHSASSPPPPRALQAESRQLVDLYKVLESRGSDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAPLADSLEELEPQHISLSVFPSSSLHPLTFSCGDKLTLDQLKMRCDSLML (ICD of IL12Rβ)

[0145] Sequence number 13 SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAPLADSLEELEPQ (Fragment of ICD of IL12Rβ)

[0146] Sequence number 14 YRVDLVLFYRHLTRRDETLTDGKTYDAFVSYLKECRPENGEEHTFAVEILPRVLEKHFGYKLCIFERDVVPGGAVVDEIHSLIEKSRRLIIVLSKSYMSNEVRYELESGLHEALVERKIKIILIEFTPVTDFTFLPQSLKLLKSHRVLKWKADKSLSYNSRFWKNLLYLMPAKTVKPGRDEPEVLPVLSES (ICD of IL8Rβ)

[0147] SEQ ID NO: 15 SLKTHPLWRLWKKIWAVPSPERFFMPLYKGCSGDFKKWVGAPFTGSSLELGPWSPEVPSTLEVYSCHPPRSPAKRLQLTELQEPAELVESDGVPKPSFWPTAQNSGGSAYSEERDRPYGLVSIDTVTVLDAEGPCTWPCSCEDDGYPALDLDAGLEPSPGLEDPLLDAGTTVLSCGCVSAGSPGLGGPLGSLLDRLKPPLADGEDWAGGLPWGGRSPGGVSESEAGSPLAGLDMDTFDSGFVGSDCSSPVECDFTSPGDEGPPRSYLRQWVVIPPPLSSPGPQAS (ICD of IL21Rβ)

[0148] In some embodiments, the signal transduction receptor comprises the extracellular domain of TGFβR or a fragment thereof, and the intracellular domain of cytokine receptor IL2Rβ or a fragment thereof, thereby forming a TGFβR2-IL2Rβ signal transduction redirector receptor. In some embodiments, the signal transduction receptor comprises the extracellular domain of TGFβR or a fragment thereof, and the intracellular domain of cytokine receptor IL12Rβ or a fragment thereof, thereby forming a TGFβR2-IL12Rβ signal transduction redirector receptor. In some embodiments, the signal transduction receptor comprises the extracellular domain of TGFβR or a fragment thereof, and the intracellular domain of cytokine receptor IL18Rβ or a fragment thereof, thereby forming a TGFβR2-IL18Rβ signal transduction redirector receptor. In some embodiments, the signal transduction receptor comprises the extracellular domain of TGFβR or a fragment thereof, and the intracellular domain of cytokine receptor IL21R or a fragment thereof, thereby forming a TGFβR2-IL21R signal transduction redirector receptor.

[0149] In some embodiments, the TGFβR2-IL12Rβ signal transduction redirector receptor comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 97%, 98%, or 99% sequence identity to the sequence represented by SEQ ID NO: 16 (specifically referred to as TGFβR2-trIL12Rβ throughout this application). In some embodiments, the TGFβR2-IL12Rβ signal transduction redirector receptor comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 16. In some embodiments, the TGFβR2-IL12Rβ signal transduction redirector receptor comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 16. In some embodiments, the TGFβR2-IL12Rβ signal transduction redirector receptor comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the transmembrane domain (TM) sequence represented by SEQ ID NO: 17 contained within SEQ ID NO: 16 may vary in sequence or length, or may be replaced by the transmembrane domain of another transmembrane protein.

[0150]

Table 1

[0151] SEQ ID NO: 17 VTGISLLPPLGVAISVIIIFYCYRVN (Exemplary variable portion of TGFβR2-trIL12Rβ)

[0152] Thus, in various embodiments, any of the TGFβ-SRRs provided herein may be introduced into iPSCs using one or more of the above-described construct designs, or may be introduced into their derived cells during iPSC differentiation. In addition to induced pluripotent stem cells (iPSCs), cloned iPSCs, cloned iPS cell lines, or iPSC-derived cells containing at least one engineered modality disclosed herein are provided. Also provided is a master cell bank comprising sorted single cells and expanded cloned engineered iPSCs having at least the TGFβ-SRR as described in this section, the cell bank providing a platform for further iPSC engineering and a renewable source for manufacturing ready-to-use engineered homogeneous cell therapy products that can be mass-produced on a large scale in a manner that is compositionally defined, uniform, and cost-effective.

[0153] Accordingly, in some embodiments, the invention provides immune cells, iPSCs, and iPSC-derived cells comprising a solid tumor-targeting backbone comprising a polynucleotide encoding a TGFβ redirector receptor (the “TGFβ-SRR” in Table 4), among other gene modalities, and cells such as derived T cells and derived NK cells are useful for overcoming or reducing tumor microenvironment suppression associated with tumors, particularly solid tumors. In some embodiments, iPSCs and their derived cells comprise a solid tumor-targeting backbone comprising a polynucleotide encoding a C-X-C motif chemokine receptor or a variant thereof, a polynucleotide encoding a TGFβ redirector receptor, and / or two or more of one or more additional genome edits described herein, without adversely affecting the differentiation ability of the iPSCs and the functions of derived effector cells such as derived T cells and derived NK cells.

[0154] Also provided is a master cell bank comprising sorted single cells and expanded clonally engineered iPSCs having at least an exogenously introduced polynucleotide encoding a TGFβ redirector receptor and optionally a polynucleotide encoding a C-X-C motif chemokine receptor or a variant thereof, which cell bank provides a platform for further iPSC engineering and a renewable source for manufacturing ready-to-use engineered homogeneous cell therapy products that can be mass-produced on a large scale in a manner that is clearly defined, homogeneous, and cost-effective.

[0155] 3. Allogeneic immune defense receptor (ADR) expression Undesirable activation of T cells and NK cells often promotes an allogeneic immune response that results in the development of graft-versus-host disease (GvHD). Although several steps can be taken to reduce the reactivity of allogeneic cells in the recipient individual, such cells can still be targeted by the recipient's immune system (primarily T cells and NK cells), recognized as foreign, leading to rejection and limiting the therapeutic benefit. On the other hand, lymphodepletion by chemotherapy, such as Cy / Flu (cyclophosphamide / fludarabine), for example, often results in associated hematotoxicities, including increased susceptibility to severe infections due to indiscrimitive lymphocyte depletion and the resulting severely impaired immune system, by modulating the host immune system to reduce the allogeneic immune response. To control the pathogenic conditions resulting from undesirable activation of the immune system, in various embodiments, the present application provides a solid tumor-targeting scaffold comprising, among other components, an allogeneic immune defense receptor (ADR). Another aspect of the present application is to preserve resting cells in the recipient while effecting, among other edits contemplated and described herein, effector cell augmentation and selective depletion of alloreactive host NK cells and T cells with upregulated expression of 4-1BB and / or CD38 (the latter including pathogenic T cells and regulatory T cells), by providing immune cells, iPSCs, and iPSC-derived effector cells genetically engineered to comprise a 4-1BB or CD38-specific allogeneic immune defense receptor (ADR).

[0156] In some embodiments of an ADR specific for 4-1BB (also known as CD137, "41BB"), the ADR comprises an extracellular domain that targets 4-1BB, which is upregulated on activated host T cells or NK cells, and a signaling domain that promotes effector cell activation. For example, the 41BB-ADR extracellular domain can comprise any suitable ligand for 4-1BB that includes 4-1BBL, an antibody (or a functional fragment thereof) that targets 4-1BB, a fusion of Fc and 4-1BBL, or a functional derivative or fragment thereof. In some embodiments, the 41BB-ADR extracellular domain comprises 4-1BBL, or a fragment thereof that is effective to bind 4-1BB. In another embodiment, the 41BB-ADR extracellular domain comprises an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO: 145. In some embodiments, the 41BB-ADR extracellular domain comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 145. In some embodiments, the 41BB-ADR extracellular domain comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 145. In some embodiments, the 41BB-ADR extracellular domain comprises the amino acid sequence of SEQ ID NO: 145.

[0157] SEQ ID NO: 145 GLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE

[0158] In one embodiment of the CD38-specific ADR, the CD38-ADR comprises an extracellular domain comprising a CD38-binding domain or a fragment thereof. In some embodiments, the CD38-binding domain or a fragment thereof is derived from an anti-CD38 antibody. In some embodiments, the anti-CD38 antibody comprises a murine antibody, a human antibody, a humanized antibody, a camelid Ig, a single variable new antigen receptor (VNAR), a shark heavy chain only antibody (Ig NAR), a chimeric antibody, a recombinant antibody, or an antibody fragment thereof. Non-limiting examples of an antibody-binding domain or a fragment thereof include Fab, Fab’, F(ab’)2, F(ab’)3, Fv, single-chain antigen-binding fragment (scFv), (scFv)2, disulfide-stabilized Fv (dsFv), minibody, diabody, triabody, tetrabody, single-domain antigen-binding fragment (sdAb, nanobody), recombinant heavy chain only antibody (VHH), and other antibody fragments that maintain the binding specificity of the whole antibody.

[0159] In some embodiments, the CD38-binding domain or fragment thereof included in the CD38-ADR comprises a heavy chain variable region and / or a light chain variable region represented by an amino acid sequence that is at least about 99%, about 98%, about 96%, about 95%, about 90%, about 85%, and / or at least about 80% identical to each of SEQ ID NOs: 146 and 147, each of SEQ ID NOs: 148 and 149, each of SEQ ID NOs: 150 and 151, each of SEQ ID NOs: 152 and 153, each of SEQ ID NOs: 154 and 155, each of SEQ ID NOs: 156 and 157, each of SEQ ID NOs: 158 and 159, each of SEQ ID NOs: 160 and 161, each of SEQ ID NOs: 162 and 163, each of SEQ ID NOs: 164 and 165, each of SEQ ID NOs: 166 and 167, each of SEQ ID NOs: 168 and 169, each of SEQ ID NOs: 170 and 171, each of SEQ ID NOs: 172 and 173, each of SEQ ID NOs: 174 and 175, each of SEQ ID NOs: 176 and 177, each of SEQ ID NOs: 178 and 179, each of SEQ ID NOs: 180 and 181, each of SEQ ID NOs: 182 and 183, each of SEQ ID NOs: 184 and 185, each of SEQ ID NOs: 186 and 187, or each of SEQ ID NOs: 188 and 189. The selected VH and VL sequences of the exemplary CD38-binding domain are provided as numbered pairs 1-23 in Table 1A. In some embodiments, the CD38-ADR extracellular domain comprises an amino acid sequence having at least about 90% sequence identity to any of the VH and / or VL sequences of pairs 1-23 in Table 1A. In some embodiments, the CD38-ADR extracellular domain comprises an amino acid sequence having at least about 95% sequence identity to any of the VH and / or VL sequences of pairs 1-23 in Table 1A. In some embodiments, the CD38-ADR extracellular domain comprises the amino acid sequence of any of the VH and / or VL sequences of pairs 1-23 in Table 1A.

[0160]

Table 2-1

[0161]

Table 2-2

[0162] In some embodiments, the extracellular domain of 41BB-ADR or CD38-ADR may be operably linked to one or more signaling domains that mediate downstream signaling upon effector cell activation upon binding of alloreactive host immune cells to 4-1BB or CD38, respectively. In certain embodiments, the ADR comprises CD3ζ represented by an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO: 60 or a functional fragment thereof, or a CD3ζ derivative (e.g., CD3ζ1XX represented by an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO: 61 or a functional fragment thereof). In some embodiments, CD3ζ comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 60. In some embodiments, CD3ζ comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 60. In some embodiments, CD3ζ comprises the amino acid sequence of SEQ ID NO: 60. In some embodiments, the CD3ζ derivative comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 61. In some embodiments, the CD3ζ derivative comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 61. In some embodiments, the CD3ζ derivative comprises the amino acid sequence of SEQ ID NO: 61. CD3ζ mediates downstream ITAM-derived signaling during effector T or NK cell activation. Other ITAM-containing signaling domains may include those derived from DAP12, Fc receptors, and other CD3 subunits.

[0163] SEQ ID NO: 60 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (CD3ζ)

[0164] Array number 61 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR (CD3ζ1XX containing two mutations in ITAM1)

[0165] In some embodiments, the intracellular domain of the ADR comprising a signaling domain further comprises one, two, three, or more co-stimulatory domains that enhance cytokine production from effector cells expressing the ADR. The co-stimulatory domain can be derived from the intracellular signaling domain of a co-stimulatory protein including, but not limited to, CD28, CD27, 4-1BB, OX40, ICOS, CD30, HVEM, CD40, etc. In some embodiments, the ADR comprising CD3ζ further comprises a co-stimulatory domain derived from the 4-1BB intracellular domain. In some embodiments, the intracellular domain is represented by an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO: 190 or a functional fragment thereof. In some embodiments, the intracellular domain comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 190. In some embodiments, the intracellular domain comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 190. In some embodiments, the intracellular domain comprises the amino acid sequence of SEQ ID NO: 190. In one embodiment, when the ADR comprises 4-1BBL in its extracellular domain, the co-stimulatory domain of the ADR is not derived from 4-1BB.

[0166] [Table 3] (41BB internal - CD3ζ)

[0167] The intracellular domain of the ADR may be non-covalently bound to the extracellular domain of the ADR via a transmembrane domain. In some embodiments, the ADR may be of any type as long as the CD3ζ component of the ADR is located intracellularly and the extracellular domain targeting 4-1BB or CD38 is located extracellularly, and includes a transmembrane domain. In other cases, the ADR is a soluble protein that can promote cytotoxicity by binding to each ligand on activated T cells and crosslinking the TCR (e.g., ADR-CD3 T cell engager protein). When the extracellular domain is derived from a surface protein having a transmembrane domain (e.g., CD40), the ADR may include a transmembrane domain derived from its corresponding endogenous molecule. In some embodiments where the ADR molecule includes one or more co-stimulatory domains, the transmembrane domain (TM) may be derived from the same endogenous molecule having the co-stimulatory domain. Non-limiting examples of TM include those derived from CD3, CD8a, CD27, CD28, 4-1BB, OX40, and CD4.

[0168] In some embodiments, the ADR comprises a spacer between the extracellular protein and the transmembrane domain. In some embodiments, the spacer may be inert or may comprise a sequence that contributes substantially little or not at all to any function the ADR may have. In other cases, however, the spacer may comprise a sequence that enhances the function of the ADR and / or makes it detectable and / or targetable for inhibition. In certain embodiments, the spacer comprises a coded protein sequence that facilitates the detection of cells expressing the ADR. For example, the spacer may encode an Fc region or a fragment thereof that enables surface detection of cells expressing the ADR, such as by using an anti-Fc antibody. In certain embodiments, the spacer provides a separation between the ligand-binding extracellular domain and the membrane to avoid potential steric hindrance. As will be appreciated by those skilled in the art, the spacer may vary in sequence and / or length, whether or not functions other than physical separation are intended. Exemplary spacers that may be included in the ADR are generally known in the art and include, but are not limited to, IgG4 spacers, CD28 spacers, CD8 spacers, or combinations of two or more spacers. The length of the spacer may also vary from about 15 amino acids (a.a.) to about 300 amino acids or more. Non-limiting exemplary spacer peptides are represented by amino acid sequences that are at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 140 or 141. In some embodiments, the spacer peptide comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 140 or 141. In some embodiments, the spacer peptide comprises an amino acid sequence that is at least about 95% identical to SEQ ID NO: 140 or 141. In some embodiments, the spacer peptide comprises the amino acid sequence of SEQ ID NO: 140. In some embodiments, the spacer peptide comprises the amino acid sequence of SEQ ID NO: 141.

[0169] SEQ ID NO: 140 ESKYGPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL (88 amino acids)

[0170] SEQ ID NO: 141 ESKYGPPCPPCPGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKKDPK (123 - amino acid IgG4 hinge - IgG1 CH3)

[0171] In one embodiment of the 4 - 1BB - specific ADR, the 41BB - ADR is represented by an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO: 18 or SEQ ID NOs: 142 - 144. In some embodiments, the 41BB - ADR comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 18 or SEQ ID NOs: 142 - 144. In some embodiments, the 41BB - ADR comprises an amino acid sequence that is at least about 95% identical to SEQ ID NO: 18 or SEQ ID NOs: 142 - 144. In some embodiments, the 41BB - ADR comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the 41BB - ADR comprises the amino acid sequence of SEQ ID NO: 142. In some embodiments, the 41BB - ADR comprises the amino acid sequence of SEQ ID NO: 143. In some embodiments, the 41BB - ADR comprises the amino acid sequence of SEQ ID NO: 144.

[0172] [Table 4] Signal peptide - 41BBL - Spacer - CD28(TM) - CD3z (The signal peptide, spacer and TM / transmembrane domain may vary.)

[0173] [Table 5] Signal peptide - 41BBL - spacer - CD28(TM) - CD28(ICD) - CD3z (The signal peptide, spacer, and TM / transmembrane domain may vary.)

[0174] [Table 6] Signal peptide - 41BBL - spacer - CD28(TM) - CD3z1xx (The signal peptide, spacer, and TM / transmembrane domain may vary.)

