Genetically engineered cells having chimeric antigen receptors with CD30 endodomains, and uses thereof

Abstract

Provided are genetically engineered induced pluripotent stem cells (iPSCs) and derivative cells thereof expressing an anti-Nectin4, anti-CD19, and / or anti-CD22 chimeric antigen receptor (CAR) having a costimulatory domain comprising CD30 or a fragment thereof, and methods of using the same. Also provided are compositions, polypeptides, vectors, and methods of manufacturing.

Description

[0001] Docket No. CNTY-033-WO-01

[0002] GENETICALLY ENGINEERED CELLS HAVING CHIMERIC ANTIGEN RECEPTORS WITH CD30 ENDODOMAINS, AND USES THEREOF

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U. S. Provisional Patent Application No.

[0004] 63 / 728.956 filed December 6, 2024, which is incorporated by reference herein in its entirety.

[0005] TECHNICAL FIELD

[0006] This application provides genetically engineered induced pluripotent stem cells (iPSCs) and derivative cells thereof. Also provided are uses of the iPSCs or derivative cells thereof to express a chimeric antigen receptor for allogenic cell therapy. Also provided are related vectors, polynucleotides, and pharmaceutical compositions.

[0007] REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0008] This application contains a sequence listing, which is submitted electronically via EFS-Web as an XML formatted sequence listing w ith a file name “SequenceListing_ST26'’ having a file size of 557 kilobytes, and a creation date of December 5, 2025. The sequence listing submitted via EFS-Web is part of the specification and is herein incorporated by reference in its entirety.

[0009] BACKGROUND

[0010] Cancer remains a significant global health challenge, with a critical need for innovative and adaptable immunotherapeutic approaches. Chimeric antigen receptor (CAR) cell therapies have emerged as a promising strategy to target specific malignancies, offering the potential for more precise and effective cancer treatment. However, current CAR-T cell therapies face substantial limitations, including limited persistence, variable efficacy, and manufacturing challenges. Induced pluripotent stem cell (iPSC)-derived CAR cell therapies represent a promising solution to address these limitations. By utilizing iPSCs as a cellular source, researchers can generate an off-the-shelf, allogeneic cell therapy platform with several key advantages. These include the ability to generate large-scale, genetically uniform cell populations. Docket No. CNTY-033-WO-01

[0011] implement precise genetic modifications, and potentially overcome the manufacturing constraints associated with autologous CAR-T cell therapies.

[0012] For example, Nectin-4, a cell adhesion molecule overexpressed in multiple solid tumors, including lung, breast, and bladder cancers, presents an attractive target for CAR-based immunotherapies. However, existing CAR designs have struggled to maintain long-term anti-tumor efficacy and cellular persistence necessary for comprehensive cancer treatment. The incorporation of the CD30 endodomain into CAR constructs targeting Nectin-4 offers a novel approach to enhancing cellular functionality. CD30, a member of the tumor necrosis factor receptor superfamily, provides unique signaling capabilities that may overcome current limitations in CAR cell therapies. Specifically, the CD30 endodomain has demonstrated potential to enhance T cell proliferation, improve cytokine production, increase resistance to cellular exhaustion, and / or promote more sustained anti-tumor activity.

[0013] By combining the versatility of iPSC-derived cellular platforms with a CD30-optimized CAR, embodiments the present disclosure can provide a more robust, persistent, and effective cell therapy approach. This strategy addresses critical challenges in current cancer immunotherapies, including limited cellular persistence, variable efficacy, and the need for personalized, scalable treatment options.

[0014] BRIEF SUMMARY

[0015] The present disclosure provides genetically engineered induced pluripotent stem cells (iPSCs) and derivative cells thereof designed for enhanced cancer immunotherapy. The cells incorporate novel combinations of genetic modifications that address key limitations in cunent cell-based therapeutics, including limited tumor targeting, inadequate persistence, immune rejection, and safety concerns. In particular, the disclosure provides iPSCs and derivative cells engineered with chimeric antigen receptors (CARs) comprising a CD30 costimulatory domain, along with strategic genomic modifications to enhance therapeutic efficacy, enable allogeneic administration, and provide safety controls.

[0016] Current CAR-based cellular immunotherapies face several technical challenges that limit their clinical utility. These include: (1) insufficient CAR T cell persistence and proliferation in vivo, leading to limited durability of response; (2) immunogenicity of allogeneic cells due to expression of major histocompatibility complex (MHC) molecules, necessitating HLA-matching and limiting off-the-shelf Docket No. CNTY-033-WO-01

[0017] availability; (3) tumor antigen escape through downregulation or loss of single target antigens; (4) limited ability to target solid tumors due to the immunosuppressive tumor microenvironment; (5) inability to leverage antibody-dependent cellular cytotoxicity (ADCC) mechanisms; and (6) lack of effective safety mechanisms to eliminate therapeutic cells in case of adverse events. The present disclosure addresses these challenges through a multi-pronged engineering approach applied at the iPSC stage, enabling scalable production of engineered therapeutic cells with enhanced properties.

[0018] In one general aspect, provided is an induced pluripotent stem cell (iPSC) or a derivative cell thereof comprising: one or more exogenous polynucleotides encoding a chimeric antigen receptor (CAR) comprising (i) a costimulatory domain comprising CD30 or a fragment thereof, and (ii) one or more antigen binding domains each targeting a tumor antigen independently selected from the group consisting of a Nectin-4 antigen, a CD 19 antigen, and a CD22 antigen; and at least one of: (i) a deletion or reduced expression of one or more of B2M, TAPI, TAP2. Tapasin, RFXANK, CIITA, RFX5, RFXAP genes; (ii) an exogenous polynucleotide encoding a natural killer (NK) cell receptor immunoglobulin gamma Fc region receptor III (FcyRIII), cluster of differentiation 16 (CD 16) and / or an NKG2D protein; (iii) a deletion or reduced expression of one or more of NKG2A or CD70, CD38, and CD33 genes; (iv) an exogenous polynucleotide encoding a cytokine: (v) an exogenous polynucleotide encoding a safety switch: (vi) an exogenous polynucleotide encoding a PSMA cell tracer; and (vii) an exogenous polynucleotide encoding a membrane bound IL- 12 polypeptide.

[0019] The incorporation of a CD30 costimulatory domain in the CAR represents a significant advancement over conventional CAR designs. CD30, a member of the tumor necrosis factor receptor superfamily, provides potent costimulatory signals that enhance T cell activation, proliferation, and cytokine production. Unlike traditional costimulatory domains such as CD28 or 4-1BB, the CD30 costimulatory domain can provide sustained signaling that promotes long-term cell persistence while maintaining appropriate activation thresholds to avoid excessive toxicity. In certain embodiments, the CD30 comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 395. In certain embodiments, the one or more exogenous polynucleotides encoding the CAR comprising the costimulatory domain comprising CD30 comprises nucleotides having Docket No. CNTY-033-WO-01

[0020] at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 396.

[0021] The disclosure provides CARs targeting clinically important tumor antigens. Nectin-4 is a cell adhesion molecule that is overexpressed in multiple solid tumor ty pes including bladder, breast, lung, pancreatic, and ovarian cancers, while showing limited expression in normal tissues, making it an ideal target for CAR-based therapy. CD 19 and CD22 are B cell lineage markers that are expressed on B cell malignancies including acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and non-Hodgkin lymphoma (NHL). In certain embodiments, the CAR is a dualtargeting CAR, wherein the one or more antigen binding domains comprises a first antigen binding domain targeting Nectin-4, and wherein the one or more antigen binding domains comprises an additional antigen-binding domain that specifically binds to an antigen selected from the group consisting of CD70, Folate Receptor alpha, FSHR, mesothelin, and SLITRK6. Dual -targeting CARs provide significant advantages by reducing the risk of tumor escape through antigen loss or downregulation, as tumor cells must lose both target antigens to evade CAR-mediated killing. In certain embodiments, the CAR is a dual-targeting CAR, wherein the one or more antigen binding domains comprises a first antigen binding domain targeting CD 19, and wherein the one or more antigen binding domains comprises an additional antigen-binding domain that specifically binds to CD22, which is particularly advantageous for treating B cell malignancies where CD19 escape variants have been observed following CD19-directed therapy.

[0022] In certain embodiments, the one or more antigen binding domains comprises an anti-Nectin4 VHH domain. VHH domains, also known as single-domain antibodies or nanobodies, are derived from heavy -chain-only antibodies naturally occurring in camelids. VHH domains offer several advantages over conventional scFv-based antigen binding domains, including: smaller size (approximately 15 kDa versus 25-30 kDa for scFvs). enabling better tissue penetration; increased stability and resistance to aggregation; the ability to recognize epitopes not accessible to conventional antibodies, such as cryptic or concave epitopes; and reduced immunogenicity. These properties make VHH-based CARs particularly suitable for targeting solid tumors where tissue penetration and stability in the tumor microenvironment are critical factors for therapeutic efficacy. Docket No. CNTY-033-WO-01

[0023] To enhance the persistence and proliferative capacity of the engineered cells, certain embodiments comprise an exogenous polynucleotide encoding a cytokine. In certain embodiments, the cytokine comprises interleukin- 15 (IL-15). IL-15 is a critical cytokine for the maintenance, proliferation, and survival of lymphocytes, including T cells and NK cells. Unlike IL-2, which can promote activation-induced cell death and expansion of regulatory T cells, IL- 15 promotes memory T cell formation and longterm persistence without these undesirable effects. In certain embodiments, the IL- 15 comprises an IL-15 and an IL-15 receptor alpha (IL-15Ra) fusion polypeptide, which provides enhanced stability and bioavailability through trans-presentation mechanisms that mimic physiological IL- 15 signaling. The IL-15 / IL-15Ra fusion can function in both autocrine and paracrine manners to support not only the engineered cells themselves but also endogenous immune cells in the tumor microenvironment. In certain embodiments, the IL-15 comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 72. In certain embodiments, the iPSC or derivative cell further comprises an inactivated cell surface receptor that comprises a monoclonal antibody-specific epitope, wherein the inactivated cell surface receptor and the IL- 15 are operably linked by an autoprotease peptide. This configuration allows for coordinate expression of the IL- 15 and a safety mechanism (described below) from a single genetic construct.