[0175] [Table 7] Signal peptide - 41BBL - spacer - CD28(TM) - CD28(ICD) - CD3z1xx (The signal peptide, spacer, and TM / transmembrane domain may vary.)

[0176] In one embodiment of the CD38 - specific ADR, the CD38 - specific ADR is represented by an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO: 191. In some embodiments, the CD38 - ADR comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 191. In some embodiments, the CD38 - ADR comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 191. In some embodiments, the CD38 - ADR comprises the amino acid sequence of SEQ ID NO: 191.

[0177] [Table 8] Signal peptide - anti - CD38 VH - linker - anti - CD38 VL - CD8(TM) - 41BB(intra) - CD3z(intra) (The signal peptide, linker, and TM / transmembrane domain may vary.)

[0178] Accordingly, in some embodiments, the present application provides a solid tumor targeting scaffold that includes a polynucleotide encoding an ADR specific for 4-1BB or CD38, among other components of the scaffold, to confer on allogeneic effector cells the ability to selectively deplete activated host immune cells while enhancing effector cells due to increased proliferation in the tumor environment. Also provided in the present application are immune cells, iPSCs, and iPSC-derived effector cells that include a solid tumor targeting scaffold that includes a polynucleotide encoding a 4-1BB-specific ADR or a CD38-specific ADR, among other selected components, and effector cells including genetically engineered T cells and NK cells have alloreactive resistance to the host immune system associated with the allogeneic use of effector cells for the treatment of tumors and infectious diseases in patients.

[0179] 4. CD38 Knockout The cell surface molecule CD38 is highly upregulated in multiple hematologic malignancies, both lymphoid and myeloid, including multiple myeloma and CD20-negative B-cell malignancies, making it an attractive target for antibody therapy to deplete cancer cells. Antibody-mediated depletion of cancer cells usually results from a combination of direct induction of cell apoptosis and activation of immune effector mechanisms such as ADCC (antibody-dependent cell cytotoxicity). In addition to ADCC, immune effector mechanisms associated with therapeutic antibodies can also include antibody-dependent cell-mediated phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC).

[0180] In addition to being highly expressed in malignant cells, CD38 is also expressed in plasma cells as well as NK cells, activated T cells, and B cells. During hematopoiesis, CD38 is expressed on CD34 +It is expressed in stem cells and progenitor cells committed to the lymphoid, erythroid, and myeloid lineages, as well as in the final stages of maturation up to the plasma cell stage. CD38, a type II transmembrane glycoprotein, serves cellular functions both as a receptor and a multifunctional enzyme involved in the production of nucleotide metabolites. As an enzyme, CD38 mediates the synthesis and upregulation of NAD + CD38 catalyzes the hydrolysis of ribosomal CADPR to ADP-ribose, thereby generating the second messengers CADPR and NAADP, which stimulate calcium release from the endoplasmic reticulum and lysosomes, which are important for calcium-dependent cell adhesion processes. CD38 recognizes CD31 as a receptor and regulates cytokine release and cytotoxicity of activated NK cells. CD38 associates with cell surface proteins in lipid rafts and mediates the release of cytoplasmic Ca. 2+ It has also been reported to regulate flux and mediate signaling in lymphoid and myeloid cells.

[0181] In the treatment of malignancies, the systemic use of T cells transduced with the CD38 antigen-binding receptor has been shown to inhibit the expression of CD34 + CD38 on hematopoietic progenitor cells, monocytes, NK cells, T cells, and B cells + Fraction lysis and impaired immune effector cell function in the recipient lead to incomplete therapeutic responses and reduced or eliminated efficacy. Furthermore, in multiple myeloma patients treated with the CD38-specific antibody daratumumab, a reduction in NK cells was observed in both bone marrow and peripheral blood, whereas other immune cell types such as T and B cells were unaffected despite their expression of CD38 (Casneuf et al., Blood Advances. 2017;1(23):2105-2114).

[0182] Although not bound by theory, the present application maximally utilizes the potential of CD38-targeted cancer therapy by knocking out CD38 in effector cells, thereby providing a strategy to overcome the depletion or reduction of effector cells induced by CD38-specific antibodies and / or CD38 antigen-binding domains through fratricide. Further, since CD38 is upregulated in activated lymphocytes such as T cells and B cells, the use of CD38-specific antibodies such as daratumumab in recipients of allogeneic effector cells suppresses the activation of these recipient lymphocytes, thereby reducing and / or preventing host allogeneic rejection reactions against these effector cells, and thereby increasing the survival rate and persistence of effector cells. Accordingly, CD38-specific antibodies, secreted CD38-specific engagers, or CD38-CAR (chimeric antigen receptor) against the activation of recipient T, Treg, NK, and / or B cells can be used as an alternative to lymphodepletion using chemotherapy such as Cy / Flu (cyclophosphamide / fludarabine) prior to adoptive cell transfer.

[0183] In addition, in the presence of an anti-CD38 antibody or a CD38 inhibitor, CD38 - effector cells are used to target CD38 + When targeting T and pbNK cells, CD38 + Depletion of alloreactive cells increases NAD + (nicotinamide adenine dinucleotide, a substrate of CD38) availability and reduces NAD + consumption-related cell death, which, among other advantages, boosts effector cell responses in the immunosuppressive tumor microenvironment and supports cellular rejuvenation in aging, degenerative, or inflammatory diseases.

[0184] Furthermore, the strategies provided herein, namely, CD38 knockout, are compatible with other components and processes contemplated for establishing the solid tumor targeting framework disclosed in this application, thereby providing immune cells, iPSCs, and effector cells differentiated therefrom that contain CD38 knockout with additional framework editing. As disclosed herein, in various embodiments, the solid tumor targeting framework contained in an iPSC line or its derivatives comprises at least two of a C-X-C-motif chemokine receptor or its variant, TGFβ-SRR, and an ADR specific to 4-1BB, and CD38 knockout. In some embodiments, the provided CD38 陰 iPSC lines optionally include one or more additional engineered modalities as described herein and shown in Table 4. Accordingly, these CD38s that contain the solid tumor targeting framework 陰 Derivative effector cells, when the CD38-targeted therapeutic moiety is used with the effector cells, are protected from fratricide and allogeneic rejection, among other advantages, including improved metabolic fitness, increased resistance to oxidative stress, and induction of a protein expression program in effector cells that enhances cell activation and effector function. In addition, anti-CD38 monoclonal antibody therapy significantly depletes a patient's activated immune system without adversely affecting the patient's hematopoietic stem cell compartment. CD38 陰 Derivative cells have the ability to resist CD38 antibody-mediated depletion and can be effectively administered in combination with anti-CD38 antibodies or CD38-CARs without using toxic conditioning agents, thus reducing and / or replacing chemotherapy-based lymphocyte depletion.

[0185] In one embodiment provided herein, the CD38 knockout in the iPSC line is a biallelic knockout. In another embodiment, knocking out CD38 while inserting one or more transgenes comprising a C-X-C-motif chemokine receptor or variant thereof, TGFβ-SRR, and / or an ADR specific for 4-1BB or CD38 at a selected position in CD38 can be achieved, for example, by a CD38-targeted knock-in / knockout (CD38-KI / KO) construct. In some embodiments of the construct, the construct comprises a pair of CD38-targeting homology arms for site-specific insertion into the CD38 locus. In some embodiments, the pre-selected targeting site is within an exon of CD38. The CD38-KI / KO constructs provided herein allow the transgene to be expressed either under the CD38 endogenous promoter or under an exogenous promoter included in the construct. When two or more transgenes are inserted at a selected position in the CD38 locus, a linker sequence, such as a 2A linker or an IRES, is placed between any two transgenes. The 2A linker encodes self-cleaving peptides derived from FMDV, ERAV, PTV-I, and TaV (referred to as "F2A", "E2A", "P2A", and "T2A", respectively), enabling the production of separate proteins from a single translation. In some embodiments, the construct includes an insulator to reduce the risk of transgene and / or exogenous promoter silencing. The exogenous promoter included in the CD38-KI / KO construct can be a CAG, or other constitutive, inducible, temporal-specific, tissue-specific, or cell-type-specific promoter including, but not limited to, CMV, EF1α, PGK, and UBC.

[0186] In various embodiments, the iPSC differentiates as directed to mesodermal cells with definitive hematopoietic endothelial (HE) potential, definitive HE, CD34 +It is possible to produce functional induced hematopoietic cells including, but not limited to, hematopoietic cells, hematopoietic stem cells and progenitor cells, multipotent progenitor (MPP) cells, T cell progenitor cells, NK cell progenitor cells, myeloid cells, neutrophil progenitor cells, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, and macrophages. In some embodiments, the CD38-negative effector cells are NK lineage cells derived from iPSCs. In some embodiments, the CD38-negative effector cells are T lineage cells derived from iPSCs. In some embodiments, the iPSCs and their derivative cells are CD38 陰 comprise a solid tumor-targeting backbone comprising at least two of a polynucleotide encoding a C-X-C motif chemokine receptor or a variant thereof, a polynucleotide encoding TGFβ-SRR, and a polynucleotide encoding 41BB-ADR, and optionally comprise one or more additional genome edits described herein.

[0187] 5. CD16 knock-in CD16 has been identified as two isoforms, the Fc receptor FcγRIIIa (CD16a; NM_000569.6) and FcγRIIIb (CD16b; NM_000570.4). CD16a is a transmembrane protein expressed by NK cells that binds to monomeric IgG attached to target cells to activate NK cells and promote antibody-dependent cell cytotoxicity (ADCC). CD16b is exclusively expressed by human neutrophils. As used herein, "high-affinity CD16", "non-cleavable CD16", or "high-affinity non-cleavable CD16" (abbreviated as hnCD16) refers to various CD16 variants. Wild-type CD16 has low affinity and is subject to extracellular domain shedding, a proteolytic cleavage process that regulates the cell surface density of various cell surface molecules on leukocytes when NK cells are activated. F176V (also called F158V in some publications) is an exemplary CD16 polymorphic variant with high affinity, while the S197P variant is an example of a genetically engineered non-cleavable version of CD16. Engineered CD16 variants containing both F176V and S197P have high affinity and are non-cleavable, which is described in more detail in International Publication No. WO 2015 / 148926, the complete disclosure of which is incorporated herein by reference. In addition, chimeric CD16 receptors in which the extracellular domain of CD16 is essentially replaced by at least a portion of the extracellular domain of CD64 can also achieve the desirable properties of high affinity and non-cleavability of CD16 receptors that can perform ADCC. In some embodiments, the substituted extracellular domain of the chimeric CD16 comprises one or more of the EC1, EC2, and EC3 exons of CD64 (UniPRotKB_P12314 or an isoform or polymorphic variant thereof).

[0188] Thus, various embodiments of exogenous CD16 introduced into cells include functional CD16 variants and chimeric receptors thereof. In some embodiments, the functional CD16 variant is a high-affinity non-cleavable CD16 receptor (hnCD16). In some embodiments, hnCD16 includes both F176V and S197P, and in some embodiments, includes F176V and the cleavage region is excluded. In some embodiments, hnCD16 has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage therebetween of identity when compared to any of SEQ ID NOs: 19, 20, and 21 (each of which includes at least a portion of the CD64 extracellular domain). In some embodiments, hnCD16 includes an amino acid sequence that is at least 90% identical to any of SEQ ID NOs: 19-21, and optionally, one or more of F176V, S197P, and at least a portion of the CD64 extracellular domain. In some embodiments, hnCD16 includes an amino acid sequence that is at least 95% identical to any of SEQ ID NOs: 19-21, and optionally, one or more of F176V, S197P, and at least a portion of the CD64 extracellular domain. In some embodiments, hnCD16 includes the amino acid sequence of SEQ ID NO: 19. In some embodiments, hnCD16 includes the amino acid sequence of SEQ ID NO: 20. In some embodiments, hnCD16 includes the amino acid sequence of SEQ ID NO: 21.

[0189]

Table 9

[0190]

Table 10

[0191]

Table 11

[0192] Accordingly, effector cells or iPSCs genetically engineered to contain a solid tumor targeting scaffold comprising exogenous CD16 or a variant thereof are provided herein, among other edits contemplated and described herein. The effector cells can be cells derived from a primary source, or cells derived from iPSC differentiation, or the genetically engineered iPSCs can differentiate into derivative effector cells containing exogenous CD16 or a variant thereof introduced into the iPSCs. In some embodiments, the exogenous CD16 is high-affinity non-cleavable CD16 (hnCD16). In some embodiments, the exogenous CD16 comprises at least a portion of the CD64 extracellular domain. In some embodiments, the exogenous CD16 is in the form of a CD16-based chimeric Fc receptor (CFcR) that includes a transmembrane domain, a stimulatory domain, and / or a signaling domain not derived from CD16.

[0193] In some embodiments, the primary-derived or derived effector cells comprising exogenous CD16 or a variant thereof are NK lineage cells. In some embodiments, the primary-derived or derived effector cells comprising exogenous CD16 or a variant thereof are T lineage cells. In some embodiments, the exogenous CD16 or a functional variant thereof included in the iPSC or effector cells has high affinity in such binding to a ligand that induces downstream signaling upon binding. Non-limiting examples of ligands that bind to exogenous CD16 or a functional variant thereof include not only ADCC antibodies or fragments thereof, but also bispecific, trispecific, or multispecific engagers or binders that recognize the extracellular binding domain of CD16 or CD64 of the exogenous CD16. Examples of bispecific, trispecific, or multispecific engagers or binders are further described below in this application. Thus, at least one of the aspects of this application is exogenous CD16 expressed on derived effector cells comprising a solid tumor targeting scaffold in an amount sufficient for therapeutic use in the treatment of a condition, disease, or infection further detailed in this application, via which one or more pre-selected ADCC antibodies are pre-loaded, and the exogenous CD16 comprises the extracellular binding domain of CD64 or CD16 having F176V and S197P.

[0194] In some other embodiments, the exogenous CD16 comprises a chimeric Fc receptor (CFcR) based on CD16 or a variant thereof. The chimeric Fc receptor (CFcR) is produced to include a non-natural transmembrane domain, a non-natural stimulatory domain, and / or a non-natural signaling domain by modifying or replacing the native CD16 transmembrane and / or intracellular domain. As used herein, the term "non-natural" means that the transmembrane domain, stimulatory domain, or signaling domain is derived from a different receptor other than the receptor providing the extracellular domain. In the exemplification herein, the CD16-based or its variant-based CFcR does not have a transmembrane domain, stimulatory domain, or signaling domain derived from CD16. In some embodiments, the exogenous CD16-based CFcR comprises a non-natural transmembrane domain derived from CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA4, PD1, LAG3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, or a T cell receptor polypeptide. In some embodiments, the exogenous CD16-based CFcR comprises a non-natural stimulatory / inhibitory domain derived from CD27, CD28, 4-1BB, OX40, ICOS, PD1, LAG3, 2B4, BTLA, DAP10, DAP12, CTLA4, or NKG2D polypeptide. In some embodiments, the exogenous CD16-based CFcR comprises a non-natural signaling domain derived from CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137 (4-1BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or NKG2D polypeptide. In one embodiment of the CD16-based CFcR, the chimeric Fc receptor provided comprises a transmembrane domain and a signaling domain both derived from one of IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, and NKG2D polypeptides.One embodiment of the CD16-based chimeric Fc receptor comprises the transmembrane domain of NKG2D, the stimulatory domain of 2B4, and the signaling domain of CD3ζ, wherein the extracellular domain of the chimeric Fc receptor is derived from the full-length or partial sequence of the extracellular domain of CD64 or CD16, and the extracellular domain of CD16 comprises F176V and S197P. Another exemplary embodiment of the CD16-based chimeric Fc receptor comprises the transmembrane domain and the signaling domain of CD3ζ, wherein the extracellular domain of the chimeric Fc receptor is derived from the full-length or partial sequence of the extracellular domain of CD64 or CD16, and the extracellular domain of CD16 comprises F176V and S197P.

[0195] The various embodiments of the CD16-based chimeric Fc receptor as described above can bind with high affinity to the Fc region of an antibody or its fragment, or to a bispecific, trispecific, or multispecific engager or binder. Upon binding, the stimulatory domain and / or signaling domain of the chimeric receptor enables effector cell activation and cytokine secretion, and the killing of tumor cells targeted by the antibody or a bispecific, trispecific, or multispecific engager or binder having a tumor antigen-binding component and an Fc region. Without being limited by theory, through the non-native transmembrane, stimulatory, and / or signaling domains of the CD16-based chimeric Fc receptor, or through the binding of an engager to the extracellular domain, the CFcR can contribute to the killing ability of effector cells and increase the proliferation and / or potential for proliferation of effector cells. Antibodies and engagers can bring tumor cells expressing an antigen and effector cells expressing CFcR into proximity, which also contributes to enhanced tumor cell killing. Exemplary tumor antigens for bispecific, trispecific, multispecific engagers or binders include, but are not limited to, B7H3, BCMA, CD10, CD19, CD20, CD22, CD24, CD30, CD33, CD34, CD38, CD44, CD79a, CD79b, CD123, CD138, CD179b, CEA, CLEC12A, CS-1, DLL3, EGFR, EGFRvIII, EPCAM, FLT-3, FOLR1, FOLR3, GD2, gpA33, HER2, HM1.24, LGR5, MSLN, MCSP, MICA / B, PSMA, PAMA, P-cadherin, and ROR1. Some non-limiting exemplary bispecific, trispecific, multispecific engagers or binders suitable for binding effector cells expressing a CD16-based CFcR when attacking tumor cells include CD16 (or CD64)-CD30, CD16 (or CD64)-BCMA, CD16 (or CD64)-IL15-EPCAM, and CD16 (or CD64)-IL15-CD33.

[0196] Unlike endogenous CD16, which is cleaved from the cell surface following NK cell activation in primary NK cells, various non-cleavable versions of CD16 in derived NK cells avoid CD16 shedding and maintain a certain level of expression. In induced NK cells, non-cleavable CD16 increases the expression of TNFα and CD107a, which are indicators of improved cell function. Non-cleavable CD16 also enhances antibody-dependent cell cytotoxicity (ADCC) and the binding of bispecific, trispecific, or multispecific engagers. ADCC is a mechanism of NK cell-mediated lysis via the binding of CD16 to antibody-coated target cells. The additional high-affinity properties of hnCD16 introduced into derived NK cells also enable in vitro loading of ADCC antibodies onto NK cells via hnCD16 before administering the cells to a subject in need of cell therapy. As presented herein, hnCD16 may, in some embodiments, include F176V and S197P, or may include a full-length or partial-length extracellular domain derived from CD64 as exemplified by SEQ ID NO: 19, 20, or 21, or may further include at least one of a non-native transmembrane domain, a stimulatory domain, and a signaling domain. As disclosed, the present application also provides derived NK cells or a population of such cells comprising a solid tumor-targeting scaffold pre-loaded with one or more pre-selected ADCC antibodies in an amount sufficient for therapeutic use in the treatment of a condition, disease, or infection, as will be described in more detail in the present application. In some embodiments, the pre-loaded CD38 antibody is daratumumab. In some embodiments, induced NK cells comprising a solid tumor-targeting scaffold comprising exogenous CD16 or a variant thereof further comprise at least two of the ADRs specific for C-X-C-motif chemokine receptor or a variant thereof, TGFβ-SRR, and 4-1BB provided herein. In some embodiments, induced NK cells comprising a solid tumor-targeting scaffold comprising at least two of the ADRs specific for C-X-C-motif chemokine receptor or a variant thereof, TGFβ-SRR, and 4-1BB, and exogenous CD16 or a variant thereof further comprise a CAR.In some embodiments, the induced NK cells are pre-loaded with one or more of an anti-HER2 antibody (e.g., trastuzumab, pertuzumab), an anti-EGFR antibody (e.g., cetuximab), or an anti-PDL1 antibody (e.g., avelumab).

[0197] Unlike primary NK cells, mature T cells derived from a primary source (i.e., a natural / native source such as peripheral blood, cord blood, or other donor tissue) do not express CD16. It was previously unexpected that iPSCs containing expressed exogenous non-cleavable CD16 could not only express exogenous CD16 but also differentiate into functional derived T cells that can perform functions through the acquired ADCC mechanism without impairing the developmental biology of T lineage cells. This ADCC acquired in the derived T lineage cells can be further used as an approach to rescue antigen escape, which often occurs in dual targeting and / or CAR-T cell therapy, where tumors relapse with reduced or lost expression of the CAR-T target antigen, or mutant antigens that avoid recognition by the CAR (chimeric antigen receptor). When the induced T lineage cells contain ADCC acquired through the expression of exogenous CD16 (including functional variants and CD16-based CFcRs), and the antibody targets a tumor antigen different from the tumor antigen targeted by the CAR, the antibody can be used to rescue CAR-T antigen escape and reduce or prevent recurrence or relapse of the target tumor, which is often seen in CAR-T therapy. Such strategies to reduce and / or prevent antigen escape while achieving dual targeting are equally applicable to NK cells expressing one or more CARs.

[0198] Accordingly, the present application provides derived T lineage cells comprising a solid tumor targeting scaffold comprising exogenous CD16 or a variant thereof. In some embodiments, the solid tumor targeting scaffold derivative comprised in the T lineage cells obtained herein comprises exogenous CD16 and at least two of an ADR specific for a C-X-C-motif chemokine receptor or a variant thereof, TGFβ-SRR, and 4-1BB. In other embodiments, the derived T lineage cells obtained herein comprise a CAR in addition to the solid tumor targeting scaffold. In some embodiments, the exogenous CD16 comprised in the solid tumor targeting scaffold comprised in the derived T lineage cells is hnCD16 comprising F176V and S197P. In some other embodiments, the hnCD16 comprised in the solid tumor targeting scaffold comprises a complete or partial extracellular domain derived from CD64 as exemplified by SEQ ID NO: 19, 20, or 21, or may further comprise at least one of a non-native transmembrane domain, a stimulatory domain, and a signaling domain. As described herein, such derived T lineage cells have an acquired mechanism of targeting tumors with monoclonal antibodies mediated by ADCC to enhance the therapeutic effect of the antibodies. As disclosed, the present application also provides derived T lineage cells or a cell population thereof comprising a solid tumor targeting scaffold pre-loaded with one or more pre-selected ADCC antibodies in an amount sufficient for therapeutic use in the treatment of a condition, disease, or infection, as further described below.

[0199] As further provided, a solid tumor targeting scaffold, optionally a CAR, and an exogenous CD16 or a variant thereof (''CD16'' in Table 4) 外Cells or populations thereof comprising ") and may further comprise one or more additional engineered modalities described herein and / or shown in Table 4. Further provided in this application is a master cell bank comprising sorted single cells and expanded clones of engineered iPSCs having at least one phenotype as provided herein, including but not limited to a solid tumor targeting backbone comprising exogenous CD16 or a variant thereof. This cell bank provides a platform for further iPSC engineering and a renewable source for manufacturing off-the-shelf engineered homogeneous cell therapy products, including but not limited to induced NK cells and T cells, which can be produced in large quantities at a significant scale in a manner that is clearly defined, homogeneous in composition, and cost-effective.

[0200] 6. Exogenously introduced cytokine signaling complex By avoiding systemic high-dose administration of clinically appropriate cytokines, the risk of dose-limiting toxicity due to such actions is reduced and cytokine-mediated cellular autonomy is established. To achieve lymphocyte autonomy without the need for additional administration of soluble cytokines, a cytokine signaling complex comprising one or more partial-length or full-length peptides of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and / or their corresponding receptors is introduced into cells as part of a solid tumor-targeting scaffold to enable cytokine signaling with or without the expression of the cytokine itself, thereby reducing the risk of cytokine toxicity and maintaining or improving cell growth, proliferation, expansion, and / or effector function. In some embodiments, the introduced cytokine and / or its respective native or modified receptor (signaling complex) for cytokine signaling is expressed on the cell surface. In some embodiments, cytokine signaling is constitutively activated. In some embodiments, activation of cytokine signaling is inducible. In some embodiments, activation of cytokine signaling is transient and / or temporary. In some embodiments, transient / temporary expression of cell surface cytokine / cytokine receptor is via an expression construct carried by a retrovirus, Sendai virus, adenovirus, episome, minicircle, or RNA including mRNA.

[0201] Provided herein are various construct designs for introducing into a cell a protein complex for the signaling of one, two, or more cytokines, including but not limited to IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21. For example, in embodiments where the signaling complex is for IL15, the transmembrane (TM) domain can be native to the IL15 receptor or modified or replaced with the transmembrane domain of any other membrane-bound protein. In various embodiments, the cytokine signaling complex comprises an IL15 receptor fusion (IL15RF) that includes the full-length or partial-length of IL15 and the full-length or partial-length of the IL15 receptor (IL15 receptor, IL15R). In some embodiments, IL15 and IL15Rα are co-expressed using a self-cleaving peptide that mimics trans-presentation of IL15 without eliminating cis-presentation of IL15. In other embodiments, IL15Rα is fused to IL15 at the C-terminus via a linker, mimicking trans-presentation without eliminating cis-presentation of IL15 and ensuring that IL15 is membrane-bound. In other embodiments, IL15Rα with a truncated intracellular domain is fused to IL15 at the C-terminus via a linker, mimicking trans-presentation of IL15, maintaining membrane-binding of IL15, and eliminating cis-presentation and / or other possible signaling pathways mediated by normal IL15R via its intracellular domain. In other embodiments, IL15Rα is fused to IL15 lacking an intracellular domain (IL15Δ) as described in International Publication Nos. 2019 / 191495 and 2019 / 126748 (the entire disclosures of each are incorporated herein by reference).

[0202] In various embodiments, the shortened construct comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 22. In some embodiments, the IL15 / IL15Rα comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 22. In some embodiments, the IL15 / IL15Rα comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 22. In some embodiments, the IL15 / IL15Rα comprises the amino acid sequence of SEQ ID NO: 22. In one embodiment of the shortened IL15 / IL15Rα, the construct does not include the last 4 amino acid residues (KSRQ) of SEQ ID NO: 22 and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 23. In some embodiments, the IL15 / IL15Rα comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 23. In some embodiments, the IL15 / IL15Rα comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 23. In some embodiments, the IL15 / IL15Rα comprises the amino acid sequence of SEQ ID NO: 23.

[0203]

Table 12

[0204]

Table 13

[0205] In yet other embodiments, the cytoplasmic domain of IL15Rα can be omitted without adversely affecting the autonomous properties of effector cells armed with IL15. In other embodiments, all of IL15Rα is removed, except that at one end it is fused to IL15 and at the other to a transmembrane domain (mb-Sushi), optionally with a linker between the Sushi domain and the transmembrane domain. The fused IL15 / mb-Sushi is expressed on the cell surface via the transmembrane domain of the membrane-bound protein. When only the desirable trans-presentation of IL15 is retained, unwanted signaling via IL15Rα, including cis-presentation, is eliminated. In some embodiments, the component comprising IL15 fused to the Sushi domain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 24. In some embodiments, the component comprising IL15 fused to the Sushi domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 24. In some embodiments, the component comprising IL15 fused to the Sushi domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 24. In some embodiments, the component comprising IL15 fused to the Sushi domain comprises the amino acid sequence of SEQ ID NO: 24.

[0206]

Table 14

[0207] In other embodiments, the native or modified IL15Rβ is fused to IL15 at the C-terminus via a linker, enabling constitutive signaling and maintaining IL15 membrane binding and trans-presentation. In other embodiments, the native or modified common receptor γC is fused to IL15 at the C-terminus via a linker for constitutive cytokine signaling and membrane-bound trans-presentation. The common receptor γC is also referred to as the common gamma chain or CD132 and is also known as interleukin 2 receptor subunit gamma or IL2RG. γC is a cytokine receptor subunit common to the receptor complexes of many interleukins, including but not limited to, the IL2, IL4, IL7, IL9, IL15 and IL21 receptors. In other embodiments, engineered IL15Rβ that forms homodimers in the absence of IL15 is useful for generating constitutive cytokine signaling.

[0208] In various other embodiments, the cytokine signaling complex comprises an IL7 receptor fusion (IL7RF) that includes the full length or partial length of IL7, and the full length or partial length of the IL7 receptor. The transmembrane (TM) domain can be native to the IL7 receptor or may be modified or substituted with the transmembrane domain of any other membrane-bound protein. In one embodiment, the native (or wild-type) or modified IL7R may be fused to IL7 at the C-terminus via a linker, allowing for constitutive signaling and maintaining membrane-bound IL7. In some embodiments, such constructs include an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 25, and the transmembrane domain, signal peptide and linker are flexible and can vary in length and / or sequence. In some embodiments, the IL7 construct includes an amino acid sequence that is at least 90% identical to SEQ ID NO: 25, and the transmembrane domain, signal peptide and linker are flexible and can vary in length and / or sequence. In some embodiments, the IL7 construct includes an amino acid sequence that is at least 95% identical to SEQ ID NO: 25, and the transmembrane domain, signal peptide and linker are flexible and can vary in length and / or sequence. In some embodiments, IL7 includes the amino acid sequence of SEQ ID NO: 25.

[0209]

Table 15

[0210] In another embodiment, the native or modified common receptor γC is fused to IL7 at the C-terminus via a linker for a constitutive and membrane-bound cytokine signaling complex. Additionally, engineered IL7R that forms homodimers in the absence of IL7 is also useful for generating constitutive signaling of the cytokine.

[0211] One of ordinary skill in the art will understand that the signal peptides and linker sequences described above are exemplary and in no way limit the variations suitable for use as signal peptides or linkers. There are many suitable signal peptides or linker sequences known and available to those of ordinary skill in the art. One of ordinary skill in the art will understand that the signal peptide and / or linker sequence can be replaced with another sequence without altering the activity of the functional peptide directed by the signal peptide or linked by the linker.

[0212] In iPSCs and derivative cells therefrom that contain both a CAR and an exogenous cytokine and / or cytokine receptor signaling (cytokine signaling complex, or "IL"), the CAR and IL can be expressed as separate constructs or co-expressed in a bicistronic construct that contains both the CAR and IL. In some further embodiments, the signaling complex can be linked to either the 5' or 3' end of the CAR expression construct via a self-cleaving 2A coding sequence. Thus, the IL signaling complex (e.g., the IL7 signaling complex) and the CAR can be within a single open reading frame (ORF). In one embodiment, the signaling complex is included in a CAR-2A-IL or IL-2A-CAR construct. When CAR-2A-IL or IL-2A-CAR is expressed, the self-cleaving 2A peptide enables the expressed CAR and IL to dissociate, and then the dissociated IL can be presented on the cell surface with the transmembrane domain fixed to the cell membrane. The bicistronic design of CAR-2A-IL or IL-2A-CAR enables the coordinated expression of the CAR and the IL signaling complex under the same regulatory mechanism that can be selected for incorporation, such as an inducible promoter or a promoter with temporal or spatial specificity for the expression of a single ORF, in terms of both timing and quantity. Self-cleaving peptides are found in members of the Picornaviridae family, including the Aphthovirus genus such as foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV), Thosea asigna virus (TaV), and porcine tescho virus-1 (PTV-I) (Donnelly, ML, et al, J. Gen. Virol, 82, 1027-101(2001); Ryan, MD, et al., J. Gen. Virol., 72, 2727-2732(2001)), as well as the Cardiovirus genus such as Theiler's murine encephalomyelitis virus and encephalomyocarditis virus.The 2A peptides derived from FMDV, ERAV, PTV-I, and TaV are sometimes referred to as "F2A", "E2A", "P2A", and "T2A", respectively.

[0213] The bicistronic CAR-2A-IL or IL-2A-CAR disclosed herein also contemplates the expression of any other cytokine provided herein, such as IL2, IL4, IL6, IL9, IL10, IL11, IL12, IL18, and IL21. In some embodiments, the bicistronic CAR-2A-IL or IL-2A-CAR is for the expression of one or more of IL2, IL4, IL7, IL9, IL15, and IL21.

[0214] In some embodiments, iPSCs and their derived effector cells comprising any one of the genotypes of Table 4 may further comprise disruption of at least one of TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, RFX5, RFXAP, RAG1, and any gene within the chromosomal region 6p21, or the introduction of at least one of HLA-E, 4-1BBL, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A R, TCR, Fc receptor, antibody, and surface trigger receptor for coupling with bispecific, multispecific, or universal engagers.

[0215] Thus, in various embodiments, the cytokine IL15 or IL7 and / or their receptors may be introduced into iPSCs using one or more of the above construct designs, or introduced into their derived cells upon iPSC differentiation. Further provided herein are induced pluripotent cells (iPSCs), clonal iPSCs, clonal iPSC lines, or iPSC-derived cells comprising a solid tumor-targeting backbone comprising two or more of a polynucleotide encoding a C-X-C motif chemokine receptor or a variant thereof, a polynucleotide encoding TGFβ-SRR, and a polynucleotide encoding 41BB-ADR, and optionally two or more of a polynucleotide encoding a cytokine signaling complex and / or one or more engineered modalities as disclosed herein. Also provided is a master cell bank comprising sorted single cells and expanded clonal engineered iPSCs having a solid tumor-targeting backbone comprising two or more of a polynucleotide encoding a C-X-C motif chemokine receptor or a variant thereof, a polynucleotide encoding TGFβ-SRR, and a polynucleotide encoding 41BB-ADR, and optionally two or more of a polynucleotide encoding a cytokine signaling complex and / or a polynucleotide encoding one or more engineered modalities, wherein the cytokine signaling complex comprises a partial or complete peptide of an exogenously expressed cytokine and / or its receptor on the cell surface, and the cell bank provides a platform for further iPSC engineering and a renewable source for manufacturing off-the-shelf engineered homogeneous cell therapy products that can be mass-produced at a substantial scale in a manner that is well-defined, uniform, and cost-effective.