[0024] A critical challenge for allogeneic cell therapy is immune rejection mediated by recipient immune cells recognizing donor MHC molecules. To address this, certain embodiments comprise a deletion or reduced expression of one or more of B2M, TAPI, TAP2, Tapasin. RFXANK. CIITA. RFX5, and RFXAP genes. B2M (beta-2-microglobulin) is an essential component of MHC class I molecules; deletion or disruption of B2M prevents surface expression of MHC class I, thereby avoiding recognition and rejection by recipient cytotoxic T cells. CIITA (class II major histocompatibility complex trans activator) is a master regulator of MHC class II expression; deletion or disruption of CIITA prevents MHC class II expression, avoiding recognition by recipient CD4+ T cells. TAPI, TAP2, Tapasin, RFXANK, RFX5, and RFXAP are components of the MHC class I and / or class II antigen presentation machinery'; their deletion or disruption provides alternative or complementary approaches to reducing immunogenicity. In certain embodiments, one or more of the exogenous polynucleotides are integrated at one or more loci on the Docket No. CNTY-033-WO-01

[0025] chromosome of the cell selected from genes encoding these MHC-related components, such that the integration results in a deletion or reduced expression of the gene. In certain embodiments, the iPSC or derivative cell has a deletion or reduced expression of one or more of B2M or CIITA genes. In certain embodiments, the iPSC or derivative cell comprises the deletion or reduced expression of B2M and CIITA genes, providing elimination of both MHC class I and class II expression.

[0026] To further enhance the therapeutic potential of the engineered cells by enabling antibody-dependent cellular cytotoxicity (ADCC) and enhanced tumor recognition, certain embodiments comprise an exogenous polynucleotide encoding a natural killer (NK) cell receptor immunoglobulin gamma Fc region receptor III (FcyRIII. cluster of differentiation 16, CD16) and / or anNKG2D protein. CD16 is an Fc receptor that enables cells to recognize and kill antibody -coated target cells through ADCC. This allows the engineered cells to leverage therapeutic antibodies already approved for cancer treatment, such as trastuzumab (for HER2+ cancers), cetuximab (for EGFR+ cancers), and rituximab (for CD20+ B cell malignancies), thereby providing dual targeting mechanisms: CAR-mediated killing and antibody-mediated killing. NKG2D is an activating receptor that recognizes stress-induced ligands (such as MICA, MICB, and ULBP family members) that are upregulated on tumor cells and cells undergoing stress or transformation. Expression of NKG2D provides an additional. CAR-independent mechanism for tumor recognition and killing that is particularly valuable when tumor cells downregulate the CAR target antigen.

[0027] In certain embodiments, the CD16 is a CD16 variant protein designed to enhance ADCC activity. In certain embodiments, the CD 16 variant protein is a high affinity CD 16 variant that exhibits increased binding affinity for the Fc region of antibodies. The natural CD16 protein contains a polymorphism at position 158, with individuals expressing either a phenylalanine (F158) or valine (V158) at this position. The VI 58 variant exhibits approximately 3 -fold higher affinity for IgGl compared to the Fl 58 variant. In certain embodiments, the CD 16 variant protein comprises wild-tjpe CD 16 having one or more amino acid substitutions selected from the group consisting of Fl 58V, Fl 76V, S197P, D205A, S219A, T220A. The F158V substitution provides enhanced binding to IgG antibodies. In certain embodiments, the CD 16 variant protein is anon-cleavable CD 16 variant that resists proteolytic cleavage from the cell surface, thereby maintaining high levels of surface CD 16 expression. Docket No. CNTY-033-WO-01

[0028] Wild-type CD 16 is subject to metalloproteinase-mediated cleavage that results in shedding of the extracellular domain, reducing ADCC capacity over time. The S197P substitution eliminates a primary cleavage site, creating a non-cleav able variant that maintains persistent surface expression and sustained ADCC function. In certain embodiments, the CD 16 variant protein comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any¬ one of SEQ ID NOS: 187 and 188.

[0029] In certain embodiments, the iPSC or derivative cell comprises an exogenous polynucleotide encoding the CD 16 protein and the NKG2D protein, wherein the CD 16 protein and the NKG2D protein are operably linked by an autoprotease peptide. Autoprotease peptides, also known as self-cleaving peptides or 2A peptides, enable the expression of multiple proteins from a single open reading frame. During translation, the 2A peptide undergoes a ribosomal "skipping" event that results in separation of the upstream and downstream proteins, producing equimolar amounts of both proteins from a single promoter. This approach provides several advantages: (1) simplified genetic engineering by requiring insertion of only a single genetic cassette; (2) coordinate regulation ensuring both proteins are expressed at similar levels; and (3) conservation of genetic cargo space in viral vectors or chromosomal integration sites. In certain embodiments, the NKG2D protein is a wildtype NKG2D protein. In certain embodiments, the NKG2D protein comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 190. In certain embodiments, the autoprotease peptide is selected from the group consisting of a porcine teschovirus-1 2 A (P2A) peptide, a foot-and-mouth disease virus 2A (F2A) peptide, an Equine Rhinitis A Virus (ERAV) 2A (E2A) peptide, a Thosea asigna virus 2A (T2A) peptide, a cytoplasmic polyhedrosis virus 2A (BmCPV2A) peptide, and a Flacherie Virus 2A (BmIFV2A) peptide. In certain embodiments, the autoprotease peptide is a P2A peptide comprising amino acids having at least 90% sequence identity to SEQ ID NO: 192. In certain embodiments, the exogenous polynucleotide encoding the CD 16 protein and the NKG2D protein comprises polynucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 193.

[0030] To further optimize the therapeutic properties of the engineered cells, certain embodiments comprise a deletion or reduced expression of one or more inhibitory receptors or checkpoint molecules. In certain embodiments, the iPSC or derivative Docket No. CNTY-033-WO-01

[0031] cell comprises a deletion or reduced expression of one or more of NKG2A or CD70, CD38, and CD33 genes. NK. G2A is an inhibitory receptor that recognizes HLA-E; deletion of NKG2A prevents inhibitory signaling that would otherwise suppress cell activity in the presence of HLA-E-expressing cells, which include many tumor cells that upregulate HLA-E as an immune evasion mechanism. CD38 is expressed on many immune cells and its deletion can prevent fratricide (self-killing) in contexts where the CAR or other receptors might recognize CD38. CD70 and CD33 are additional targets whose deletion can provide advantages depending on the specific therapeutic application and tumor ty pe being targeted.

[0032] A key advantage of the present disclosure is the strategic integration of exogenous polynucleotides at specific genomic loci to achieve multiple objectives simultaneously: (1) targeted disruption of genes whose elimination is desirable (such as B2M or CIITA to reduce immunogenicity); (2) integration at "safe harbor" loci that allow robust transgene expression without disrupting essential genes; and (3) knockdown of genes encoding inhibitory’ receptors or checkpoint molecules. In certain embodiments, one or more of the exogenous polynucleotides are integrated at one or more loci on the chromosome of the cell selected from the group consisting of AAVS1, CLYBL, CCR5, ROSA26, collagen, HTRP, Hll, GAPDH, RUNX1, B2M, TAPI, TAP2, Tapasin. NLRC5, RFXANK, CIITA, RFX5, RFXAP, TCR a or constant region, NKG2A, NKG2D, CD33, CD38. CD70, TRAC, CIS, CBL-B.

[0033] SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT genes, provided at least one of the exogenous polynucleotides is integrated at a locus of a gene selected from the group consisting of B2M, TAPI, TAP2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes to thereby result in a deletion or reduced expression of the gene.

[0034] The AAVS1 locus (adeno-associated virus integration site 1) is a well-characterized safe harbor site in the human genome that allows stable, long-term transgene expression without silencing or disruption of essential genes. Integration at AAV S 1 provides reliable expression of the integrated transgene across multiple cell divisions and through differentiation processes. In certain embodiments, one or more of the exogenous polynucleotides are integrated at the loci of the AAVS1 and B2M genes, enabling both stable CAR expression (at AAVS1) and elimination of MHC class I expression (through B2M disruption). In certain embodiments, the iPSC or derivative cell has a deletion or reduced expression of one or more of B2M or CIITA genes. In certain embodiments, the iPSC or derivative cell comprises the deletion or Docket No. CNTY-033-WO-01

[0035] reduced expression of B2M and CIITA genes, providing a cell product with dramatically reduced immunogenicity suitable for allogeneic administration.

[0036] In a specific configuration that exemplifies the multi-pronged engineering approach, certain embodiments comprise exogenous polynucleotides integrated at the following loci: (i) the one or more exogenous polynucleotides encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain targeting a Nectin4 antigen is integrated at a locus of the AAVS1 gene; (ii) the exogenous polynucleotide encoding an NK cell receptor immunoglobulin gamma Fc region receptor III (FcyRIII, cluster of differentiation 16 (CD16)) and / or an NKG2D is integrated at a locus of the CD70 gene; (iii) the exogenous polynucleotide encoding the cytokine is integrated at the locus of the NK. G2A gene; (iv) the exogenous polynucleotide encoding a safety switch is integrated at the locus of the CLYBL gene; and (v) there is a deletion or reduced expression of the CIITA gene. This configuration provides: stable CAR expression (AAVS1 integration); ADCC capability and enhanced tumor recognition (CD16 / NKG2D at CD70 locus, with simultaneous CD70 disruption); enhanced persistence through autocrine cytokine support (IL- 15 at NKG2A locus, with simultaneous NKG2A disruption to remove inhibitory signaling); safety controls (safety switch at CLYBL safe harbor); and reduced immunogenicity (CIITA deletion eliminating MHC class II expression, plus B2M disruption if the CD16 / NKG2D cassette is integrated at B2M instead of CD70 in alternative embodiments).

[0037] The source of the iPSCs can significantly impact the properties of the derivative cells. In certain embodiments, the iPSC is reprogrammed from whole peripheral blood mononuclear cells (PBMCs). PBMCs are readily obtained from blood draws and represent a heterogeneous population including T cells, B cells, NK cells, and monocytes. In certain embodiments, the iPSC is derived from a reprogrammed T cell. T cell-derived iPSCs can retain epigenetic memory that facilitates differentiation back to T cell lineages, potentially providing derivative T cells with enhanced functional properties. iPSCs derived from T cells that have undergone antigen-specific selection and expansion may retain T cell receptor (TCR) rearrangements, providing additional antigen recognition capabilities in the derivative cells.

[0038] The iPSC or derivative cell comprises a CAR with a structure optimized for function. In certain embodiments, the CAR comprises: (i) a signal peptide; (ii) an extracellular domain comprising a binding domain that specifically binds the Nectin4 Docket No. CNTY-033-WO-01

[0039] antigen, the CD19 antigen, and / or the CD22 antigen; (iii) a hinge region; (iv) a transmembrane domain; (v) an intracellular signaling domain; and / or (vi) the coslimulatory domain comprising CD30 or a fragment thereof.

[0040] The signal peptide directs the nascent CAR protein to the endoplasmic reticulum for proper folding, glycosylation, and trafficking to the cell surface. In certain embodiments, the signal peptide comprises a GMCSFR (granulocytemacrophage colony-stimulating factor receptor) signal peptide or a MARS signal peptide, both of which are well-characterized signal sequences that provide efficient CAR surface expression.

[0041] The extracellular domain determines the target specificity of the CAR. In certain embodiments, the extracellular domain comprises a VHH single domain antibody that specifically binds the Nectin4 antigen. In certain embodiments, the extracellular domain comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 105-130, 379-381, and 384-388, providing a range of anti-Nectin4 VHH domains with varying affinities and epitope specificities. In certain embodiments, the extracellular domain is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 131-156, 382, 383, and 389-392. In certain embodiments, the CAR is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 171-184, and 394, providing complete CAR constructs optimized for expression and function.

[0042] For dual-targeting CARs, the iPSC or derivative cell comprises an additional CAR or additional antigen binding domains within a single CAR construct. In certain embodiments, the additional CAR comprises: (i) a signal peptide; (ii) an additional extracellular domain comprising a binding domain that specifically binds to an antigen selected from the group consisting of CD70, Folate Receptor alpha, FSHR, mesothelin, and SLITRK6; (iii) a hinge region; (iv) a transmembrane domain; (v) an intracellular signaling domain; and (vi) a co-stimulatory domain. In certain embodiments, the additional extracellular domain comprises a VHH or an scFv that specifically binds to an antigen selected from the group consisting of CD70, Folate Receptor alpha, FSHR, mesothelin, and SLITRK6. These additional targets are expressed on various solid tumors: CD70 is expressed on renal cell carcinoma and certain hematological malignancies; Folate Receptor alpha is overexpressed in Docket No. CNTY-033-WO-01

[0043] ovarian, endometrial, and lung cancers; FSHR (follicle stimulating hormone receptor) is expressed on ovarian cancers; mesothelin is expressed on mesothelioma, ovarian, pancreatic, and lung cancers; and SLITRK6 is expressed on certain solid tumors.

[0044] The hinge region provides spacing between the antigen binding domain and the cell membrane, affecting the flexibility and reach of the CAR. In certain embodiments, the hinge region for each of the CAR and the additional CAR are independently selected from the group consisting of a CD28 hinge region, an IgG4 hinge region, and a CD8 hinge region. The choice of hinge can be optimized based on the size and location of the target antigen epitope; longer hinges may be advantageous for epitopes close to the target cell membrane, while shorter hinges may be suitable for more distal epitopes.

[0045] The transmembrane domain anchors the CAR in the cell membrane and can influence CAR stability and signaling. In certain embodiments, the transmembrane domain for each of the CAR and the additional CAR are independently selected from the group consisting of a CD28 transmembrane domain and a CD8 transmembrane domain. The CD28 transmembrane domain can promote CAR dimerization and may enhance signaling, while the CD8 transmembrane domain provides a well-characterized alternative.

[0046] The intracellular signaling domain provides the primary activation signal upon CAR engagement with its target antigen. In certain embodiments, the intracellular signaling domain comprises a CD3^ intracellular domain. CD3^ is the primary signaling component of the T cell receptor complex and contains three immunoreceptor tyrosine-based activation motifs (IT AMs) that, when phosphorylated following antigen engagement, recruit and activate kinases that initiate the T cell activation cascade.

[0047] The co-stimulatory domain provides secondary signals that enhance T cell activation, proliferation, survival, and effector function. In certain embodiments, the co-stimulatory domain for each of the CAR and the additional CAR are independently selected from the group consisting of a CD28 signaling domain, a 4 IBB signaling domain, and a DAP 10 signaling domain. CD28 provides rapid but potentially shorter-lived activation signals, while 4- IBB provides sustained signaling that promotes longterm persistence. DAP 10 provides alternative costimulation that can be advantageous in certain contexts. The inclusion of the CD30 costimulatory domain, either alone or Docket No. CNTY-033-WO-01

[0048] in combination with these conventional costimulatory domains, provides enhanced signaling properties as discussed above.

[0049] In certain embodiments, the CAR comprises specific sequence elements optimized for function: (i) the signal peptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 1, 97, or 98; (ii) the extracellular domain comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity' to SEQ ID NOs: 105-130, 379-381, and 384-388, or the extracellular domain is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 131-156, 382, 383. and 389-392; (iii) the hinge region comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity' to SEQ ID NO: 21 or 96; (iv) the transmembrane domain comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 23 or 24; (v) the intracellular signaling domain comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 6, or the intracellular signaling domain is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 101; and (vi) the costimulatory domain comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 395.

[0050] In certain embodiments, the CAR comprises exact reference sequences: (i) the signal peptide comprises amino acids having the sequence of SEQ ID NO: 1, 97, or 98; (ii) the extracellular domain comprises amino acids having the sequence of one of SEQ ID NOs: 105-130; (iii) the hinge region comprises amino acids having the sequence of SEQ ID NO: 21 or 96; (iv) the transmembrane domain comprises amino acids having the sequence of SEQ ID NO: 23 or 24; (v) the intracellular signaling domain comprises amino acids having the sequence of SEQ ID NO: 6, or the intracellular signaling domain is encoded by the polynucleotide having the sequence of SEQ ID NO: 101; and (vi) the co-stimulatory domain comprises amino acids having the sequence of SEQ ID NO: 395.

[0051] Safety is a paramount concern in cellular immunotherapy, particularly given cases of severe toxicity observed with CAR T cell therapies. To provide clinicians with the Docket No. CNTY-033-WO-01

[0052] ability to eliminate the therapeutic cells if necessary, certain embodiments comprise an exogenous polynucleotide encoding a safety switch. In certain embodiments, the safety switch comprises an exogenous polynucleotide encoding an inactivated cell surface receptor that comprises a monoclonal antibody-specific epitope. This safety sw itch operates on the principle of targeted antibody-mediated depletion: the engineered cells express a cell surface epitope that can be recognized by an approved therapeutic antibody, allowing selective elimination of the engineered cells by administering the corresponding antibody, which triggers antibody -dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and / or antibodydependent cellular phagocytosis (ADCP) to eliminate the engineered cells. The cell surface receptor is "inactivated" in that it lacks functional intracellular signaling domains, thus serving solely as an epitope tag without affecting cell function unless the cognate antibody is administered.

[0053] In certain embodiments, the inactivated cell surface receptor is selected from the group of monoclonal antibody specific epitopes selected from epitopes specifically recognized by ibritumomab, tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, alemtuzumab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, polatuzumab vedotin, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, avelumab, ofatumumab, panitumumab, and ustekinumab. These antibodies are approved therapeutics with w ell-characterized safety profiles, providing a readily available means for eliminating engineered cells if needed. In certain embodiments, the inactivated cell surface receptor is a truncated epithelial growth factor receptor (tEGFR) variant that retains the epitope recognized by cetuximab but lacks the intracellular tyrosine kinase domain, thereby avoiding unwanted signaling. In certain embodiments, the tEGFR variant consists of amino acids having at least 90%, 91%, 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99% or 100% sequence identity to SEQ ID NO: 71.