[0216] 7. Deficiency of HLA-I- and HLA-II- To avoid the problem of allogeneic rejection, multiple HLA class I and class II proteins need to match for histocompatibility of allogeneic recipients. Provided herein are iPSC cell lines in which the expression of HLA class I and / or HLA class II proteins is eliminated or substantially reduced, and their derived cells differentiated therefrom. HLA class I deficiency can be achieved by functional deletion of any region of the HLA class I locus (chromosome 6p21), or deletion or disruption of HLA class I-related genes including, but not limited to, the beta-2 microglobulin (B2M) gene, TAP1 gene, TAP2 gene, and tapasin. For example, the B2M gene encodes a common subunit essential for cell surface expression of all HLA class I heterodimers. B2M-negative cells are HLA-I deficient. HLA class II deficiency can be achieved by functional deletion or disruption of HLA class II-related genes including, but not limited to, RFXANK, CIITA, RFX5, and RFXAP. CIITA is a transcriptional coactivator and functions through activation of the transcription factor RFX5 required for expression of class II proteins. CIITA-negative cells are HLA-II deficient. Thus, the present application provides, for example, iPSCs and their derived cells comprising HLA-I and / or HLA-II deficiency due to lack of expression of B2M and / or CIITA, and the obtained derived effector cells enable allogeneic cell therapy by eliminating the need for MHC (major histocompatibility complex) matching and avoiding recognition and killing by host (allogeneic) T cells.

[0217] Furthermore, the lack of HLA class I expression triggers lysis by host NK cells. Thus, in addition to the above-described approach of CD38 conditioning to remove activated CD38-expressing host NK cells, HLA-E, HLA-G, or other non-classical HLA-I proteins can be optionally knocked in to overcome this "self-loss" response and avoid the killing of HLA-I-deficient effector cells derived from iPSCs that are recognized and manipulated by NK cells. In one embodiment, the provided HLA-I-deficient iPSCs and their derived cells further comprise an HLA-G knock-in.

[0218] Alternatively, in one embodiment, the provided HLA-I-deficient iPSCs and their derived cells further comprise one or both of a CD58 knockout and a CD54 knockout. CD58 (or LFA-3) and CD54 (or ICAM-1) are adhesion proteins that initiate signal-dependent cell interactions and promote the migration of cells, including immune cells. It has previously been shown that disruption of CD58 and / or CD54 effectively reduces the sensitivity of HLA-I-deficient iPSC-derived effector cells to allogeneic NK cell killing. CD58 knockout results in higher efficiency in reducing allogeneic NK cell activation than CD54 knockout, and double knockout of both CD58 and CD54 has been shown to result in the most enhanced reduction of NK cell activation. In some observations, the CD58 and CD54 double knockout is more effective than HLA-G overexpression on HLA-I-deficient cells in overcoming the "self-loss" effect.

[0219] As provided herein, in some embodiments, iPSCs and their derivative cells comprise a solid tumor targeting scaffold that includes two or more of C-X-C-motif chemokine receptor or a variant thereof, TGFβ-SRR, and 41BB-ADR, and the cells are HLA-I and / or HLA-II deficient. In some embodiments, the HLA-I and / or HLA-II deficient iPSCs and their derivative cells are CD58 negative. In some other embodiments, the HLA-I and / or HLA-II deficient iPSCs and their derivative cells are CD54 negative. In still some other embodiments, the HLA-I and / or HLA-II deficient iPSCs and their derivative cells are CD54 negative and CD58 negative. Further, in some embodiments of iPSCs and their derivative cells that include the solid tumor targeting scaffold described herein, the cells are HLA-I and / or HLA-II deficient and have an exogenous polynucleotide encoding HLA-G. In some embodiments of iPSCs and their derivative cells that include the solid tumor targeting scaffold described herein, the cells are HLA-I and / or HLA-II deficient and CD54 negative. In still some other embodiments of iPSCs and their derivative cells that include the solid tumor targeting scaffold as described herein, and optionally CD38 knockout, exogenous CD16 or a variant thereof, and CAR, the cells are HLA-I and HLA-II deficient and both CD58 negative and CD54 negative.

[0220] In some embodiments, by expressing an inactivated CAR that targets surface proteins upregulated in activated recipient immune cells, the manipulation for HLA-I and / or HLA-II deficiency can be bypassed or left intact to avoid allogeneic rejection. In some embodiments, surface proteins upregulated in activated recipient immune cells include, but are not limited to, CD38, CD25, CD69, CD44, 4-1BB, OX40, or CD40L. When a cell expresses such an inactivated CAR, it is preferred that the cell does not express or has a knockout of the same surface protein targeted by the CAR. In some embodiments, the inactivated CAR includes at least one of CD38-CAR, CD25-CAR, CD69-CAR, CD44-CAR, 4-1BB-CAR, OX40-CAR, and CD40L-CAR.

[0221] Further provided in the present application is a master cell bank comprising sorted single cells and expanded clonally engineered iPSCs having at least one phenotype as provided herein, including but not limited to the solid tumor targeting scaffolds and HLA modifications (Table 4, "HLA": HLA-E or HLA-G knock-in with or without, or HLA-I and / or HLA-II deficiency with knockout of one or both of CD58 and CD54) described herein. This cell bank provides a platform for further iPSC engineering and a renewable source for manufacturing off-the-shelf engineered homogeneous cell therapy products, including but not limited to derived NK and T cells, which can be mass-produced on a significant scale in a manner that is clearly defined, homogeneous in composition, and cost-effective.

[0222] 8. Cell Surface Chimeric Fusion Receptor (CFR) Effector cells engineered to express CFR are enabled to initiate appropriate signaling cascades through CFR binding to agonists selected for enhancing the therapeutic properties of the effector cells. Such enhanced effector cell therapeutic properties include, but are not limited to, increased activation and cytotoxicity, acquired dual targeting ability, extended persistence, improved trafficking and tumor infiltration, enhanced ability to prime, activate or mobilize bystander immune cells at the tumor site, enhanced ability to resist immunosuppression, improved ability to rescue tumor antigen escape, and / or controlled cell signaling feedback, metabolism and apoptosis.

[0223] Accordingly, in some embodiments, the present application provides iPSCs and derived cells therefrom that include, among other edits, a chimeric fusion receptor (CFR) that includes an extracellular domain, a transmembrane domain, and an intracellular domain, where the extracellular domain, transmembrane domain, and intracellular domain do not include any endoplasmic reticulum (ER) retention signals or endocytosis signals. The extracellular domain of the CFR is for initiating signal transduction upon binding to an engager, the transmembrane domain is for membrane anchoring of the CFR, and the intracellular domain includes at least one signaling domain that modulates (i.e., activates or inactivates) a signaling pathway selected to enhance cell therapy properties including, but not limited to, tumor killing, persistence, motility, differentiation, neutralization of the tumor microenvironment (TME), and / or controlled apoptosis. Elimination of the ER retention signal from the CFR enables cell surface presentation of the CFR by itself when expressed, and elimination of the endocytosis signal from the CFR reduces CFR internalization and sub-surface control. It is important to select domain components that have neither an ER retention nor an endocytosis signal, or to use molecular engineering tools to remove ER retention or endocytosis signals from selected components of the CFR. Additionally, the domains of the CFR provided by some embodiments herein are modular, which means that for a given intracellular domain of the CFR, the extracellular domain of the CFR is switchable depending on the binding specificity of the selected agonist, such as an antibody, BiTE, TRikE, or any other type of engager, used with the CFR, and for a given extracellular domain and specificity-matched agonist, the intracellular domain is switchable depending on the signaling pathway desired to be activated. Additionally, the transmembrane domain according to some embodiments is switchable for a given extracellular domain and / or a given intracellular domain as long as the transmembrane domain does not include any ER retention signals or endocytosis signals.

[0224] In some embodiments, the extracellular domain of the CFR applicable to the cells described herein comprises the full or partial length of the extracellular portion of a protein involved in cell-cell signaling or interaction. In some embodiments, the extracellular domain of the CFR comprises the full or partial length of the extracellular portion of CD3ε, CD3γ, CD3δ, CD28, CD5, CD16, CD64, CD32, CD33, CD89, NKG2C, NKG2D, or any functional variant thereof, or a combination and chimeric form. In some embodiments, the extracellular domain of the CFR is recognized by at least an agonist, for example, an antibody or an engager (e.g., BiTE, BiKE or TriKE) comprising a binding domain specific for an epitope contained in the extracellular domain of the CFR. In some embodiments, the antibody or engager used with the CFR-expressing cells binds to at least one extracellular epitope of the CFR, and the CFR comprises the full or partial length of the extracellular portion of CD3ε, CD3γ, CD3δ, CD28, CD5, CD16, CD64, CD32, CD33, CD89, NKG2C, NKG2D, or any functional variant or combination thereof / chimeric form. In some embodiments, the engager recognizes at least one tumor antigen comprising B7H3, BCMA, CD10, CD19, CD20, CD22, CD24, CD30, CD33, CD34, CD38, CD44, CD79a, CD79b, CD123, CD138, CD179b, CEA, CLEC12A, CS-1, DLL3, EGFR, EGFRvIII, EPCAM, FLT-3, FOLR1, FOLR3, GD2, gpA33, HER2, HM1.24, LGR5, MSLN, MCSP, MICA / B, PSMA, PAMA, P-cadherin, or ROR1. In some embodiments of the CFR extracellular domain, neither an ER retention signal nor an endocytosis signal is present, or is removed or excluded from the CFR extracellular domain using genetic engineering methods.

[0225] In some embodiments, the extracellular domain of CFR comprises the full-length or partial-length of the extracellular portion of CD3ε, CD3γ, CD3δ or any functional variant or combination / chimeric form thereof to utilize CD3-based agonists. Non-limiting exemplary CD3-based agonists including, but not limited to, antibodies or engagers are CD3×CD19, CD3×CD20, CD3×CD33, blinatumomab, catumaxomab, ertumaxomab, RO6958688, AFM11, MT110 / AMG110, MT111 / AMG211 / MEDI-565, AMG330, MT112 / BAY2010112, MOR209 / ES414, MGD006 / S80880, MGD007, and / or FBTA05. In some embodiments, the extracellular domain of CFR comprises the full-length or partial-length of the extracellular portion of NKG2C or any functional variant thereof to utilize NKG2C-based agonists. Non-limiting exemplary NKG2C-based agonists including, but not limited to, antibodies or engagers are NKG2C-IL15-CD33, NKG2C-IL15-CD19, and / or NKG2C-IL15-CD20 trispecific engagers. In some other embodiments, the extracellular domain of CFR comprises the full-length or partial-length of the extracellular portion of CD28 or any functional variant thereof to utilize CD28-based agonists. Non-limiting exemplary CD28-based agonists including, but not limited to, antibodies or engagers include at least one of 15E8, CD28.2, CD28.6, YTH913.12, 37.51, 9D7 (TGN1412), 5.11A1, ANC28.1 / 5D10, and / or 37407.

[0226] In some embodiments, the extracellular domain of the CFR comprises the full-length or partial-length of the extracellular portion of CD16, CD64, or any functional variant or combination / chimeric form thereof, to utilize CD16- or CD64-based agonists. Non-limiting exemplary CD16- or CD64-based agonists, including but not limited to antibodies or engagers, include IgG antibodies, or CD16- or CD64-based engagers. When the Fc portion of an IgG antibody binds to a CD16- or CD64-based CFR, it activates antibody-dependent cell-mediated cytotoxicity (ADCC) in CFR-expressing cells, along with other enhanced therapeutic properties conferred by the signaling domain contained in the intracellular domain of the CFR. Non-limiting exemplary CD16- or CD64-based agonists (including but not limited to antibodies or engagers) include at least one of CD16×CD30, CD64×CD30, CD16×BCMA, CD64×BCMA, CD16-IL-EPCAM or CD64-IL-EPCAM, CD16-IL-CD33, or CD64-IL-CD33, wherein "IL" in TriKE comprises all or part of at least one cytokine including IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, or any functional variant or combination / chimeric form thereof.

[0227] Generally, a transmembrane domain is a three-dimensional protein structure that is thermodynamically stable in a membrane such as the phospholipid bilayer of a biological membrane (e.g., the membrane of a cell or a cell vesicle). Thus, in some embodiments, the transmembrane domain of the CFR of the present invention comprises a single alpha helix, a stable complex of several transmembrane alpha helices, a transmembrane beta barrel, the beta helix of gramicidin A, or any combination thereof. In various embodiments, the transmembrane domain of the CFR comprises all or part of a "transmembrane protein" or "membrane protein" that is within the membrane. As used herein, a "transmembrane protein" or "membrane protein" is a protein that is located on and / or within a membrane. Examples of transmembrane proteins suitable for providing the transmembrane domain included in the CFR according to some embodiments of the present invention include, but are not limited to, receptors, ligands, immunoglobulins, glycophorins, or combinations thereof. In some embodiments, the transmembrane domain included in the CFR comprises all or part of the transmembrane domain of CD3ε, CD3γ, CD3δ, CD3ζ, CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD137, CD166, FcεRIγ, 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, T cell receptor (e.g., TCRα and / or TCRβ), nicotinic acetylcholine receptor, GABA receptor, or combinations thereof. In some embodiments, the transmembrane domain comprises all or part of the transmembrane domain of IgG, IgA, IgM, IgE, IgD, or combinations thereof. In some embodiments, the transmembrane domain comprises all or part of the transmembrane domain of glycophorin A, glycophorin D, or combinations thereof. In some embodiments of the CFR transmembrane domain, both the ER retention and endocytosis signals are absent or removed using genetic engineering.In various embodiments, either both the ER retention signal and the endocytosis signal are absent or are removed or excluded from the CFR transmembrane domain using genetic engineering methods. In some embodiments, the transmembrane domain comprises all or a portion of the transmembrane domain of CD3ε, CD28, CD27, CD8, ICOS, or CD4.

[0228] In some embodiments, the intracellular domain of the CFR described herein includes at least one signaling domain that activates a selected intracellular signaling pathway. In various embodiments of the CFR intracellular domain, both ER retention and endocytosis signals are absent or removed or excluded therefrom using genetic engineering methods. In some embodiments, the intracellular domain includes at least a cytotoxic domain. In some other embodiments, the intracellular domain may optionally include, in addition to the cytotoxic domain, one or more of a costimulatory domain, a persistent signaling domain, a death-inducing signaling domain, a tumor cell control signaling domain, or any combination thereof. In some embodiments, the cytotoxic domain of the CFR includes at least the full length or a portion of a polypeptide of CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137 (4-1BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or NKG2D. In one embodiment, the cytotoxic domain of the CFR includes an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to at least one ITAM (immunoreceptor tyrosine-based activation motif) of CD3ζ. In one embodiment, the cytotoxic domain of the CFR includes a modified CD3ζ represented by an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 26. In some embodiments, the cytotoxic domain of the CFR includes an amino acid sequence having at least about 90% identity to SEQ ID NO: 26. In some embodiments, the cytotoxic domain of the CFR includes an amino acid sequence having at least about 95% identity to SEQ ID NO: 26. In some embodiments, the cytotoxic domain of the CFR includes the amino acid sequence of SEQ ID NO: 26.

[0229]

Table 16

[0230] In some embodiments, the CFR comprises an intracellular domain that further comprises a co-stimulatory domain in addition to a cytotoxic signaling domain. Co-stimulatory domains suitable for use in the CFR include, but are not limited to, the full length or at least a portion of a polypeptide of CD2, CD27, CD28, CD40L, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or NKG2D, or any combination thereof. In some embodiments of the CFR, the co-stimulatory domain comprises the full length or at least a portion of a polypeptide of CD28, 4-1BB, CD27, CD40L, ICOS, CD2, or a combination thereof. In some embodiments, the CFR comprises an intracellular domain that comprises a co-stimulatory domain of CD28 and a cytotoxic domain of CD3ζ (also referred to as "28ζ"). In some embodiments, the -CD28-CD3ζ portion of the intracellular domain of the CFR is represented by an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 27. In some embodiments, the -CD28-CD3ζ portion of the intracellular domain of the CFR comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 27. In some embodiments, the -CD28-CD3ζ portion of the intracellular domain of the CFR comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 27. In some embodiments, the -CD28-CD3ζ portion of the intracellular domain of the CFR comprises the amino acid sequence of SEQ ID NO: 27.

[0231] SEQ ID NO: 27 RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQ LYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGE RRRGKGHDGLFQGLSTATKDTFDALHMQALPPR (153 amino acid CD28 co-stimulation + CD3ζ ITAM)

[0232] In some embodiments, the CFR comprises an intracellular domain that further comprises a sustained signaling domain in addition to the cytotoxic signaling domain and / or co-stimulatory domain. Suitable sustained signaling domains for use in the CFR include, but are not limited to, all or part of the intracellular domain of cytokine receptors such as IL2R, IL7R, IL15R, IL18R, IL12R, IL23R, or combinations thereof. In addition, the intracellular domain of a receptor tyrosine kinase (RTK) such as EGFR provides tumor cell control, or a tumor necrosis factor receptor (TNFR) such as FAS provides controlled cell death.