[0054] Alternative safety switch mechanisms are also provided. In certain embodiments, the safety switch comprises an intracellular domain having a herpes simplex vims thymidine kinase (HSV-TK). HSV-TK phosphorylates nucleoside analogs such as ganciclovir, converting them into toxic metabolites that cause cell Docket No. CNTY-033-WO-01

[0055] death. Administration of ganciclovir to a patient whose engineered cells express HSV-TK results in selective killing of those cells. This approach provides an alternative safety mechanism that does not rely on antibody-mediated depletion.

[0056] The disclosure also provides combined safety switch and cell tracking capabilities. In certain embodiments, the iPSC or derivative cell comprises an exogenous polynucleotide encoding a PSMA cell tracer, wherein the PSMA cell tracer comprises an extracellular domain comprising a PSMA (prostate-specific membrane antigen) extracellular domain or fragment thereof. PSMA can be detected using approved PSMA-targeted imaging agents, allowing non-invasive tracking of engineered cell biodistribution and persistence in patients. In certain embodiments, the iPSC or derivative cell comprises a combined artificial cell death / reporter system polypeptide comprising an intracellular domain having a herpes simplex virus thymidine kinase (HSV-TK) and a linker, a transmembrane region, and an extracellular domain comprising the PSMA extracellular domain or fragment thereof. This combined construct provides both imaging capability (via PSMA detection) and safety switch functionality (via HSV-TK-mediated killing upon ganciclovir administration) in a single molecule.

[0057] In certain embodiments, the HSV-TK comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 229 or 230. In certain embodiments, the combined artificial cell death / reporter system polypeptide comprises the HSV -TK fused to a truncated variant PSMA polypeptide via the linker. In certain embodiments, the truncated variant PSMA polypeptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 231. In certain embodiments, the linker comprises an autoprotease peptide sequence selected from the group consisting of P2A peptide sequence, T2A peptide sequence, E2A peptide sequence, and F2A peptide sequence, providing coordinate expression of the HSV-TK and PSMA domains. In certain embodiments, the artificial cell death / reporter system polypeptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 232. In certain embodiments, the artificial cell death / reporter system polypeptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%. 99% or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOS: 233-235. In certain embodiments, the artificial cell Docket No. CNTY-033-WO-01

[0058] death / reporter system polypeptide is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%. 99% or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOS: 236-238.

[0059] In certain embodiments, the complete genetic configuration of the engineered iPSC or derivative cell is defined by specific sequence identities for each component: (i) the one or more exogenous polynucleotides encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain targeting a Nectin4 antigen comprises nucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more selected from the group consisting of SEQ ID NOs: 171-184 and 396; (ii) the exogenous polynucleotide encoding an NK cell receptor immunoglobulin gamma Fc region receptor III (FcyRIII. cluster of differentiation 16 (CD16)) and / or an NK. G2D protein comprises nucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more of SEQ ID NOs: 185, 189, and 191; (iii) the exogenous polynucleotide encoding a cytokine comprises nucleotides having at least 90%, 91%.

[0060] 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99% or 100% sequence identity to SEQ ID NO: 239; (iv) the exogenous polynucleotide encoding a safety switch comprises nucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more of SEQ ID NO: 236-238; and / or (v) the exogenous polynucleotide encodes a PSMA cell tracer, and the PSMA cell tracer comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 231.

[0061] In certain embodiments using exact reference sequences: (i) the one or more exogenous polynucleotides encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain targeting a Nectin4 antigen comprises nucleotides having a sequence selected from the group consisting of SEQ ID NOs: 171-184 and 396; (ii) the exogenous polynucleotide encoding an NK cell receptor immunoglobulin gamma Fc region receptor III (FcyRIII, cluster of differentiation 16 (CD 16)) and / or an NKG2D protein comprises nucleotides having a sequence of SEQ ID NO: 185, 189, or 191; (iii) the exogenous polynucleotide encoding the cytokine comprises nucleotides having a sequence of SEQ ID NO: 239; and / or (iv) the exogenous polynucleotide encoding the safety switch comprises nucleotides having a sequence of one of SEQ ID NOs: 236-238. Docket No. CNTY-033-WO-01

[0062] In certain embodiments, the strategic integration sites for the various genetic components are specified: the exogenous polynucleotides are integrated into a gene locus independently selected from the group consisting of an AAVS1 locus, a B2M locus, a CIITA locus, a CCR5 locus, a CD70 locus, a CLYBL locus, an NKG2A locus, an NKG2D locus, a CD33 locus, a CD38 locus, a TRAC locus, a TRBC1 locus, a ROSA26 locus, an HTRP locus, a GAPDH locus, a RUNX1 locus, a TAP1 locus, a TAP2 locus, a TAPBP locus, an NLRC5 locus, a RFXANK locus, a RFX5 locus, a RFXAP locus, a CISH locus, a CBLB locus, a SOCS2 locus, a PD1 locus, a CTLA4 locus, a LAG3 locus, a TIM3 locus, and a TIGIT locus. These integration sites provide various advantages: safe harbor loci (AAVS1, CLYBL. ROSA26, HTRP) allow stable transgene expression without disrupting essential genes; integration at immunogenicity-related loci (B2M, CIITA, TAPI, TAP2, TAPBP, NLRC5, RFXANK, RFX5, RFXAP) simultaneously provides transgene expression and reduces immunogenicity; integration at inhibitory receptor loci (NKG2A, PD1, CTLA4, LAG3, TIM3, TIGIT) simultaneously provides transgene expression and removes checkpoint inhibition; and integration at TCR loci (TRAC, TRBC1) can eliminate endogenous TCR expression in embodiments where TCR-independent T cell function is desired.

[0063] The engineered iPSCs can be differentiated into various therapeutic cell types. In certain embodiments, the derivative cell is a natural killer (NK) cell or a T cell. NK cells are innate immune cells with potent cytotoxic activity against tumor cells and infected cells. NK cells do not require HLA matching and can recognize stressed or transformed cells through a balance of activating and inhibitory receptors. The engineered NK cells of the present disclosure combine the natural tumor-killing capabilities of NK cells with CAR-mediated targeting, enhanced ADCC through CD 16 variants, and improved persistence through cytokine expression. In certain embodiments, the derivative cell is a T cell. T cells are adaptive immune cells that provide potent and specific immune responses. The engineered T cells of the present disclosure provide CAR-mediated tumor targeting combined with enhanced properties as described herein. In certain embodiments, the T cell is a gamma delta T cell.

[0064] Gamma delta T cells represent a unique T cell subset that bridges innate and adaptive immunity. Unlike conventional alpha beta T cells that recognize peptide antigens presented by MHC molecules, gamma delta T cells can recognize antigens in an MHC-independent manner, including stress-induced molecules and phosphoantigens. Docket No. CNTY-033-WO-01

[0065] This MHC -independent recognition makes gamma delta T cells particularly suitable for allogeneic therapy. In certain embodiments, the T cell is a gamma delta VyO / Vo I T cell or Vy9 / V52 T cell, which represent specific gamma delta T cell subsets with distinct functional properties.

[0066] The disclosure also provides CD34+ hematopoietic progenitor cells (HPCs) derived from the engineered iPSCs. CD34+ HPCs represent an intermediate stage of differentiation that retains multi-lineage potential while being committed to hematopoietic lineages. CD34+ HPCs can be expanded and differentiated into various mature blood cell types, including T cells, NK cells, and myeloid cells. The CD34+ HPCs comprise the same genetic modifications as the parent iPSCs and thus inherit all engineered properties. In certain embodiments, the CD34+ HPC comprises: (i) the CD30 comprising amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 395; or (ii) the one or more exogenous polynucleotides encoding the CAR comprising the costimulatory domain comprising CD30 comprises nucleotides having at least 90%, 91%, 92%, 93%. 94%. 95%. 96%. 97%. 98%. 99% or 100% sequence identity to SEQ ID NO: 396. The CD34+ HPCs can be cryopreserved and stored, providing an "off-the-shelf' cell therapy product that can be thawed and differentiated into mature therapeutic cells on demand.

[0067] The disclosure also provides isolated CAR polypeptides independent of cells. In certain embodiments, provided is a chimeric antigen receptor (CAR) polypeptide comprising (i) an extracellular domain comprising an antigen binding domain that specifically binds to Nectin4, CD19, and / or CD22, and (ii) a costimulatory domain comprising CD30. These CAR polypeptides can be used for various purposes including generating engineered cells using alternative methods (such as mRNA transfection or transposon-based integration), studying CAR structure-function relationships, or developing next-generation CAR designs. In certain embodiments, the CAR is a dual -targeting CAR, and wherein the extracellular domain comprises an additional antigen-binding domain that specifically binds to an antigen selected from the group consisting of CD70, Folate Receptor alpha, FSHR, mesothelin, and SLITRK6.

[0068] The disclosure also provides pharmaceutical compositions comprising the derivative cells. In certain embodiments, provided is a pharmaceutical composition comprising the derivative cell according to any of the embodiments described herein. Docket No. CNTY-033-WO-01

[0069] The pharmaceutical compositions are formulated for therapeutic administration to patients and can include pharmaceutically acceptable carriers, excipients, stabilizers, and cryoprotectants suitable for cellular therapies. In certain embodiments, the composition further comprises or is used in combination with, one or more therapeutic agents selected from the group consisting of a peptide, a cytokine, a checkpoint inhibitor, a mitogen, a growth factor, a small RNA, a dsRNA (double stranded RNA), siRNA, oligonucleotide, mononuclear blood cells, a vector comprising one or more polynucleic acids of interest, an antibody, a chemotherapeutic agent or a radioactive moiety, or an immunomodulatory drug (IMiD). Combination with checkpoint inhibitors (such as anti-PD-1, anti-PD-Ll, or anti-CTLA-4 antibodies) can enhance the anti-tumor activity of the engineered cells by blocking inhibitor}' signals in the tumor microenvironment. Combination with chemotherapeutic agents can provide lymphodepletion that creates space for the engineered cells to expand and may enhance their activity. Combination with therapeutic antibodies that target tumor antigens leverages the CD16-mediated ADCC capabilities of the engineered cells.

[0070] The disclosure further provides methods of treating cancer in a subject in need thereof. In certain embodiments, provided is a method of treating cancer in a subject in need thereof, comprising administering the derivative cell according to any of the embodiments described herein, or the composition, to a subject in need thereof. The engineered cells can be administered by various routes including intravenous infusion, intra-arterial infusion, intratumoral injection, or other routes appropriate for the cancer type and location. In certain embodiments, the cancer is selected from the group consisting of leukemias, such as AML (acute myeloid leukemia), CML (chronic myeloid leukemia), ALL (acute lymphoblastic leukemia) and CLL (chronic lymphocytic leukemia), lymphomas, such as Hodgkin lymphoma, non-Hodgkin lymphoma and multiple myeloma, and solid cancers such as sarcomas, skin cancer, melanoma, bladder cancer, brain cancer, breast cancer, uterus cancer, ovarian cancer, prostate cancer, lung cancer, colorectal cancer, cervical cancer, liver cancer, head and neck cancer, esophageal cancer, pancreatic cancer, renal cancer, adrenal cancer, stomach cancer, testicular cancer, cancer of the gall bladder and biliary tracts, thyroid cancer, thymus cancer, cancer of bone, and cerebral cancer, as well as cancer of unknown primary (CUP). In certain embodiments, the cancer is selected from the group consisting of bladder, breast, lung, pancreatic, ovarian, head & neck, and Docket No. CNTY-033-WO-01

[0071] esophageal cancers, which represent solid tumor ty pes where Nectin-4 expression is prevalent and where current CAR-based therapies have shown limited efficacy.

[0072] In certain embodiments, the subject has minimal residual disease (MRD) after an initial cancer treatment. MRD refers to small numbers of cancer cells that remain in a patient after treatment and that may not be detectable by conventional methods but can lead to relapse. The engineered cells of the present disclosure are particularly suited for eliminating MRD due to their multiple targeting mechanisms (CAR, CD16 / ADCC, NKG2D), enhanced persistence, and ability to function in an allogeneic setting. In certain embodiments, the subject has no minimal residual disease (MRD) after one or more cancer treatments or repeated dosing, indicating achievement of complete remission.

[0073] In certain embodiments, the method further comprises administering to the subject a therapeutic agent selected from the group consisting of ibritumomab, tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, alemtuzumab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, polatuzumab vedotin, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, avelumab. ofatumumab, panitumumab, and ustekinumab. These therapeutic antibodies can serve multiple purposes: (1) providing tumor targeting that is leveraged by the CD16-expressing engineered cells for ADCC; (2) blocking inhibitory' pathways (such as PD-L1 blockade by avelumab); or (3) if the safety' switch utilizes an epitope recognized by one of these antibodies, providing a means for eliminating the engineered cells if necessary. In certain embodiments, the therapeutic agent is avelumab, an anti-PD-Ll antibody that can both enhance antitumor immunity and, in embodiments where the safety switch includes an epitope recognized by avelumab, provide a depletion mechanism. In certain embodiments, the cell and the therapeutic agent are administered concurrently, providing immediate combined activity. In certain embodiments, the cell and the therapeutic agent are administered sequentially, allowing for staged treatment approaches such as antibody administration following cell engraftment.

[0074] The disclosure also provides methods of manufacturing the derivative cells. In certain embodiments, provided is a method of manufacturing the derivative cell comprising differentiating the iPSC according to any of the embodiments described Docket No. CNTY-033-WO-01

[0075] herein under conditions for cell differentiation to thereby obtain the derivative cell. iPSC differentiation protocols for generating T cells and NK cells are well-established and can be optimized for the specific engineered iPSC lines. The use of iPSCs as the starting material provides significant advantages for manufacturing: (1) unlimited self-renewal capacity enabling generation of large master cell banks; (2) clonal selection ensuring uniformity’ of genetic modifications across all derivative cells; (3) extensive characterization and quality control at the iPSC stage before committing to differentiation; (4) ability to generate multiple different derivative cell types (NK cells, T cells, CD34+ HPCs) from a single engineered iPSC clone; and (5) scalability' to produce therapeutic quantities of cells for treating multiple patients from a single master cell bank.

[0076] In certain embodiments, the iPSC is obtained by genetically engineering an unmodified iPSC, wherein the genetic engineering comprises targeted editing of the genome of the iPSC. This approach allows use of well-characterized, clinical-grade iPSC lines as starting material, with genetic modifications introduced subsequently. In certain embodiments, the targeted editing comprises deletion, insertion, or indel (insertion / deletion) carried out by CRISPR (clustered regularly interspaced short palindromic repeats), ZFN (zinc finger nuclease), TALEN (transcription activator-like effector nuclease), homing nuclease, homology recombination, or any other functional variation of these methods. These genome editing technologies enable precise introduction of the desired genetic modifications, including targeted integration at specific loci and knockout of specific genes. CRISPR-Cas9 systems are particularly advantageous due to their simplicity, efficiency, and ability to multiplex (introducing multiple modifications simultaneously).

[0077] The disclosure represents a comprehensive platform for generating nextgeneration cellular immunotherapies that address multiple limitations of current approaches through strategic genetic engineering at the iPSC stage, enabling scalable production of enhanced therapeutic cells suitable for allogeneic administration in treating a broad range of cancers.

[0078] BRIEF DESCRIPTION OF THE DRAWINGS

[0079] The foregoing summary', as well as the following detailed description of preferred embodiments of the present application, will be better understood when read Docket No. CNTY-033-WO-01

[0080] in conjunction with the appended drawings. It should be understood, however, that the application is not limited to the precise embodiments shown in the drawings.

[0081] FIGs. 1A-C show (A) an endodomain panel of 37 endodomains there were generated representing key signaling group types relevant to T cell activation, costimulation, and cytokine support. Unless otherwise noted, all CARs were formatted with a aNectin4 VHH, CD8 hinge, and CD28 TMD, leaving only the endodomain as a variable region; (B) a schematic representation of CAR formats and variable regions. CARs were formatted with an oNectin4 VHH binder to target Nectin4 on tumor cells. CAR cassettes were cloned into a lentiviral bicistronic vector with t2a-PuromcyinR to allow for selection during differentiation of iPSC derived yd-iT cells; and (C) CARs that were transduced via lentivirus into Day 21 yS-iT cells and differentiated to day 35, with selection starting at Day 28 to provide >90% CAR positivity (data not shown). CAR-iT cells were screened for serial killing function and expansion potential as described in Example 1.

[0082] FIGs. 2A-C show (A) a CAR-iT endodomain panel screened for serial killing across multiple Nectin4-positive tumor lines at the indicated E: T ratio. CAR #5034 (CD30-CD3z endodomain) exhibited improved serial killing performance compared to other endodomains and the benchmark 41BB-CD3z endodomain, and exhibited a slow but persistent cytotoxic phenotype; (B) a CTV dilution assay to assess CAR-mediated expansion 5 days after stimulation with T47D target lines. CAR #5034 (CD30-CD3z endodomain) mediated greatest dilution of CTV labeling, indicating greater relative compared to other endodomains and the benchmark 41BB-CD3z endodomain; and (C) a spheroid assay comparing CAR mediated cytotoxicity and expansion of CAR gd-iT (41BB-CD3z vs CD30-CD3z) and CAR primary T cells (41BB-CD3z) against T47D tumor (Nectin4 positive breast cancer) cells over 7 days. Imaging of increased cell mass size at Day7 of spheroid assay gives visualization of CAR mediated expansion (top). Quantified expansion via Count Bright beads demonstration an approximate 5 -fold increase in expansion of CD30-CD3z CARs over 41BB-CD3z in gd-iT (middle). Quantified spheroid killing curves showing CD30-CD3z provides slower but complete killing kinetics on solid tumor targets (bottom).

[0083] FIGs. 3A-B show (A) a spheroid assay comparing CAR mediated cytotoxicity and expansion of CAR gd-iT (41BB-CD3z vs CD30-CD3z) and CAR primary T cells (41BB-CD3z) against Nalm6 B-cell lymphoma lines overexpressing Nectin4 (Nalm6- Docket No. CNTY-033-WO-01

[0084] N4) over 7 days at 1: 1 and 1:5 E: T. Imaging of increased cell mass size at Day7 of spheroid assay provided visualization of CAR mediated expansion (top left).

[0085] Quantified expansion via Count Bright beads demonstrated an approximate 5-fold increase in expansion of CD30-CD3z CARs over 41BB-CD3z in gd-iT (top right). Quantified spheroid killing curves showed CD30-CD3z provides improved killing kinetics on Nalm6-N4targets compared to both 41BB-CD3z CAR gd-iT and primary T cells (bottom); and (B) a serial killing assay comparing CAR mediated cytotoxicity and persistence of CAR gd-iT (41BB-CD3z vs CD30-CD3z) and CAR primary T cells (41BB-CD3z) against Nalm6 B-cell lymphoma lines overexpressing Nectin4 (Nalm6-N4) at 1:1 and 1:5 E: T. Quantified spheroid killing curves showed CD30-CD3z provides improved cytotoxic persistence on Nalm6-N4targets compared to both 41BB-CD3z CAR gd-iT and primary T cells.