[0233] In some exemplary designs, the CFR comprises the extracellular domain of one CD3 subunit, and in some other designs, the CFR comprises a single-chain heterodimer extracellular domain comprising the extracellular domain of CD3ε linked to the extracellular domain of CD3δ or CD3γ (SEQ ID NO: 28 or SEQ ID NO: 29, respectively). The type and length of the linker in the single-chain heterodimer extracellular domain can vary. In some embodiments, the extracellular domain comprises an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 28 or 29. In some embodiments, the extracellular domain comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 28 or 29. In some embodiments, the extracellular domain comprises an amino acid sequence that is at least about 95% identical to SEQ ID NO: 28 or 29. In some embodiments, the extracellular domain comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, the extracellular domain comprises the amino acid sequence of SEQ ID NO: 29.

[0234]

Table 17

[0235]

Table 18

[0236] Cell surface-expressed CFRs (including CD3-based CFRs, also called cs-CD3) in the various constructs described herein can function as cell surface trigger receptors for binding to molecules having a selected binding specificity, which molecules include antibodies, engagers, and / or CARs (chimeric antigen receptors). As described herein, in some embodiments, cells comprising a solid tumor targeting backbone comprising a polynucleotide encoding two or more of a C-X-C-motif chemokine receptor or a variant thereof, TGFβ-SRR, and an ADR specific for 4-1BB optionally comprise a polynucleotide encoding one or more CFRs. The cells can be any type of cell, including human and non-human cells, pluripotent or non-pluripotent cells, immune or immunomodulatory cells, APCs (antigen presenting cells) or feeder cells, cells from a primary source (e.g., PBMC), or cells from cultured or engineered cells (e.g., cell lines, cells, and / or derivative cells differentiated from iPSCs). In some embodiments, cells comprising a solid tumor targeting backbone as described herein, and optionally CD38 knockout, exogenous CD16 or a variant thereof, HLA-I and / or HLA-II deficiency, and one or more CFRs are primary or derivative CD34 cells, hematopoietic stem progenitor cells, hematopoietic pluripotent progenitor cells, T cell precursors, NK cell precursors, T lineage cells, NKT lineage cells, NK lineage cells, or B lineage cells. In some embodiments, derivative cells comprising a polynucleotide encoding one or more of the gene modalities described herein are effector cells obtained by differentiating iPSCs comprising a polynucleotide encoding one or more of the gene modalities described herein.

[0237] Also provided in the present application is a master cell bank comprising sorted single cells and expanded cloned engineered iPSCs having at least one phenotype as provided herein, including but not limited to solid tumor targeting scaffolds and CFRs as described herein among other gene modalities, said cell bank providing a platform for further iPSC engineering and a renewable source for manufacturing off-the-shelf engineered homogeneous cell therapy products including but not limited to induced NK and T cells that can be produced in large scale in a cost-effective manner with well-defined and homogeneous composition.

[0238] 9. Chimeric Antigen Receptor (CAR) Expression What is applicable to genetically engineered immune cells, iPSCs, and their derived effector cells can be any CAR design known in the art. A CAR is generally a fusion protein comprising an extracellular domain that includes a target-binding region (e.g., an antigen recognition domain), a transmembrane domain, and an intracellular domain. In some embodiments, the extracellular domain can further include a signal peptide or leader sequence and / or a spacer. In some embodiments, the intracellular domain can further include a signaling peptide that activates the effector cell expressing the CAR. In some embodiments, the signaling peptide of the intracellular domain (or intracellular domains) includes the full length or at least a portion of the polypeptide of 2B4, CD2, CD3ζ, CD3ζ1XX, CD8, CD28, CD28H, CD137 (4-1BB), CS1, DAP10, DAP12, DNAM1, FcERIγ, IL2Rγ, IL7R, IL21R, IL2Rβ (IL15Rβ), IL21, IL7, IL12, IL15, IL21, KIR2DS2, NKp30, NKp44, NKp46, NKG2C, or NKG2D. In one embodiment, the signaling peptide of the CAR includes an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to at least one ITAM (immunoreceptor tyrosine-based activation motif) of CD3ζ. Exemplary N-terminal signal peptides include MALPVTALLLPLALLLHA (SEQ ID NO: 30, CD8asp) or MDFQVQIFSFLLISASVIMSR (SEQ ID NO: 31, IgKsp), or any signal peptide sequence known in the art or a functional variant thereof.

[0239] In some embodiments, the antigen recognition domain can specifically bind to an antigen. In some embodiments, the CAR is suitable for activating T cells, NK cells, or NKT cells that express the CAR. In some embodiments, the CAR is NK cell-specific and includes NK-specific signaling components. In some embodiments, the CAR is NKT cell-specific and includes NKT-specific signaling components. In certain embodiments, the T cells are derived from CAR-expressing iPSCs that contain a solid tumor targeting backbone as described herein, and the derived T cells can include T helper cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, αβ T cells, γδ T cells, or combinations thereof. In one embodiment, the NK cells are derived from CAR-expressing iPSCs that contain a solid tumor targeting backbone as described herein. In one embodiment, the NKT cells are derived from CAR-expressing iPSCs that contain a solid tumor targeting backbone as described herein.

[0240] In various embodiments, the antigen recognition region may be a mouse antibody, a human antibody, a humanized antibody, a camel IgG, a single variable new antigen receptor (VNAR), a shark heavy chain antibody (Ig-NAR), a chimeric antibody, a recombinant antibody, a single domain antibody (dAb), an anti-idiotype antibody, a bispecific, multispecific, or multimeric antibody, or a fragment thereof. An anti-idiotype antibody is specific for binding to the idiotype of another antibody, and the idiotype is the antigen determinant of the antibody. The bispecific antibody may be a BiTE (bispecific T cell engager) or a BiKE (bispecific killer cell engager), and the multispecific antibody may be a TriKE (trispecific killer cell engager). Non-limiting examples of antibody fragments include Fab, Fab’, F(ab’)2, F(ab’)3, Fv, Fabc, pFc, Fd, single-chain antigen-binding fragment (scFv), tandem scFv (scFv)2, single-chain Fab (scFab), disulfide-stabilized Fv (dsFv), minibody, diabody, triabody, tetrabody, single domain antigen-binding fragment (sdAb), camel heavy chain IgG and Nanobody® fragment, recombinant heavy chain only antibody (VHH), and other antibody fragments that maintain the binding specificity of the whole antibody. In some embodiments, the antigen-binding domain of the CAR comprises the CDR1, CDR2, and CDR3 (H-CDR) of the heavy chain of an antibody or a fragment thereof. In some embodiments, the antigen-binding domain of the CAR comprising the H-CDR of the antibody further comprises the CDR (L-CDR) of the light chain of the antibody.

[0241] In some embodiments, the antigen recognition domain of the CAR specifically binds an antigen associated with a disease or pathogen. In some embodiments, the disease-associated antigen is a tumor antigen, and the tumor may be a liquid tumor or a solid tumor. In some embodiments of the CAR, the CAR targets antigens of hematological malignancies including, but not limited to, acute and chronic leukemias (acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), lymphoma, non-Hodgkin lymphoma (NHL), Hodgkin's disease, multiple myeloma, and myelodysplastic syndromes).

[0242] In some embodiments of CARs targeting solid cancer antigens, the antigens are associated with sarcomas and carcinomas. In some embodiments, solid cancers suitable for CAR targeting include, but are not limited to, bladder cancer, bone cancer, brain / CNS cancer, breast cancer, breast lung cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric / stomach cancer, head and neck cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, metastatic cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, kidney cancer, salivary gland cancer, skin cancer, testicular tumors, thyroid tumors, urothelial cancer, and uterine / endometrial cancer. More specifically, in some embodiments, the CAR targets antigens associated with adenocarcinoma, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bronchiogenic carcinoma, cholangiocarcinoma, chondrosarcoma, choriocarcinoma, colon cancer, Ewing's tumor, fibrosarcoma, gallbladder cancer, hepatocellular carcinoma, liver cancer, leiomyosarcoma, liposarcoma, lymphoid malignancies, myeloid cancer, medullary thyroid cancer, melanoma, mesothelioma, myxosarcoma, non-small cell lung cancer, osteosarcoma, papillary adenocarcinoma, papillary carcinoma, papillary thyroid cancer, pheochromocytoma sebaceous gland carcinoma, peritoneal cancer, renal cell carcinoma, rhabdomyosarcoma, sarcoma, seminoma, squamous cell carcinoma, sweat gland carcinoma, synovial sarcoma, synovial tumor, and Wilms tumor. In some embodiments, the CAR targets antigens of CNS tumors including, but not limited to, acoustic neuroma, astrocytoma, CNS lymphoma, ependymoma, hemangioblastoma, germ cell tumor, glioma (including brainstem glioma and mixed glioma), glioblastoma (also known as glioblastoma multiforme), medulloblastoma, meningioma, neuroblastoma, oligodendroglioma, pineal tumor, retinoblastoma, schwannoma craniopharyngioma, and brain metastases.

[0243] Non-limiting examples of antigens that can be targeted by CARs include tumor fetal antigens (h5T4), 8H9, 9D7, ACPP, alpha-actinin-4 (actinin-4, ACTN4), ADAM12, ADRB3, ADGRE2 / EMR2, AFP, AKAP-4, ALK, ALPP, ALPPL2, androgen receptor, ASGR1 (asialoglycoprotein receptor 1), ASGR2 (asialoglycoprotein receptor 2), AXL, B7H3, B7H6, BAGE, beta-catenin, BCR, BCR-ABL, Bigh3, BING-4, BORIS, BRCA1 / 2, BST2, carbonic anhydrase IX (CAIX / CA9), CA125, C-C motif chemokine receptor 1 (CCR1), CCR4, carcinoembryonic antigen (CEA / CECAM5), calcium-activated chloride channel 2 (CLCA4), carbohydrate (Le), CD3, CD4, CD5, CD7, CD8, CD10, CD19, CD20, CD22, CD24, CD30, CD33, CD34, CD37, CD38, CD41, CD44, CD44V6, CD44v7 / 8, CD47, CD49f, CD52, CD56, CD70, CD72, CD74, CD79a, CD79b, CD97, CD99, CD123, CD133, CD138, CD171, CD179a, CD207, CD269 (BCMA), CD300LF, CDCl27, CDH3 (p-cadherin), CDH6, cadherin 19 (CDH19), CDK4, CFC1, CLCA1, CLDN6, CLDN18.2, CLEC12A, CLL-1, c-MET, CML66, antigens of cytomegalovirus (CMV)-infected cells (e.g., cell surface antigens), CR1L, CS-1, CSPG4, CXCR2, CXCR5, CXORF61, cyclin B1 (CCNB1), CYP1B1, DLL3, EFNA4, EGFR (or erbB-1), EGFRvIII, EGF1R, epithelial cell adhesion molecule / epithelial glycoprotein-2 (EpCAM / EGP2), epithelial glycoprotein-40 (EGP40), ELF2M, ENPP3, EphA2, EphA3, EphB2, ERBB2 (or HER2 / neu), ERBB3, ERBB4, ERG (TMPRSS2ETS fusion gene), ETA, ETV6-AML, FAP, folate-binding protein (FBP), FCAR, FCRL5, fetal acetylcholine receptor (AChR),Fibronectin, FLT3, folate receptor-α (FR-α / FOLR1), folate receptor beta (FR-β / FOLR2), FOLR3, Fos-related antigen 1 (FOSL1), FRcc, FZD10, GAGE, gangliosides (GM1, FucGM1, GM2, GM3, GD2, o-acetyl-GD2, GD3), GloboH, GpA33, Gp75, Gp100, glypican-1 (Glypican-1, GPC1), glypican-2 (Glypican-2, GPC2), glypican-3 (Glypican-3, GPC3), GPNMB, GPR20, GPR27, GPR35, GPR119, GPRC5D, guanylate cyclase C (GC-C), GUCY2C, HAVCR1, HERV envelope protein, HLA-A1, HM1.24, HMWMAA, HPV E6, HPV E7, human telomerase reverse transcriptase (hTERT), IGFr / IGF1R, IGLL1 (CD179b), IL11Rα, interleukin 13 receptor subunit alpha-2 (IL13Rα2), IL13Rcc2, immature laminin receptor (iLRP), integrin αVβ3, integrin α5β, integrin B7, intercellular adhesion molecule 1 (ICAM1), intestinal carboxyl esterase (iCE), κ light chain, kinase insert domain receptor (KDR), KIT, KISS1R, LAIR1, LAGE-1a, LAMP-1, LCK, legumain, Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (L1-CAM), LILRA2, LILRB2, LIV-1, LMP2, LRRC15, LY6K, LY75, LYPD3, MAD-CT-1, MAD-CT-2, melanoma antigen family A1 (MAGE-A1), MC1R, MelanA / MART1, MART2, melanoma-associated chondroitin sulfate proteoglycan (MCSP), c-Met, MICA / B, mesothelin (MSLN), ML-IAP, MR1Multidrug resistance-associated protein 3 (MRP3), MS4A12, Mucin 1 (MUC1, tMUC1), MUC2, MUC5A, MUC12, MUC16, MUC17, MUC21, Mud, MUM1, MUM2, MUM3, mut hsp70-2, MYCN, NA17, NA88-1, NCAM, nectin 4, NKCSI, NKG2D ligand, NPM, NY-BR-1, cancer-testis antigen NY-ESO-1, OA1, OGT, OR51E2, OY-TES1, p53, p53 variant, PANX3, PAP, PAX3, PAX5, PCTA-1 / galectin 8, PDGFR-beta, PDL1, periostin, PLAC1, PRAME, PRLR, prostate-specific antigen (KLK2, KLK4), prostasin (P501S), PRSS21, polysialic acid (PSA), prostate stem cell antigen (PSCA), PSC1, PRAME prostate-specific membrane antigen (PSMA / FOLH1), PTK7, QRFPR, RAGE-1, RANKL, Ras, Ras variant, RCC, RhoC, Ron kinase, ROR1, RU1, RU2, SAGE, SAP1, sarcoma translocation breakpoint, SART3, SIGLEC-15, sialoepitope CA6, SLC6A3, SLC12A3, SLC13A5, SLC22A1, SLC22A7, SLC30A4, SLC30A8, SLC34A2, SLC45A3, sLe, SLITRK6, SPARC, sperm protein 17 (SP17), SSEA-4, SSTR1, SSX2, STRAP, sTN, Survivin, tumor-associated glycoprotein 72 (TAG72), TARP, TEM1 / CD248, TEM7R, TEM, telomerase, TGF-beta receptor, TGS5, Tie2, tissue factor (TF), TIM-3, TMEFF2 (TENB2), TMEM238, TMPRSS11B, TMPRSS11E, Tn Ag, TNC, TP-3, TRAILR1, TRAILR2, TRBC1, TRBC2, TRF2, TRG, TROP2, TRP1, TRP2, TSHR, TSTA, tyrosinase, UGT1A1, UPK1B, UPK2, VEGF, VEGFR, vascular endothelial growth factor R2 (VEGF-R2)Examples include VTCN1 (B7H4), Wilms' tumor protein (WT1), XAGE1, and various pathogen antigens known in the art. Non-limiting examples of pathogens include viruses, bacteria, fungi, parasites, and protozoa that may cause disease.