[0086] FIGs. 4A-C show (A) Spheroid assay comparing cytotoxicity and expansion of CD30-CD3z CARs with alternative binders to M8 VHH (Enfortimab and FMC63) compared to 41BB-CD3z counterparts against Nalm6 B-cell lymphoma lines overexpressing Nectin4 (Nalm6-N4) over 7 days at 1: 1 E: T. Imaging of increased cell mass size at Day7 of spheroid assay provided visualization of CAR mediated expansion and cytotoxicity (top left). Schematic representation of CD30-CD3z CARs formatted with three different binders (M8, Enformtimab, FMC63) (bottom); (B) Quantification of cell expansion from the spheroid assay in Figure 4A after 7 days. All binders in CD30-CD3z endodomain format (#5034) exhibited similar and ~5-fold improved expansion compared to the respective 41BB-CD3z endodomain controls; and (C) quantification of CAR gd-iT cell mediated cytotoxicity from the spheroid assay in Figure 4A after 7 days. All binders in CD30-CD3z endodomain format (#5034) exhibited improved cytotoxicity compared to the respective 41BB-CD3z endodomain controls.

[0087] FIGs. 5A-C show (A) a schematic representation of CD30-CD3z CAR (p5034) vs a modified CAR (p5374 - DAP10-CD30-CD3z) with an additional costimulatory endodomain (DAP 10) that stimulates a complimentary signaling pathway to CD30 co-stimulation; (B) a spheroid assay comparing cytotoxicity and expansion of CD30-CD3z (#5034), DAP10-CD30-CD3z (#5374) and 41BB-CD3z against Nalm6 B-cell lymphoma lines overexpressing Nectin4 (Nalm6-N4) over 7 days at 1: 1 and 1:5 E: T. Imaging of increased cell mass size at Day 7 of the spheroid assay provided visualization of further improved CAR mediated expansion and cytotoxicity Docket No. CNTY-033-WO-01

[0088] from DAP10-CD30-CD3z endodomain (#5374) compared to CD30-CD3z (#5034) and 41BB-CD3z; and (C) quantification of spheroid size over time from the spheroid assay in Figure 5B, as determine by spheroid size masking and quantification using Incucyte analysis software. DAP10-CD30-CD3z endodomain (#5374) yielded greater cell expansion compared to CD30-CD3z (#5034) and 41BB-CD3z.

[0089] DETAILED DESCRIPTION

[0090] Various publications, articles and patents are cited or described in the background and throughout the specification; each of these references is herein incorporated by reference in its entirety. Discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is for the purpose of providing context for the invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any inventions disclosed or claimed.

[0091] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this application pertains. Otherwise, certain terms used herein have the meanings as set forth in the specification.

[0092] It must be noted that as used herein and in the appended claims, the singular forms “a.” “an / ’ and “the” include plural reference unless the context clearly dictates otherwise.

[0093] Unless otherwise stated, any numerical values, such as a concentration or a concentration range described herein, are to be understood as being modified in all instances by the term “about.” Thus, a numerical value typically includes ± 10% of the recited value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Likewise, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). As used herein, the use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise.

[0094] Unless otherwise indicated, the term “at least” preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation. Docket No. CNTY-033-WO-01

[0095] many equivalents to the specific embodiments of the application described herein. Such equivalents are intended to be encompassed by the application.

[0096] As used herein, the terms “comprises,’’ “comprising,” “includes,” “including,” “has,” “having,” “contains” or “containing,” or any other variation thereof, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers and are intended to be nonexclusive or open-ended. For example, a composition, a mixture, a process, a method, an article, or an apparatus that comprises a list of elements is not necessarily limited to only those elements but can include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, “or” refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present), and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[0097] As used herein, the conjunctive term “and / or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and / or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and / or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and / or.”

[0098] As used herein, the term “consists of,” or variations such as “consist of’ or “consisting of,” as used throughout the specification and claims, indicate the inclusion of any recited integer or group of integers, but that no additional integer or group of integers can be added to the specified method, structure, or composition.

[0099] As used herein, the term “consists essentially of.” or variations such as “consist essentially of’ or “consisting essentially of,” as used throughout the specification and claims, indicate the inclusion of any recited integer or group of integers, and the optional inclusion of any recited integer or group of integers that do not materially change the basic or novel properties of the specified method, structure or composition. See M. P. E. P. § 2111.03. Docket No. CNTY-033-WO-01

[0100] As used herein, “subject'’ means any animal, preferably a mammal, most preferably a human. The term “mammal” as used herein, encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., more preferably a human.

[0101] It should also be understood that the terms “about.” “approximately,” “generally,” “substantially,” and like terms, used herein when referring to a dimension or characteristic of a component of the preferred invention, indicate that the described dimension / characteristic is not a strict boundary or parameter and does not exclude minor variations therefrom that are functionally the same or similar, as would be understood by one having ordinary skill in the art. At a minimum, such references that include a numerical parameter would include variations that, using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc ), would not vary the least significant digit.

[0102] The term "chimeric antigen receptor” or “CAR” refers to engineered receptors, which are grafted onto cells. In general, a CAR of the present disclosure comprises one or more extracellular domains comprising the antigen binding domain(s), one or more intracellular domains comprising one or more costimulatory and / or signaling domains, and a scaffold comprising multiple transmembrane domains and intracellular or extracellular loops, at which the one or more extracellular or intracellular domains are disposed. The antigen binding domain of the CAR targets specific antigens. The targeting regions may comprise full length heavy chain, Fab fragments, scFvs, divalent single chain antibodies or diabodies, each of which are specific to the target antigen (e.g., Nectin4 or DSG1). The antigen binding domain can be derived from the same species or a different species for or in which the CAR will be used in.

[0103] The terms “binder” or “specifically binds” or “specific for” with respect to an antigen-binding domain of a ligand like an antibody, of a fragment thereof or of a CAR refer to an antigen-binding domain which recognizes and binds to a specific antigen, but does not substantially recognize or bind other molecules in a sample. An antigen-binding domain that binds specifically to an antigen from one species may bind also to that antigen from another species. This cross-species reactivity is not contrary to the definition of that antigen-binding domain as specific. An antigen- Docket No. CNTY-033-WO-01

[0104] binding domain that specifically binds to an antigen may bind also to different allelic forms of the antigen (allelic variants, splice variants, isoforms etc.). This cross reactivity is not contrary to the definition of that antigen-binding domain as specific.

[0105] The terms “engineered cell” and “genetically modified cell” as used herein can be used interchangeably. The terms mean containing and / or expressing a foreign gene or nucleic acid sequence which in turn modifies the genotype or phenotype of the cell or its progeny. Especially, the terms refers to cells, preferentially T cells which are manipulated by recombinant methods well known in the art to express stably or transiently peptides or proteins which are not expressed in these cells in the natural state. For example, T cells are engineered to express an artificial construct such as a chimeric antigen receptor on their cell surface. For example, the sequences encoding the CAR may be delivered into cells using a retroviral or lentiviral vector.

[0106] The term “target” as used herein refers to an antigen or epitope associated with a cell that should be recognized specifically by an antigen binding domain, e.g. an antigen binding domain of an antibody or of a CAR. The antigen or epitope for antibody recognition can be bound to the cell surface but also be secreted, part of the extracellular membrane, or shed from the cell.

[0107] As used herein, the term “dual-targeting” refers to a protein (e.g., a chimeric protein) capable of binding to two different antigens. Specifically, a dual-targeting protein of the present disclosure (e.g., a CAR having two or more tumor or cancer antigen binding domains) does not naturally occur and is produced by a genetic engineering method or other method. In one embodiment, a primary cell, an engineered iPSC or derivative cell of the present disclosure can comprise one or more exogenous polynucleotides encoding a CAR having a first antigen binding domain that specifically binds Nectind and a second antigen binding domain that specifically binds DSG1. This is m contrast with other examples of the present disclosure wherein a primary cell, an engineered iPSC or derivative cell comprises one or more polynucleotides encoding a first CAR having a first antigen binding domain that specifically binds Nectind and a second CAR having a second antigen binding domain that specifically binds DSG1.

[0108] The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences (e.g., CAR polypeptides and the CAR polynucleotides that encode them), refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides Docket No. CNTY-033-WO-01

[0109] that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.

[0110] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[0111] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat’l. Acad. Set. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA. and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally, Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.. (1995 Supplement) (Ausubel)).

[0112] Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Docket No. CNTY-033-WO-01

[0113] Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity’ X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N= -4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0114] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see. e.g., Karlin & Altschul, Proc. Nat ’I. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity’ provided by the BLAST algorithm is the smallest sum probability’ (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0115] A further indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the tw o molecules hybridize to each other under stringent conditions.

[0116] As used herein, the term “isolated” means a biological component (such as a nucleic acid, peptide, protein, or cell) has been substantially separated, produced apart from, or purified away from other biological components of the organism in which the Docket No. CNTY-033-WO-01

[0117] component naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, proteins, cells, and tissues. Nucleic acids, peptides, proteins, and cells that have been ‘'isolated” thus include nucleic acids, peptides, proteins, and cells purified by standard purification methods and purification methods described herein.

[0118] “Isolated” nucleic acids, peptides, proteins, and cells can be part of a composition and still be isolated if the composition is not part of the native environment of the nucleic acid, peptide, protein, or cell. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.

[0119] As used herein, the term “polynucleotide,” synonymously referred to as “nucleic acid molecule,” “nucleotides” or “nucleic acids,” refers to any polyribonucleotide or poly deoxyribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. “Polynucleotides” include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and doublestranded regions, single- and double-stranded RNA. and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that can be single-stranded or, more typically, double-stranded or a mixture of single-and double-stranded regions. In addition, “polynucleotide” refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. “Polynucleotide” also embraces relatively short nucleic acid chains, often referred to as oligonucleotides.

[0120] A “construct” refers to a macromolecule or complex of molecules comprising a polynucleotide to be delivered to a host cell, either in vitro or in vivo. A “vector,” as used herein refers to any nucleic acid construct capable of directing the delivery or transfer of a foreign genetic material to target cells, where it can be replicated and / or expressed. The term “vector"’ as used herein comprises the construct to be delivered. A vector can be a linear or a circular molecule. A vector can be integrating or nonintegrating. The major types of vectors include, but are not limited to, plasmids. Docket No. CNTY-033-WO-01

[0121] episomal vector, viral vectors, cosmids, and artificial chromosomes. Viral vectors include, but are not limited to, adenovirus vector, adeno-associated virus vector, retrovirus vector, lentivirus vector, Sendai virus vector, and the like.

[0122] By “integration” it is meant that one or more nucleotides of a construct is stably inserted into the cellular genome, i.e., covalently linked to the nucleic acid sequence within the cell's chromosomal DNA. By “targeted integration” it is meant that the nucleotide(s) of a construct is inserted into the cell's chromosomal or mitochondrial DNA at a pre-selected site or “integration site”. The term “integration” as used herein further refers to a process involving insertion of one or more exogenous sequences or nucleotides of the construct, with or without deletion of an endogenous sequence or nucleotide at the integration site. In the case, where there is a deletion at the insertion site, “integration” can further comprise replacement of the endogenous sequence or a nucleotide that is deleted with the one or more inserted nucleotides.

[0123] As used herein, the term “exogenous” is intended to mean that the referenced molecule or the referenced activity is introduced into, or non-native to, the host cell. The molecule can be introduced, for example, by introduction of an encoding nucleic acid into the host genetic material such as by integration into a host chromosome or as non- chromosomal genetic material such as a plasmid. Therefore, the term as it is used in reference to expression of an encoding nucleic acid refers to introduction of the encoding nucleic acid in an expressible form into the cell. The term “endogenous” refers to a referenced molecule or activity that is present in the host cell in its native form. Similarly, the term when used in reference to expression of an encoding nucleic acid refers to expression of an encoding nucleic acid natively contained within the cell and not exogenously introduced.

[0124] As used herein, a “gene of interest” or “a polynucleotide sequence of interest” is a DNA sequence that is transcribed into RNA and in some instances translated into a polypeptide in vivo when placed under the control of appropriate regulatory’ sequences. A gene or polynucleotide of interest can include, but is not limited to, prokary otic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and synthetic DNA sequences. For example, a gene of interest may encode an miRNA, an shRNA, a native polypeptide (i.e. a polypeptide found in nature) or fragment thereof; a variant polypeptide (i.e. a mutant of the native polypeptide having less than 100% sequence identity’ with the Docket No. CNTY-033-WO-01

[0125] native polypeptide) or fragment thereof; an engineered polypeptide or peptide fragment, a therapeutic peptide or polypeptide, an imaging marker, a selectable marker, and the like.

[0126] “Operably-linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably-linked with a coding sequence or functional RNA when it is capable of affecting the expression of that coding sequence or functional RNA (i.e., the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation.

[0127] The term “expression” as used herein, refers to the biosynthesis of a gene product. The term encompasses the transcription of a gene into RNA. The term also encompasses translation of RNA into one or more polypeptides, and further encompasses all naturally occurring post-transcriptional and post-translational modifications. The expressed CAR can be within the cytoplasm of a host cell, into the extracellular milieu such as the growth medium of a cell culture or anchored to the cell membrane.

[0128] As used herein, the terms “peptide,” “polypeptide,” or “protein” can refer to a molecule comprised of amino acids and can be recognized as a protein by those of skill in the art. The conventional one-letter or three-letter code for amino acid residues is used herein. The terms “peptide,” “polypeptide,” and “protein” can be used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear or branched, it can comprise modified amino acids, and it can be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.

[0129] The peptide sequences described herein are written according to the usual convention whereby the N-terminal region of the peptide is on the left and the C-terminal region is on the right. Although isomeric forms of the amino acids are Docket No. CNTY-033-WO-01

[0130] known, it is the L-form of the amino acid that is represented unless otherwise expressly indicated.

[0131] As used herein, the term "engineered immune cell’’ refers to an immune cell, also referred to as an immune effector cell, that has been genetically modified by the addition of exogenous genetic material in the form of DNA or RNA to the total genetic material of the cell.

[0132] Induced Pluripotent Stem Cells (IPSCs) And Immune Effector Cells

[0133] IPSCs have unlimited self-renewing capacity. Use of iPSCs enables cellular engineering to produce a controlled cell bank of modified cells that can be expanded and differentiated into desired immune effector cells, supplying large amounts of homogeneous allogeneic therapeutic products.

[0134] Provided herein are genetically engineered IPSCs and derivative cells thereof. The selected genomic modifications provided herein enhance the therapeutic properties of the derivative cells. The derivative cells are functionally improved and suitable for allogenic off-the-shelf cell therapies following a combination of selective modalities being introduced to the cells at the level of iPSC through genomic engineering. This approach can help to reduce the side effects mediated by CRS / GVHD and prevent long-term autoimmunity while providing excellent efficacy.

[0135] As used herein, the term "differentiation" is the process by which an unspecialized ("uncommitted") or less specialized cell acquires the features of a specialized cell. Specialized cells include, for example, a blood cell or a muscle cell. A differentiated or differentiation- induced cell is one that has taken on a more specialized ("committed") position within the lineage of a cell. The term "committed", w hen applied to the process of differentiation, refers to a cell that has proceeded in the differentiation pathway to a point where, under normal circumstances, it will continue to differentiate into a specific cell type or subset of cell types, and cannot, under normal circumstances, differentiate into a different cell type or revert to a less differentiated cell type. As used herein, the term "pluripotent" refers to the ability’ of a cell to form all lineages of the body or soma or the embryo proper. For example, embryonic stem cells are a type of pluripotent stem cells that are able to form cells from each of the three germs layers, the ectoderm, the mesoderm, and the endoderm. Pluripotency is a continuum of developmental potencies ranging from the incompletely or partially pluripotent cell (e.g., an epiblast stem cell or EpiSC), which Docket No. CNTY-033-WO-01

[0136] is unable to give rise to a complete organism to the more primitive, more pluripotent cell, which is able to give rise to a complete organism (e.g., an embryonic stem cell).

[0137] As used herein, the terms "reprogramming" or "dedifferentiation" refers to a method of increasing the potency of a cell or dedifferentiating the cell to a less differentiated state. For example, a cell that has an increased cell potency has more developmental plasticity (i.e., can differentiate into more cell types) compared to the same cell in the non-reprogrammed state. In other words, a reprogrammed cell is one that is in a less differentiated state than the same cell in a non-reprogrammed state.

[0138] As used herein, the term "induced pluripotent stem cells" or, iPSCs, means that the stem cells are produced from differentiated adult, neonatal or fetal cells that have been induced or changed or reprogrammed into cells capable of differentiating into tissues of all three germ or dermal layers: mesoderm, endoderm, and ectoderm. The iPSCs produced do not refer to cells as they are found in nature.

[0139] The term “hematopoietic stem and progenitor cells,” “hematopoietic stem cells,” “hematopoietic progenitor cells,” or “hematopoietic precursor cells” or “HPCs” refers to cells which are committed to a hematopoietic lineage but are capable of further hematopoietic differentiation. Hematopoietic stem cells include, for example, multipotent hematopoietic stem cells (hematoblasts), myeloid progenitors, megakaryocyte progenitors, erythrocyte progenitors, and lymphoid progenitors. Hematopoietic stem and progenitor cells (HSCs) are multipotent stem cells that give rise to all the blood cell types including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells), and lymphoid lineages (T cells, B cells, NK cells). As used herein, “CD34+ hematopoietic progenitor cell” refers to an HPC that expresses CD34 on its surface.

[0140] As used herein, the term “immune cell” or “immune effector cell” refers to a cell that is involved in an immune response. Immune response includes, for example, the promotion of an immune effector response. Examples of immune cells include T cells, B cells, natural killer (NK) cells, mast cells, and myeloid-derived phagocytes.