[0244] Non-limiting examples of solid tumor antigens that can be targeted by CARs include h5T4, 8H9, 9D7, ACPP, ACTN4, ADAM12, ADRB3, AFP, AKAP-4, ALK, ALPP, ALPPL2, androgen receptor, ASGR1, ASGR2, AXL, B7H3, B7H6, BAGE, β-catenin, BCMA (CD269), BCR, BCR-ABL, Bigh3, BING-4, BORIS, BRCA1 / 2, BST2, CAIX / CA9, CA19.9, CA125, CCR1, CCR4, carbohydrate (Le), CCNB1, CD3, CD4, CD10, CD19, CD20, CD22, CD24, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD47, CD49f, CD56, CD70, CD72, CD74, CD79a, CD79b, CD97, CD99, CD123, CD133, CD138, CD171, CD179a, CD207, CD300LF, CDCl27, CDH3, CDH6, CDH19, CDK4, CEA / CECAM5, CFC1, CLCA1, CLCA4, CLDN6, CLDN18.2, CLEC12A, CLL-1, c-MET, CML66, CR1L, CS-1, CSPG4, CXCR2, CXCR5, CXORF61, CYP1B1, DLL3, EFNA4, EGFR, EGFRvIII, EGP2 / EpCAM, EGP40, ELF2M, EMR2, ENPP3, EphA2, EphA3, EphB2, ERBB2 (HER2 / neu), ERBB3, ERBB4, periostin, ERG (TMPRSS2 ETS fusion gene), ETA, ETV6-AML, FAP, FBP, FCAR, FCRL5, fetal AchR, fibronectin, FLT3, FR-α / FOLR1, FR-β / FOLR2, FOLR3, FOSL1, FRcc, FZD10, GAGE, ganglioside GM1, FucGM1, GM2, GM3, GD2, o-acetyl-GD2, GD3), GloboH, GpA33, Gp75, Gp100, GPC1, GPC2, GPC3, GPNMB, GPR20, GPR27, GPR35, GPR119, GPRC5D, GC-C, GUCY2C, HAVCR1, HER2, HERV envelope protein, HLA-A1, HM1.24, HMWMAA, HPV E6, HPVE7, hTERT, iCE, ICAM1, IGFr / IGF1R, IGLL1 (CD179b), IL-11Rα, IL13-Rα2, IL-13Rα2, iLRP, Integrin αVβ3, Integrin α5β, KDR, KIT, KISS1R, KLK2, KLK4, LAIR1, LAGE-1a, LAMP-1, LCK, Legumain, LeY, L1-CAM, LILRA2, LIV-1, LILRB2, LMP2, LRRC15, LY6K, LY75, LYPD3, MAD-CT-1, MAD-CT-2, MAGE-A1, MC1R, Melan-A / MART1, MART2, MCSP, MICA / B, ML-IAP, MR1, MRP3, MS4A12, MSLN, MUC1, tMUC1, MUC2, MUC5A, MUC12, MUC16, MUC17, MUC21, Mud, MUM1, MUM2, MUM3, mut hsp70-2, MYCN, NA17, NA88-1, NCAM, Necl-4, NKG2D Ligand, NPM, NY-BR-1, NY-ESO-1, OA1, OGT, OR51E2, OY-TES1, p53, p53 Variant, PANX3, PAP, PAX3, PAX5, PCTA-1 / Galectin 8, PDGFR-β, PDL1, PLAC1, PRAME, PRLR, P501S, PRSS21, PSA, PSCA, PSC1, PSMA / FOLH1, PTK7, QRFPR, RAGE-1, RANKL, Ras, Ras Variant, RCC, RhoC, Ron Kinase, ROR1, RU1, RU2, SAGE, SAP1, Sarcoma Translocation Breakpoint, SART3, SIGLEC-15, Sialoepitope CA6, SLC6A3, SLC12A3, SLC13A5, SLC22A1, SLC22A7, SLC30A4, SLC30A8, SLC34A2, SLC45A3, sLe, SLITRK6, SPARC, SP17, SSEA-4, SSTR1, SSX2, STEAP, sTN, Survivin, TAG72, TARP, TEM1 / CD248, TEM7R, TEM, Telomerase, TGF-β Receptor, TGS5, Tie2, Tissue Factor (TF), TIM-3, TMEFF2 (TENB2), TMEM238, TMPRSS11B, TMPRSS11E, TnExamples include Ag, TNC, TP-3, TRAILR1, TRAILR2, TRF2, TRG, TROP2, TRP1, TRP2, TSHR, TSTA, tyrosinase, UGT1A1, UPK1B, UPK2, VEGF, VEGFR, VEGFR-II, VTCN1 (B7H4), WT1, and XAGE1.

[0245] Table 1B provides non-limiting examples of solid cancers having corresponding tumor antigens.

[0246]

Table 19-1

[0247]

Table 19-2

[0248] In some embodiments, the antigen recognition domain of the CAR comprises the complementarity determining region of the heavy chain (H-CDR) of the binding domain of an antibody specific for a tumor antigen, the CDRs of both the heavy and light chains (H- and L-CDRs), the variable region of the heavy chain (VH), or a single chain of the variable regions of both the heavy and light chains (VH and VL), including those exemplified in this application. In some embodiments, the CAR is designed based on the binding domain of an antibody comprising trastuzumab, cetuximab, panitumumab, ofatumumab, belimumab, ipilimumab, pertuzumab, tremelimumab, nivolumab, pembrolizumab, atezolizumab, MDX-1105, dacetuzumab, urelumab, MPDL3280A, ranibizumab, blinatumomab, nimotuzumab, zalutumumab, ofatumumab, patritumab, crizanlizumab, sophilizumab, edrecolomab, adecatumumab, anetumab, huDS6, refastuzumab, sacituzumab, PR1A3, humanized PR1A3, humanized Ab2-3, claudiximab, AMG595, ABT806, sibrotuzumab, DS-8895a variant 1, DS-8895a variant 2, MEDI-547, nalatumomab, RG7841, farletuzumab, milatuzumab, J591 variant 1, J591 variant 2, robatupizumab, PF-06647020, radretumab, siltuximab, radretumab, huLiv1-14, Liv1-1.7A4, huLiv1-22, 4H11, 4H5, glembatumumab, oportuzumab, enfortumab, depatuxizumab, or codrituzumab.

[0249] Thus, in some embodiments, the antigen recognition domain of the CAR specifically binds to an antigen present on bladder cancer. In some embodiments, the CAR targeting a bladder cancer-related antigen specifically binds to HER2, MICA / B, CD207, EFNA4, LY6K, LYPD3, nectin 4, PTK7, SLITRK6, TIM-3, TNC, UPK1B, or UPK2. In some embodiments, the antigen recognition domain of the CAR comprises the H-CDR, H- and L-CDRs, VH, or single-chain of VH and VL of an antibody including enfortumab, trastuzumab, pertuzumab, or SLITRK6.

[0250] In some embodiments, the antigen recognition domain of the CAR specifically binds to an antigen present on bone cancer. In some embodiments, the CAR targeting a bone cancer-related antigen specifically binds to MICA / B, ADAM12, CCR1, CD99, CD248, EPHA2, GPNMB, LRRC15, or TP-3. In some embodiments, the antigen recognition domain of the CAR comprises the H-CDR, H- and L-CDRs, VH, or single-chain of VH and VL of an antibody including huM25, DS-8895a variant 1, DS-8895a variant 2, or glembatumumab.

[0251] In some embodiments, the antigen recognition domain of the CAR specifically binds to an antigen present on brain cancer. In some embodiments, the CAR targeting a brain cancer-related antigen specifically binds to MICA / B, CD133, DLL3, EGFRvIII, or TNC. In some embodiments, the antigen recognition domain of the CAR comprises the H-CDR, H- and L-CDRs, VH, or single-chain of VH and VL of an antibody including AMG595, ABT806, robatupizumab, or depatuxizumab.

[0252] In some embodiments, the antigen recognition domain of the CAR specifically binds to an antigen present on breast cancer cells. In some embodiments, the CAR specifically binds to HER2, MICA / B, ADAM12, ADGRE2 / EMR2, CCR4, CD49f, CD133, CDH3 (p-cadherin), CLDN6, c-MET, CXCR2, EFNA4, EGFR, EPCAM / EGP2, EPHA2, GPNMB, ICAM1, LAMP-1, LIV-1, LILRB2, LRRC15, LYPD3, MUC1, tMUC1, PRLR, PTK7, sialo-epitope CA6, TNC, or TROP2. In some embodiments, the antigen recognition domain of the CAR comprises the H-CDR, H- and L-CDRs, VH, or single-chain of VH and VL of an antibody comprising trastuzumab, pertuzumab, sacituzumab, ladiratuzumab, huLiv1-14, Liv1-1.7A4, huLiv1-22, huDS6, glembatumumab, PF-0664720, MEDI-547, DS-8895a variant 1, or DS-08895a variant 2.

[0253] In some embodiments, the antigen recognition domain of the CAR specifically binds to an antigen present on breast and lung cancer cells. In some embodiments, the CAR targeting breast and lung cancer-related antigens specifically binds to HER2, MICA / B, ADGRE2 / EMR2, EPCAM / EGP2, or ROR1.

[0254] In some embodiments, the antigen recognition domain of the CAR specifically binds to an antigen present on cervical / uterine / endometrial cancer. In some embodiments, the CAR targeting cervical / uterine / endometrial cancer-related antigens specifically binds to MICA / B, EFNA4, LY6K, MUC1, MUC16, LYPD3, PTK7, SLC12A3, or SSTR1. In some embodiments, the antigen recognition domain of the CAR comprises the H-CDR, H- and L-CDRs, VH, or single-chain of VH and VL of an antibody comprising PF-0664720, anetumab, 4H11, 4H5, huDS6 or sophizumab.

[0255] In some embodiments, the antigen recognition domain of the CAR specifically binds to an antigen present on cholangiocarcinoma cells. In some embodiments, the CAR targeting a cholangiocarcinoma-related antigen specifically binds to MICA / B or tMUC1.

[0256] In some embodiments, the antigen recognition domain of the CAR specifically binds to an antigen present on colorectal cancer. In some embodiments, the CAR targeting a colorectal cancer-related antigen specifically binds to HER2, MICA / B, ADAM12, CA19.9, CD3, CD49f, CD133, CEA / CECAM5, CLCA1, c-MET, EFNA4, EPHB2, GPA33, GPR35, GUCY2C, ICAM1, LGR5 / GPR49, LRRC15, MS4A12, MUC12, MUC17, TIM-3, or TMEM238. In some embodiments, the antigen recognition domain of the CAR comprises the H-CDR, H- and L-CDR, VH, or single chain of VH and VL of an antibody including huM25, PR1A3, humanized PR1A3, panitumumab, cetuximab, nimotuzumab, or zalutumumab.

[0257] In some embodiments, the antigen recognition domain of the CAR specifically binds to an antigen present on esophageal cancer cells. In some embodiments, the CAR targeting an esophageal cancer-related antigen specifically binds to HER2, MICA / B, CA19.9, CD10, CEA / CECAM5, EFNA4, EPHB2, MUC21, TMEM238, TMPRSS11B or TMPRSS11E.

[0258] In some embodiments, the antigen recognition domain of the CAR specifically binds to an antigen present on gallbladder cancer cells. In some embodiments, the CAR targeting a gallbladder cancer-related antigen specifically binds to EPCAM / EGP2.

[0259] In some embodiments, the antigen recognition domain of the CAR specifically binds to an antigen present on the stomach / stomach cancer. In some embodiments, the CAR targeting a stomach / stomach cancer-related antigen specifically binds to HER2, MICA / B, CEA / CECAM5, CLDN18.2, c-MET, CR1L, EFNA4, EPHB2, LGR5 / GPR49, MUC17, PSCA, TIM-3, or TMEM238. In some embodiments, the antigen recognition domain of the CAR comprises the H-CDR, H- and L-CDR, VH, or single chain of VH and VL of an antibody comprising trastuzumab, anetumab, pertuzumab, trastuzumab, or humanized PR1A3.

[0260] In some embodiments, the antigen recognition domain of the CAR specifically binds to an antigen present on glioma cancer cells. In some embodiments, the CAR targeting a glioma cancer-related antigen specifically binds to MICA / B, ADGRE2 / EMR2, CD49f, CD133, EGFR, EGFRvIII, EPHA2, HM1.24, or IL13-Rα2.

[0261] In some embodiments, the antigen recognition domain of the CAR specifically binds to an antigen present on head and neck cancer. In some embodiments, the CAR targeting a head and neck cancer-related antigen specifically binds to HER2, MICA / B, ADAM12, CD3, c-MET, EFNA4, LRRC15, LY6K, LYPD3, PTK7, or TNC. In some embodiments, the antigen-binding domain of the CAR comprises the H-CDR, H- and L-CDR, VH, or single chain of VH and VL of an antibody comprising cetuximab, panitumumab, nimotuzumab, PF-0664720, pantumumab, cetuximab, nimotuzumab, or zalutumumab.

[0262] In some embodiments, the antigen recognition domain of the CAR specifically binds to an antigen present on kidney cancer. In some embodiments, the CAR targeting a kidney cancer-related antigen specifically binds to MICA / B, CD70, CDH6, c-MET, ENPP3, or HAVCR1. In some embodiments, the antigen recognition domain of the CAR comprises the H-CDR, H- and L-CDRs, VH, or single-chain of VH and VL of AGS-16M8F, AGS-16C3, CDX-014, or ofatumumab.

[0263] In some embodiments, the antigen recognition domain of the CAR specifically binds to an antigen present on liver cancer. In some embodiments, the CAR targeting a liver cancer-related antigen specifically binds to MICA / B, ASGR1, ASGR2, C9 (CAIX), CA19.9, CEA / CECAM5, CCR1, CD3, CD133, EPCAM / EGP2, GPC3, ICAM1, LGR5 / GPR49, SLC13A5, SLC22A1, SLC22A7, TIM-3, TRF2, or UGT1A1. In some forms, the antigen recognition domain of the CAR comprises the H-CDR, H- and L-CDRs, VH, or single-chain of VH and VL of an antibody comprising codrituzumab, oportuzumab, or humanized PR1A3.

[0264] In some embodiments, the antigen recognition domain of the CAR specifically binds to an antigen present on lung cancer. In some embodiments, the CAR targeting a lung cancer-related antigen specifically binds to HER2, MICA / B, ADAM12, ADGRE2 / EMR2, CCR1, CCR4, CD56, CD133, CEA / CECAM5, CXCR2, DLL3, EFNA4, EGFR, EGFRvIII, FOLR1, GPC3, HM1.24, ICAM1, LILRB2, LRRC15, LY6K, LYPD3, MSLN, MUC1, MUC16, PDL1, PTK7, SLC34A2, or TIM-3. In some such embodiments, the antigen recognition domain of the CAR comprises the H-CDR, H- and L-CDR, VH, or a single chain of VH and VL of an antibody including panitumumab, cetuximab, pembrolizumab, nivolumab, atezolizumab, and nimotuzumab, rituximab, anetumab, PF-0664720, farletuzumab, robatupizumab, rituximab, sofituzumab, huDS6, ABT806, AMG595, or huM25.

[0265] In some embodiments, the antigen recognition domain of the CAR specifically binds to an antigen present on mesothelioma cells. In some embodiments, the CAR targeting a mesothelioma-related antigen specifically binds to MICA / B, FAP, or MSLN.

[0266] In some embodiments, the antigen recognition domain of the CAR specifically binds to an antigen present on metastatic cancer cells. In some embodiments, the CAR targeting a metastatic cancer cell-related antigen specifically binds to MICA / B, MSLN, or VEGFR-II.

[0267] In some embodiments, the antigen recognition domain of the CAR specifically binds to an antigen present on neuroblastoma cells. In some embodiments, the CAR targeting a neuroblastoma-related antigen specifically binds to MICA / B or GD2.

[0268] In some embodiments, the antigen recognition domain of the CAR specifically binds to an antigen present on non-small cell lung cancer (NSCLC) cells. In some embodiments, the CAR targeting a non-small cell lung cancer-related antigen specifically binds to MICA / B, c-MET, or EGFR.

[0269] In some embodiments, the antigen recognition domain of the CAR specifically binds to an antigen present on ovarian cancer. In some embodiments, the CAR targeting an ovarian cancer-related antigen specifically binds to HER2, MICA / B, CCR1, CD3, CD133, CLDN6, c-MET, EFNA4, EPCAM / EGP2, FAP, FOLR1, FOLR3, FR-α, FZD10, GPR27, GPR119, LRRC15, MSLN, MUC1, MUC16, PTK7, SLC34A2, sTnTMEM238, or VTCN1. In some embodiments, the antigen recognition domain of the CAR comprises the H-CDR, H- and L-CDR, VH, or single-chain of VH and VL of an antibody including, but not limited to, cetuximab, 4H11, 4H5, huDS6, farletuzumab, anetumab, trastuzumab, pertuzumab, PF-0664720, sibrotuzumab, huM25, or refetuzumab.

[0270] In some embodiments, the antigen recognition domain of the CAR specifically binds to an antigen present on pancreatic cancer. In some embodiments, the CAR targeting a pancreatic cancer-related antigen specifically binds to MICA / B, ADAM12, CA19.9, CFC1, EFNA4, EPCAM / EGP2, ICAM1, LILRB2, LRRC15, MSLN, MUC1, tMUC1, MUC5A, MUC16, MUC17, PSCA, PTK7 or SLC30A8. In some embodiments, the antigen recognition domain of the CAR comprises the H-CDR, H- and L-CDR, VH, or single-chain of VH and VL of an antibody including, but not limited to, PF-0664720, crizotinib, 4H11, 4H5, anetumab, huDS6, cetuximab, huM25, or RG7841.

[0271] In some embodiments, the antigen recognition domain of the CAR specifically binds to an antigen present on peritoneal cancer cells. In some embodiments, the CAR targeting a peritoneal cancer-related antigen specifically binds to FOLR3.

[0272] In some embodiments, the antigen recognition domain of the CAR specifically binds to an antigen present on prostate cancer. In some embodiments, the CAR targeting a prostate cancer-related antigen specifically binds to MICA / B, ACPP, CD10, CD49f, CD133, EFNA4, OR51E2, PSCA, PSMA / FOLH1, PTK7, SLC30A4, SLC45A3, STEAP, TIM-3 or TMEFF2 / TENB2. In some embodiments, the antigen recognition domain of the CAR comprises the H-CDR, H- and L-CDR, VH, or a single chain of VH and VL of an antibody including milatuzumab or J591 variant 1 or 2.

[0273] In some embodiments, the antigen recognition domain of the CAR specifically binds to an antigen present on kidney cancer cells. In some embodiments, the CAR targeting a kidney cancer-related antigen specifically binds to MICA / B, CD3, CD70, ICAM1, KISS1R, LILRB2, QRFPR, SLC6A3, or TIM-3.

[0274] In some embodiments, the antigen recognition domain of the CAR specifically binds to an antigen present on sarcoma. In some embodiments, the CAR targeting a sarcoma-related antigen specifically binds to MICA / B or LRRC15.

[0275] In some embodiments, the antigen recognition domain of the CAR specifically binds to an antigen present on salivary gland cancer cells. In some embodiments, the CAR targeting a salivary gland cancer-related antigen specifically binds to HER2 or MICA / B.

[0276] In some embodiments, the antigen recognition domain of the CAR specifically binds to an antigen present on cutaneous cancer. In some embodiments, the CAR targeting a cutaneous cancer-related antigen specifically binds to CCR4, CD3, CD10, or ICAM1.

[0277] In some embodiments, the antigen recognition domain of the CAR specifically binds to an antigen present on synovial sarcoma. In some embodiments, the CAR targeting a synovial sarcoma-related antigen specifically binds to CD99.

[0278] In some embodiments, the antibody specifically binds to an antigen present on thyroid cancer / tumor cells. In some embodiments, the CAR targeting a thyroid cancer / tumor-related antigen specifically binds to MICA / B, CD10, c-MET, PTK7, or TSHR.

[0279] In some embodiments, the antibody specifically binds to an antigen present on urothelial cancer cells. In some embodiments, the CAR targeting a urothelial cancer-related antigen specifically binds to MICA / B, CLDN6, EPCAM / EGP2, SIGLEC-15, TIM-3, or UPK2.

[0280] In some embodiments, the antigen recognition domain of the CAR specifically binds to an antigen present on uterine / endometrial cancer cells. In some embodiments, the CAR targeting a uterine / endometrial cancer-related antigen specifically binds to HER2, MICA / B, ALPP, ALPPL2, CCR1, CLDN6, EFNA4, EPHB2, FOLR1, LILRB2, LY6K, LYPD3, MUC1, MUC16, or PTK7. In some embodiments, the antigen recognition domain of the CAR comprises the H-CDR, H- and L-CDR, VH, or single chain of VH and VL of an antibody including PF-0664720, farletuzumab, sofituzumab, 4H11, or 4H5.

[0281] In various embodiments, the antigen recognition domain of the CAR specifically binds to tumor antigens that are known to be associated with three or more cancer types (sometimes referred to as "pan-tumor antigens"). A non-limiting set of such pan-tumor antigens includes, at least, ADAM12, ADGRE2 / EMR2, CA19.9, CCR1, CCR4, CD3, CD10, CD49f, CD133, CEA / CECAM5, CLDN6, c-MET, EFNA4, EGFR, EGFRvIII, EPHA2, EPHB2, FOLR1, HER2, ICAM1, LILRB2, LRRC15, LY6K, LYPD3, MICA / B, MSLN, MUC1, tMUC1, MUC16, MUC17, PSCA, PTK7, TIM-3, TMEM238, and TNC, as exemplified in Table 2.

[0282]

Table 20

[0283] Thus, in some embodiments, the antigen recognition domain of the CAR specifically binds to tumor-associated ADAM12, and effector cells comprising the CAR and the disclosed solid tumor targeting scaffold are useful for treating one or more cancers including, at least, bone cancer, breast cancer, colorectal cancer, head and neck cancer, lung cancer, or pancreatic cancer.

[0284] In some embodiments, the antigen recognition domain of the CAR specifically binds to tumor-associated ADGRE2 / EMR2, and effector cells comprising the CAR and the disclosed solid tumor targeting scaffold are useful for treating one or more cancers including, at least, breast cancer, breast lung cancer, glioma, or lung cancer.

[0285] In some embodiments, the antigen recognition domain of the CAR specifically binds to tumor-associated CA19.9, and effector cells comprising the CAR and the disclosed solid tumor targeting scaffold are useful for treating one or more cancers including, at least, colorectal cancer, esophageal cancer, liver cancer, or pancreatic cancer.

[0286] In some embodiments, the antigen recognition domain of the CAR specifically binds to tumor-associated CCR1, and effector cells comprising the CAR and the disclosed solid tumor targeting scaffold are useful for treating one or more cancers including at least bone cancer, liver cancer, lung cancer, ovarian cancer, or uterine / endometrial cancer.

[0287] In some embodiments, the antigen recognition domain of the CAR specifically binds to tumor-associated CCR4, and effector cells comprising the CAR and the disclosed solid tumor targeting scaffold are useful for treating one or more cancers including at least breast cancer, lung cancer, or skin cancer.

[0288] In some embodiments, the antigen recognition domain of the CAR specifically binds to tumor-associated CD3, and effector cells comprising the CAR and the disclosed solid tumor targeting scaffold are useful for treating one or more cancers including at least colorectal cancer, head and neck cancer, liver cancer, ovarian cancer, kidney cancer, or skin cancer.

[0289] In some embodiments, the antigen recognition domain of the CAR specifically binds to tumor-associated CD10, and effector cells comprising the CAR and the disclosed solid tumor targeting scaffold are useful for treating one or more cancers including at least esophageal cancer, prostate cancer, skin cancer, or thyroid tumors.

[0290] In some embodiments, the antigen recognition domain of the CAR specifically binds to tumor-associated CD49f, and effector cells comprising the CAR and the disclosed solid tumor targeting scaffold are useful for treating one or more cancers including at least breast cancer, colorectal cancer, glioma, or prostate cancer.

[0291] In some embodiments, the antigen recognition domain of the CAR specifically binds to tumor-associated CD133, and effector cells comprising the CAR and the disclosed solid tumor targeting scaffold are useful for treating one or more cancers including at least brain cancer, breast cancer, colorectal cancer, glioma, liver cancer, lung cancer, ovarian cancer, or prostate cancer.

[0292] In some embodiments, the antigen recognition domain of the CAR specifically binds to tumor-associated CEA / CECAM5, and effector cells comprising the CAR and the disclosed solid tumor targeting scaffold are useful for treating one or more cancers including at least colorectal cancer, esophageal cancer, gastric / stomach cancer, liver cancer, or lung cancer.

[0293] In some embodiments, the antigen recognition domain of the CAR specifically binds to tumor-associated CLDN6, and effector cells comprising the CAR and the disclosed solid tumor targeting scaffold are useful for treating one or more cancers including at least breast cancer, ovarian cancer, urothelial cancer, or uterine / endometrial cancer.

[0294] In some embodiments, the antigen recognition domain of the CAR specifically binds to tumor-associated c-MET, and effector cells comprising the CAR and the disclosed solid tumor targeting scaffold are useful for treating one or more cancers including at least breast cancer, colorectal cancer, gastric / stomach cancer, head and neck cancer, kidney cancer, non-small cell lung cancer, ovarian cancer, or thyroid tumors.

[0295] In some embodiments, the antigen recognition domain of the CAR specifically binds to tumor-associated EFNA4, and effector cells comprising the CAR and the disclosed solid tumor targeting scaffold are useful for treating one or more cancers including at least bladder cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric / stomach cancer, head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, or uterine / endometrial cancer.

[0296] In some embodiments, the antigen recognition domain of the CAR specifically binds to tumor-associated EGFR, and effector cells comprising the CAR and the disclosed solid tumor targeting scaffold are useful for treating one or more cancers including at least breast cancer, glioma, lung cancer, non-small cell lung cancer, or neuroblastoma.

[0297] In some embodiments, the antigen recognition domain of the CAR specifically binds to tumor-associated EGFRvIII, and effector cells comprising the CAR and the disclosed solid tumor targeting scaffold are useful for treating one or more cancers including at least brain cancer, glioma, or lung cancer.

[0298] In some embodiments, the antigen recognition domain of the CAR specifically binds to tumor-associated EPCAM / EGP2, and effector cells comprising the CAR and the disclosed solid tumor targeting scaffold are useful for treating one or more cancers including at least breast cancer, breast lung cancer, gallbladder cancer, liver cancer, ovarian cancer, or urothelial cancer.

[0299] In some embodiments, the antigen recognition domain of the CAR specifically binds to tumor-associated EPHA2, and effector cells comprising the CAR and the disclosed solid tumor targeting scaffold are useful for treating one or more cancers including at least bone cancer, breast cancer, or glioma.

[0300] In some embodiments, the antigen recognition domain of the CAR specifically binds to tumor-associated EPHB2, and effector cells comprising the CAR and the disclosed solid tumor targeting scaffold are useful for treating one or more cancers including at least colorectal cancer, esophageal cancer, stomach / stomach cancer, or uterus / endometrial cancer.

[0301] In some embodiments, the antigen recognition domain of the CAR specifically binds to tumor-associated FOLR1, and effector cells comprising the CAR and the disclosed solid tumor targeting scaffold are useful for treating one or more cancers including at least lung cancer, ovarian cancer, or uterus / endometrial cancer.

[0302] In some embodiments, the antigen recognition domain of the CAR specifically binds to tumor-associated HER2, and effector cells comprising the CAR and the solid tumor-targeting scaffold as disclosed are useful for treating one or more cancers including at least bladder cancer, breast cancer, thoracic lung cancer, colorectal cancer, esophageal cancer, gastric / stomach cancer, head and neck cancer, lung cancer, ovarian cancer, or salivary gland cancer.

[0303] In some embodiments, the antigen recognition domain of the CAR specifically binds to tumor-associated ICAM1, and effector cells comprising the CAR and the disclosed solid tumor-targeting scaffold are useful for treating one or more cancers including at least breast cancer, colorectal cancer, liver cancer, lung cancer, pancreatic cancer, kidney cancer, or skin cancer.

[0304] In some embodiments, the antigen recognition domain of the CAR specifically binds to tumor-associated LILRB2, and effector cells comprising the CAR and the disclosed solid tumor-targeting scaffold are useful for treating one or more cancers including at least breast cancer, lung cancer, pancreatic cancer, kidney cancer, or uterine / endometrial cancer.

[0305] In some embodiments, the antigen recognition domain of the CAR specifically binds to tumor-associated LRRC15, and effector cells comprising the CAR and the disclosed solid tumor-targeting scaffold are useful for treating one or more cancers including at least bone cancer, breast cancer, colorectal cancer, head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer, or kidney cancer.

[0306] In some embodiments, the antigen recognition domain of the CAR specifically binds to tumor-associated LY6K, and effector cells comprising the CAR and the disclosed solid tumor-targeting scaffold are useful for treating one or more cancers including at least bladder cancer, cervical cancer, head and neck cancer, lung cancer, or uterine / endometrial cancer.

[0307] In some embodiments, the antigen recognition domain of the CAR specifically binds to tumor-associated LYPD3, and effector cells comprising the CAR and the disclosed solid tumor targeting scaffold are useful for treating one or more cancers including at least bladder cancer, breast cancer, cervical cancer, head and neck cancer, lung cancer, or uterine / endometrial cancer.

[0308] In some embodiments, the antigen recognition domain of the CAR specifically binds to tumor-associated MICA / B, and effector cells comprising the CAR and the disclosed solid tumor targeting scaffold are useful for treating one or more cancers including at least bladder cancer, bone cancer, brain cancer, breast cancer, breast lung cancer, cervical cancer, cholangiocarcinoma, colorectal cancer, esophageal cancer, gastric / stomach cancer, glioma, head and neck cancer, kidney cancer, liver cancer, lung cancer, mesothelioma, metastatic cancer, neuroblastoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, sarcoma, salivary gland cancer, thyroid cancer, urothelial cancer, or uterine / endometrial cancer.

[0309] In some embodiments, the antigen recognition domain of the CAR specifically binds to tumor-associated MSLN, and effector cells comprising the CAR and the disclosed solid tumor targeting scaffold are useful for treating one or more cancers including at least lung cancer, metastatic cancer, mesothelioma, ovarian cancer, or pancreatic cancer.

[0310] In some embodiments, the antigen recognition domain of the CAR specifically binds to tumor-associated MUC1, and effector cells comprising the CAR and the disclosed solid tumor targeting scaffold are useful for treating one or more cancers including at least breast cancer, cervical cancer, lung cancer, ovarian cancer, pancreatic cancer, or uterine / endometrial cancer.

[0311] In some embodiments, the antigen recognition domain of the CAR specifically binds to tumor-associated tMUC1, and effector cells comprising the CAR and the disclosed solid tumor targeting scaffold are useful for treating one or more cancers including at least breast cancer, cholangiocarcinoma, or pancreatic cancer.

[0312] In some embodiments, the antigen recognition domain of the CAR specifically binds to tumor-associated MUC16, and effector cells comprising the CAR and the disclosed solid tumor targeting scaffold are useful for treating one or more cancers including at least cervical cancer, lung cancer, ovarian cancer, pancreatic cancer, or uterine / endometrial cancer.

[0313] In some embodiments, the antigen recognition domain of the CAR specifically binds to tumor-associated MUC17, and effector cells comprising the CAR and the disclosed solid tumor targeting scaffold are useful for treating one or more cancers including at least colorectal cancer, gastric / stomach cancer, or pancreatic cancer.

[0314] In some embodiments, the antigen recognition domain of the CAR specifically binds to tumor-associated PSCA, and effector cells comprising the CAR and the disclosed solid tumor targeting scaffold are useful for treating one or more cancers including at least gastric / stomach cancer, pancreatic cancer, or prostate cancer.

[0315] In some embodiments, the antigen recognition domain of the CAR specifically binds to tumor-associated PTK7, and effector cells comprising the CAR and the solid tumor targeting scaffold as disclosed are useful for treating one or more cancers including at least bladder cancer, breast cancer, cervical cancer, head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, thyroid tumor, or uterine / endometrial cancer.

[0316] In some embodiments, the antigen recognition domain of the CAR specifically binds to tumor-associated TIM-3, and effector cells comprising the CAR and the disclosed solid tumor targeting scaffold are useful for treating one or more cancers including at least bladder cancer, colorectal cancer, gastric cancer, liver cancer, lung cancer, prostate cancer, kidney cancer, or urothelial cancer.

[0317] In some embodiments, the antigen recognition domain of the CAR specifically binds to tumor-associated TMEM238, and effector cells comprising the CAR and the disclosed solid tumor targeting scaffold are useful for treating one or more cancers including at least colorectal cancer, esophageal cancer, gastric / stomach cancer, or ovarian cancer.

[0318] In some embodiments, the antigen recognition domain of the CAR specifically binds to tumor-associated TNC, and effector cells comprising the CAR and the disclosed solid tumor targeting scaffold are useful for treating one or more cancers including at least bladder cancer, brain cancer, breast cancer, or head and neck cancer.

[0319] In various embodiments, CARs applicable to the cells described herein include at least an extracellular domain, a transmembrane domain, and an intracellular domain. In some embodiments, the intracellular domain of the CAR comprises at least one signaling domain that is activated upon antigen binding. In some embodiments of the CAR intracellular domain, one or more co-stimulatory domains (often referred to as "additional signaling domains") are further included for optimized functionality. Exemplary signaling proteins suitable for CAR design include, but are not limited to, 2B4, 4-1BB, CD16, CD2, CD28, CD28H, CD3ζ / 1XX (i.e., CD3ζ or CD3ζ1XX), DAP10, DAP12, DNAM1, FcERIγ, IL21R, IL-2Rβ (IL-15Rβ), IL-2Rγ, IL-7R, KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CS1, and CD8. Descriptions of exemplary signaling proteins (including transmembrane and cytoplasmic sequences of the proteins) are provided below and further in Table 3A.

[0320] [Table 21-1]

[0321] [Table 21-2]

[0322]

Table 21-3

[0323]

Table 21-4

[0324] In some embodiments of the CARs applicable to the cells provided herein, the intracellular domain of the CAR comprises at least a first signaling domain having an amino acid sequence that has at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the cytoplasmic domain of 2B4, 4-1BB, CD16, CD2, CD28, CD28H, CD3ζ, CD3ζ1XX, DAP10, DAP12, DNAM1, FcERIγ IL21R, IL-2Rβ (IL-15Rβ), IL-2Rγ, IL-7R, KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CS1, or CD8, or a portion thereof, as represented by SEQ ID NOs: 54-76, respectively. In some embodiments, the first signaling domain comprises an amino acid sequence that is at least 90% identical to any of SEQ ID NOs: 54-76. In some embodiments, the first signaling domain comprises an amino acid sequence that is at least 95% identical to any of SEQ ID NOs: 54-76. In some embodiments, the first signaling domain comprises the amino acid sequence of any of SEQ ID NOs: 54-76. In some embodiments, the signaling domain of the CAR comprises only a portion of the cytoplasmic domain of 2B4, 4-1BB, CD16, CD2, CD28, CD28H, CD3ζ, CD3ζ1XX, DAP10, DAP12, DNAM1, FcERIγ IL21R, IL-2Rβ (IL-15Rβ), IL-2Rγ, IL-7R, KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CS1, or CD8. In some embodiments, the portion of the cytoplasmic domain selected for the CAR signaling domain comprises an amino acid sequence that has at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to an ITAM (immunoreceptor tyrosine-based activation motif), YxxM motif, TxYxxV / I motif, FcRγ, hemi-ITAM, and / or ITT-like motif.

[0325] In some embodiments of the CARs provided, the intracellular domain of the CAR comprising a first signaling domain further comprises a second signaling domain comprising an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the cytoplasmic domain of 2B4, 4-1BB, CD16, CD2, CD28, CD28H, CD3ζ, CD3ζ1XX, DAP10, DAP12, DNAM1, FcERIγ IL21R, IL-2Rβ (IL-15Rβ), IL-2Rγ, IL-7R, KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CS1, or CD8, or a portion thereof, and the second signaling domain is different from the first signaling domain. In some embodiments, the second signaling domain comprises an amino acid sequence that is at least 90% identical to any of SEQ ID NOs: 54-76. In some embodiments, the second signaling domain comprises an amino acid sequence that is at least 95% identical to any of SEQ ID NOs: 54-76. In some embodiments, the second signaling domain comprises an amino acid sequence of any of SEQ ID NOs: 54-76.

[0326] In some embodiments of the provided CARs, the intracellular domain of the CAR comprising the first and second signaling domains further comprises a third signaling domain comprising an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the cytoplasmic domain of 2B4, 4-1BB, CD16, CD2, CD28, CD28H, CD3ζ, CD3ζ1XX, DAP10, DAP12, DNAM1, FcERIγ IL21R, IL-2Rβ (IL-15Rβ), IL-2Rγ, IL-7R, KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CS1, or CD8, or a portion thereof, wherein the third signaling domain is different from the first and second signaling domains. In some embodiments, the third signaling domain comprises an amino acid sequence that is at least 90% identical to any of SEQ ID NOs: 54-76. In some embodiments, the third signaling domain comprises an amino acid sequence that is at least 95% identical to any of SEQ ID NOs: 54-76. In some embodiments, the third signaling domain comprises an amino acid sequence of any of SEQ ID NOs: 54-76. In some embodiments, the signaling proteins suitable for designing the signaling domains of the CAR intracellular domain further comprise CD27, OX40, ICOS, PD-1, LAG-3, BTLA, or CTLA-4.

[0327] In some exemplary embodiments of CARs having an intracellular domain consisting of only one signaling domain, the intracellular domain comprises an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the cytoplasmic domain of a protein including, but not limited to, DNAM1, CD28H, KIR2DS2, DAP12, or DAP10, or a portion thereof.

[0328] In some exemplary embodiments of a CAR having an intracellular domain composed of two different signaling domains, the intracellular domain comprises a fusion cytoplasmic domain or a portion thereof in a form including, but not limited to, 2B4-CD3ζ / 1XX (i.e., 2B4-CD3ζ or 2B4-CD3ζ1XX, the same applies hereinafter), 2B4-DNAM1, 2B4-FcERIγ, 2B4-DAP10, CD16-DNAM1, CD16-DAP10, CD16-DAP12, CD2-CD3ζ / 1XX, CD2-DNAM1, CD2-FcERIγ, CD2-DAP10, CD28-DNAM1, CD28-FcERIγ, CD28-DAP10, CD28-DAP12, CD28-CD3ζ / 1XX, CD28H-CD3ζ / 1XX, DAP10-CD3ζ / 1XX, DAP10-DAP12, DAP12-CD3ζ / 1XX, DAP12-DAP10, DNAM1-CD3ζ / 1XX, KIR2DS2-CD3ζ / 1XX, KIR2DS2-DAP10, KIR2DS2-2B4, or NKp46-2B4.

[0329] In some exemplary embodiments of a CAR having an intracellular domain composed of three different signaling domains, the intracellular domain comprises a fusion cytoplasmic domain or a portion thereof in a form including, but not limited to, 2B4-DAP10-CD3ζ / 1XX, 2B4-IL21R-DAP10, 2B4-IL2RB-DAP10, 2B4-IL2RB-CD3ζ / 1XX, 2B4-41BB-DAP10, CD16-2B4-DAP10, or KIR2DS2-2B4-CD3ζ / 1XX.

[0330] In some embodiments, the transmembrane domain of the CAR comprises an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the full length or a portion of the transmembrane region of CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8, CD8a, CD8b, CD16, CD27, CD28, CD28H, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA4, PD1, LAG3, 2B4, BTLA, DNAM1, DAP10, DAP12, FcERIγ, IL7, IL12, IL15, KIR2DL4, KIR2DS1, KIR2DS2, NKp30, NKp44, NKp46, NKG2C, NKG2D, CS1, or a T cell receptor polypeptide. In some other embodiments, the transmembrane domain of the CAR comprises an amino acid sequence having at least about 85%, about 90%, about...

Claims

1. A cell or a group thereof, (i) The cells are (a) immune cells, (b) induced pluripotent cells (iPSCs), or (c) derived effector cells obtained by differentiating iPSCs. (ii) The cell comprises a solid tumor targeting skeleton, and the solid tumor targeting skeleton is (a) Polynucleotides encoding a C-X-C motif chemokine receptor or a variant thereof, (b) Polynucleotides encoding a TGFβ signaling redirector receptor (TGFβ-SRR), including a partial or complete peptide of the extracellular domain (ECD) of the transformed growth factor beta receptor (TGFβR), A cell or population thereof comprising two or more of (c) polynucleotides encoding alloimmune protective receptors (ADRs).

2. The cells or population thereof according to claim 1, wherein the cells have improved transport in solid tumors, tumor microenvironment (TME) resistance, and / or alloreactivity resistance compared to corresponding cells that do not contain the solid tumor targeting scaffold.

3. The solid tumor targeting skeleton is (i) CD38 knockout, (ii) Polynucleotides encoding exogenous CD16 or its variants, and (iii) Polynucleotides encoding cytokine signaling complexes comprising partial or complete peptides of cell surface-expressed exogenous cytokines and / or their receptors, The cells or population thereof according to claim 1, further comprising:

4. The cell is (i) Chimeric antigen receptor (CAR), (ii) HLA-I deficiency and / or HLA-II deficiency, (iii) Introduction of HLA-G or non-cleaving HLA-G, (iv) Destruction of at least one of B2M, CIITA, TAP1, TAP2, Tapasin, NLRC5, RFXANK, RFX5, RFXAP, TCR, NKG2A, NKG2D, CD25, CD44, CD54, CD56, CD58, CD69, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT, (v) Introduction of at least one of the following: HLA-E, 4-1BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A2AR, antigen-specific TCR, chimeric fusion receptor (CFR), Fc receptor, antibody or functional variant or fragment thereof, checkpoint inhibitor, engager, and surface trigger receptor for coupling with an agonist, or (vi) At least one of the genotypes listed in Table 4, The cell or population thereof according to claim 1, further comprising one or more of the above.

5. The cell or population thereof according to claim 1, wherein the C-X-C motif chemokine receptor comprises CXCR2 or CXCR3.

6. The cell or population thereof according to claim 1, wherein the TGFβ-SRR further comprises a partial or complete peptide of the intracellular domain (ICD) of a cytokine receptor comprising IL2R, IL12R, IL18R, IL21R, or any combination thereof.

7. (a) The cytokine receptor is IL2Rβ, thereby forming a TGFβR2-IL2Rβ redirector receptor, wherein the intracellular domain (ICD) of IL2Rβ includes the amino acid sequence represented by SEQ ID NO: 11, or (b) The cytokine receptor is IL12Rβ, thereby forming a TGFβR2-IL12Rβ redirector receptor, and the intracellular domain (ICD) of IL12Rβ contains the amino acid sequence represented by SEQ ID NO: 12 or SEQ ID NO: 13, or (c) The cytokine receptor is IL18Rβ, thereby forming a TGFβR2-IL18Rβ redirector receptor, and the intracellular domain (ICD) of IL18Rβ contains the amino acid sequence represented by SEQ ID NO: 14, or (d) The cytokine receptor is IL21R, thereby forming a TGFβR2-IL21R redirector receptor, and the intracellular domain (ICD) of IL21Rβ contains the amino acid sequence represented by SEQ ID NO: 15, or (e) The cell or population thereof according to claim 6, wherein the extracellular domain (ECD) of TGFβR comprises the amino acid sequence represented by SEQ ID NO:

10.

8. The cell or population thereof according to claim 6, wherein the cytokine receptor is a fragment of IL2Rβ that forms a TGFβR2-trIL12Rβ redirector receptor, comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, or 97%, 98%, or 99% sequence identity with respect to the sequence represented by SEQ ID NO: 16, and the amino acid sequence represented by SEQ ID NO: 17 included in SEQ ID NO: 16 is variable.

9. The cell or population thereof according to claim 1, wherein the ADR is specific to 4-1BB or CD38.

10. The cell or population thereof according to claim 1, wherein two or more polynucleotides of the solid tumor targeting scaffold are inserted into the endogenous CD38 gene locus to knock out CD38.

11. The cell or population thereof according to claim 3, wherein the polynucleotide encoding the exogenous CD16 or a variant thereof and two or more polynucleotides of the solid tumor targeting skeleton are co-expressed within a tricistronic construct.

12. The exogenous CD16 or its variant is (a) High affinity non-cleavable CD16 (hnCD16), (b) F176V and S197P in the extracellular domain of CD16, (c) Complete or partial extracellular domains derived from CD64, (d) Non-natural (or non-CD16) transmembrane domains, (e) Non-natural (or non-CD16) intracellular domains, (f) Non-natural (or non-CD16) signaling domains, (g) unnatural irritant domains, and (h) Transmembrane domains, signaling domains, and stimulating domains that are not derived from CD16 and are derived from the same or different polypeptides, The cells or population thereof according to claim 3, comprising at least one of the above.

13. The cell further comprises the cytokine signaling complex, and the cytokine signaling complex is (a) cell surface-expressed exogenous cytokines or partial or complete peptides of their receptors, comprising IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, or at least one of their respective receptors, (b) (i) Co-expression of IL15 and IL15Rα with a self-cleaving peptide in between, (ii) Fusion protein of IL15 and IL15Rα (iii) IL15 / IL15Rα fusion protein in which the intracellular domain of IL15Rα is shortened. (iv) A fusion protein of IL15 and the membrane-bound Sushi domain of IL15Rα, (v) Fusion protein of IL15 and IL15Rβ, (vi) A fusion protein of IL15 and the common receptor γC, wherein the common receptor γC is either native or modified, and (vii) At least one of the homodimers of IL15Rβ, (b) At least one of (i) to (vii) can be co-expressed with CAR in a separate construct or in a bicistronic construct, (c) (i) A fusion protein of IL7 and IL7Rα, (ii) A fusion protein of IL7 and the common receptor γC, wherein the common receptor γC is either natural or modified, and (iii) At least one of the homodimers of IL7Rβ, (c) at least one of (i) to (iii) which is optionally co-expressed with CAR in a separate construct or in a bisistronic expression cassette, Optional, (d) The cells or population thereof according to claim 3, which are transiently expressed.

14. The cell further comprises CAR, and the CAR is (i) T cell specific or NK cell specific, (ii) bispecific antigen binding CAR; (iii) Switchable CAR, (iv) Dimerized CAR, (v) Split CAR, (vi) multi-chain CAR; (vii) inducible CAR; (viiii) Those co-expressed with other CARs, (ix) In the bisistronic construct, co-expressed with the cytokine signaling complex, (x) Co-expressed with a checkpoint inhibitor in a separate construct of any choice, or in a bisistronic construct. (xi) Specific to at least one tumor-associated antigen, including CD19, B7H3, BCMA, CD20, CD22, CD38, CD123, CD79b, CD52, EGFR, EGP2 / EpCAM, GD2, GPRC5D, HER2, KLK2, MICA / B, MSLN, VEGF-R2, PSMA, and PDL1, and / or (xi) 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, antigens of cytomegalovirus (CMV) infected cells, epithelial glycoprotein EGP-2, Epithelial glycoprotein-40, EpCAM, EGFRvIII, Receptor tyrosine protein kinase erb-B2, 3, 4, EGFIR, EGFR-VIII, ERBB folate-binding protein (FBP), Fetal acetylcholine receptor (AChR), Folate receptor-α, Ganglioside G2 (GD2), Ganglioside G3 (GD3), Human epidermal growth factor receptor 2 (HER2), Human telomerase Reverse transcriptase (hTERT), ICAM-1, integrin B7, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), κ-light chain, kinase insertion domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (L1-CAM), LILRB2, melanoma antigen family A1 (MAGE-A1), MICA / B, mucin 1 (Muc-1), mucin 16 (Muc-16), mesoserine (MSLN), NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, tumor embryonic 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 specific to at least one tumor-associated antigen, including pathogen antigens. The cells or population thereof according to claim 4.

15. The CAR is (a) An extracellular domain comprising an antigen-binding domain that recognizes the HER2 (human epidermal growth factor receptor 2) antigen, wherein the antigen-binding domain is (i) Heavy chain variable (VH) domains including heavy chain complementarity determination region 1 (H-CDR1) containing SEQ ID NO: 103, heavy chain complementarity determination region 2 (H-CDR2) containing SEQ ID NO: 104, and heavy chain complementarity determination region 3 (H-CDR3) containing SEQ ID NO: 105, and optionally, (ii) An extracellular domain comprising a light chain complementarity determination region 1 (L-CDR1) containing SEQ ID NO: 106, a light chain complementarity determination region 2 (L-CDR2) containing SEQ ID NO: 107, and a light chain variable (VL) domain containing a light chain complementarity determination region 3 (L-CDR3) containing SEQ ID NO: 108, (b) Transmembrane domain and (c) an intracellular domain comprising at least one signaling domain, The cells or population thereof according to claim 4, wherein at least one signaling domain specifically responds to the binding of the CAR to the HER2 antigen expressed on cancer cells, thereby generating a cancer antigen-specific response.

16. The antigen-binding domain of the CAR is (a) comprising a VH domain having at least 80% sequence identity with SEQ ID NO: 109, (b) comprising a VL domain having at least 80% sequence identity with SEQ ID NO: 110, (c) comprising a single-stranded variable fragment (scFV) containing a VH-linker-VL or a VL-linker-VH, wherein the linker differs in length and sequence, and optionally the linker has at least 80% sequence identity with sequence numbers 111-114. (d) comprising an scFV represented by an amino acid sequence that is at least about 99%, about 98%, about 96%, about 95%, about 90%, about 85%, or about 80% identical to SEQ ID NO: 115 or SEQ ID NO: 116, wherein each of SEQ ID NOs: 115 and 116 contains a linker of different length and sequence, and / or (e) The humanized cells or population thereof according to claim 15.

17. The cells or population thereof according to claim 1, wherein (i) the iPSC is a clonal iPSC, a single-cell dissociated iPSC, an iPSC cell line, or an iPSC master cell bank (MCB) cell, or (ii) the derived cells include derived CD34+ cells, derived hematopoietic stem progenitor cells, derived hematopoietic pluripotent progenitor cells, derived T cell precursors, derived NK cell precursors, derived T lineage cells, derived NKT lineage cells, derived NK lineage cells, or derived B lineage cells, or (iii) the derived cells include derived effector cells having one or more functional characteristics not present in the corresponding primary T, NK, NKT, and / or B cells.

18. The derived cells are compared to their corresponding primary cells obtained from peripheral blood, umbilical cord blood, or any other donor tissue that does not have the same gene editing, (i) Increased cytotoxicity, (ii) Improved persistence and / or survival rate, (iii) Enhanced ability to migrate and / or activate or mobilize bystander immune cells to tumor sites. (iv) Improved tumor infiltration, (v) Enhanced ability to reduce tumor immunosuppression, (vi) Improved ability to rescue tumor antigen escapes, (vii) controlled apoptosis, (viiii) Enhanced or acquired ADCC, and (ix) Ability to avoid flatteryside, A cell or population thereof according to claim 17, having therapeutic properties including one or more of the following.

19. The cells are NK cell lineage cells or T cell lineage cells, (i) The NK lineage cells or the T lineage cells have improved infiltration and / or retention at the tumor site, (ii) Whether the NK cell lineage can mobilize and / or migrate T cells to the tumor site, (iii) The cells or population thereof according to claim 17, wherein the NK cell lineage or the T cell lineage can reduce tumor immunosuppression in the presence of one or more checkpoint inhibitors.

20. A composition comprising the cells or population thereof described in Claim 1.

21. A therapeutic use of the composition according to claim 20, wherein the composition is introduced into a subject requiring adoptive cell therapy, the subject having an autoimmune disorder, hematological malignancy, solid tumor, cancer, or viral infection.

22. A method for improving anti-HER2 monoclonal antibody (mAb) therapy, Introducing a composition comprising effector cells containing a polynucleotide encoding CasMab250-CAR, a polynucleotide encoding CXCR2, a polynucleotide encoding TGFβ-SRR, and a polynucleotide encoding exogenous CD16 or a variant thereof, into a subject requiring the aforementioned treatment, A method comprising introducing an anti-HER2 mAb to the subject.