[0141] As used herein, the terms “T lymphocyte” and “T cell” are used interchangeably and refer to a type of white blood cell that completes maturation in the thymus and that has various roles in the immune system. A T cell can have the roles including, e.g., the identification of specific foreign antigens in the body and the activation and deactivation of other immune cells. A T cell can be any T cell, such as Docket No. CNTY-033-WO-01

[0142] a cultured T cell, e.g., a primary T cell, or a T cell from a cultured T cell line, e.g., Jurkat, SupTl, etc., or a T cell obtained from a mammal. The T cell can be CD3+ cells. The T cell can be any type of T cell and can be of any developmental stage, including but not limited to, CD4+ / CD8+ double positive T cells, CD4+ helper T cells (e.g., Thl 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, regulator T cells, gamma delta T cells (gd T cells), and the like. Additional types of helper T cells include cells such as Th3 (Treg), Thl7, Th9, or Tfh cells. Additional types of memory T cells include cells such as central memory' T cells (Tcm cells), effector memory T cells (Tern cells and TEMRA cells). The T cell can also refer to a genetically engineered T cell, such as a T cell modified to express a T cell receptor (TCR) or a chimeric antigen receptor (CAR). The T cell can also be differentiated from a stem cell or progenitor cell.

[0143] “CD4+ T cells” refers to a subset of T cells that express CD4 on their surface and are associated with cell-mediated immune response. They are characterized by the secretion profiles following stimulation, which may include secretion of cytokines such as IFN-gamma, TNF-alpha, IL2, IL4 and IL10. “CD4” are 55-kD glycoproteins originally defined as differentiation antigens on T-lymphocytes, but also found on other cells including monocytes / macrophages. CD4 antigens are members of the immunoglobulin supergene family and are implicated as associative recognition elements in MHC (major histocompatibility complex) class Il-restricted immune responses. On T-lymphocytes they define the helper / inducer subset.

[0144] “CD8+ T cells” refers to a subset of T cells which express CD8 on their surface, are MHC class I-restricted, and function as cytotoxic T cells. ’ CD8" molecules are differentiation antigens found on thymocytes and on cytotoxic and suppressor T- lymphocytes. CD8 antigens are members of the immunoglobulin supergene family and are associative recognition elements in major histocompatibility7complex class I-restricted interactions.

[0145] As used herein, the term “NK cell” or ‘'Natural Killer cell” refers to a subset of peripheral blood lymphocytes defined by the expression of CD56 and CD45 and the absence of the T cell receptor (TCR chains). The NK cell can also refer to a genetically engineered NK cell, such as aNK cell modified to express a chimeric antigen receptor (CAR). The NK cell can also be differentiated from a stem cell or progenitor cell. Docket No. CNTY-033-WO-01

[0146] As used herein, the term “genetic imprint’' refers to genetic or epigenetic information that contributes to preferential therapeutic attributes in a source cell or an iPSC, and is retainable in the source cell derived iPSCs, and / or the iPSC-derived hematopoietic lineage cells. As used herein, “a source cell” is anon-pluripotent cell that may be used for generating iPSCs through reprogramming, and the source cell derived iPSCs may be further differentiated to specific cell types including any hematopoietic lineage cells. The source cell derived iPSCs, and differentiated cells therefrom are sometimes collectively called “derived” or “derivative” cells depending on the context. For example, derivative effector cells, or derivative NK or “iNK” cells or derivative T or “iT” cells, as used throughout this application are cells differentiated from an iPSC, as compared to their primary counterpart obtained from natural / native sources such as peripheral blood, umbilical cord blood, or other donor tissues. As used herein, the genetic imprint(s) conferring a preferential therapeutic attribute is incorporated into the iPSCs either through reprogramming a selected source cell that is donor-, disease-, or treatment response- specific, or through introducing genetically modified modalities to iPSC using genomic editing.

[0147] The induced pluripotent stem cell (iPSC) parental cell lines may be generated from peripheral blood mononuclear cells (PBMCs) or T-cells using any known method for introducing re-programming factors into non-pluripotent cells such as the episomal plasmid-based process as previously described in U. S. Pat. Nos. 8,546,140; 9,644,184; 9,328,332; and 8,765,470, the complete disclosures of which are incorporated herein by reference. The reprogramming factors may be in a form of polynucleotides, and thus are introduced to the non-pluripotent cells by vectors such as a retrovirus, a Sendai virus, an adenovirus, an episome. and a mini-circle. In particular embodiments, the one or more polynucleotides encoding at least one reprogramming factor are introduced by a lentiviral vector. In some embodiments, the one or more polynucleotides introduced by an episomal vector. In various other embodiments, the one or more polynucleotides are introduced by a Sendai viral vector. In some embodiments, the iPSC’s are clonal iPSCs or are obtained from a pool of iPSCs and the genome edits are introduced by making one or more targeted integration and / or in / del at one or more selected sites. In another embodiment, the iPSC’s are obtained from human T cells having antigen specificity and a reconstituted TCR gene (hereinafter, also refer to as " T-iPS” cells) as described in US Pat. Nos. 9206394, and 10787642 hereby incorporated by reference into the present application. Docket No. CNTY-033-WO-01

[0148] According to a particular aspect, the application relates to an induced pluripotent stem cell (iPSC) cell or a derivative cell thereof comprising: (i) an exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (ii) an exogenous polynucleotide encoding a truncated epithelial growth factor (tEGFR) variant and an interleukin 15 (IL-15), wherein the tEGFR variant and IL-15 are operably linked by an autoprotease peptide sequence, such as the porcine teschovirus-1 2A (P2A); and (iii) a deletion or reduced expression of B2M and CIITA genes.

[0149] I. Chimeric Antigen Receptor (CAR) Expression

[0150] According to embodiments of the application, an iPSC or a derivative cell thereof comprises one or more exogenous polynucleotides encoding a chimeric antigen receptor (CAR), wherein the CAR comprises a cotimulatory domain comprising CD30 or a fragment thereof, and wherein the CAR targets a Nectin4 antigen, a CD19 antigen, and / or a CD22 antigen.

[0151] CD30, also know n as TNFRSF8, is a member of the tumor necrosis factor receptor superfamily that plays a crucial role in immune signaling and cellular communication. In the context of chimeric antigen receptor (CAR) design, CD30 as a costimulalory domain offers several compelling advantages for cancer and autoimmune therapy. When incorporated into CAR T-cell constructs, CD30 can provide enhanced T-cell activation, proliferation, and persistence, which are critical factors in developing effective immunotherapeutic strategies.

[0152] The unique signaling properties of CD30 make it particularly valuable in CAR design. Conventional CD28 and 41BB Costimulatory domains fail to provide robust expansion profiles in y5-iT cells, suggesting unconventional signaling mechanisms and endodomain optimization may be necessary for expansion and improved cytotoxic persistence. Unlike traditional costimulatory domains like CD28 or 4- IBB, CD30 can trigger a more robust T-cell expansion and generate a distinctive cytokine profile that can potentially improve therapeutic outcomes. CAR T-cells incorporating the CD30 costimulatory domain can demonstrate improved antitumor efficacy. The domain's ability to enhance T-cell survival and reduce exhaustion means that engineered T-cells can maintain their therapeutic potential for longer periods, potentially increasing the durability7of treatment responses. Docket No. CNTY-033-WO-01

[0153] The CD30 costimulatory domain shows promise in modulating immune responses more precisely, which can be useful in treating autoimmune diseases. Its signaling can help regulate T-cell activation thresholds, potentially allowing for more targeted interventions in conditions like rheumatoid arthritis, multiple sclerosis, and other inflammatory disorders. The domain's nuanced immune modulation capabilities suggest it could be a valuable tool in developing more sophisticated immunotherapeutic approaches that balance between effective immune response and preventing excessive inflammatory reactions.

[0154] Nectins are cell adhesion molecules (CAMs) involved in Ca2+- independent cell-cell interactions. The Nectin family includes four Nectins, including Nectins 1 -3, which are enriched in normal adult tissues; and Nectin 4, which is mostly expressed during fetal development and its expression declines in adult tissues (low expression levels in skin, bladder, placenta, oral mucosa, and tonsils).

[0155] Nectins interact with other cell surface molecules including cadherins, integrins and growth factor receptors. These interactions help modulate cell adhesion, migration and proliferation. Nectin4 dimers bind to Nectin-1 or Nectin4 on adjacent cells. Nectin4 also binds TIGIT on immune cells and this interaction leads to inhibition of NK cells.

[0156] Accordingly, Nectin4 is a suitable target for a CAR of the invention because it is expressed in high frequency in bladder, breast, lung, pancreatic, ovarian, head & neck, and esophageal cancers. The highest levels of expression of Nectin4 are seen in bladder, breast, lung and pancreatic cancers. Clinical validation of Nectin4 as a tumor target has been demonstrated by the approval of Enfortumab vedotin for the treatment of urothelial cancer

[0157] Thus in one embodiment, the CAR comprises a costimulatory domain comprising CD30 or a fragment thereof, and the CAR targets aNectin4 antigen and the targeting region (e.g., the extracellular domain) of the CAR comprises an antibody fragment (e.g, a VHH domain). In other embodiments, an iPSC or a derivative cell thereof comprises one or more first exogenous polynucleotides encoding a single CAR that comprises a costimulatory domain comprising CD30 or a fragment thereof, wherein the CAR targets a Nectin4 antigen.

[0158] In some embodiments, an iPSC or a derivative cell thereof comprises one or more first exogenous polynucleotides encoding a CAR (e.g., targeting Nectin4) and Docket No. CNTY-033-WO-01

[0159] an additional CAR targeting another antigen. In some embodiments, the antigen targeted by the additional CAR is selected from the group consisting of CD70, Folate Receptor alpha, FSHR, mesothelin, and SLITRK6. In other embodiments, an iPSC or a derivative cell thereof comprises one or more first exogenous polynucleotides encoding a dual-targeting CAR targeting a Nectin4 antigen and an another antigen selected from the group consisting of CD70. Folate Receptor alpha. FSHR, mesothelin, and SLITRK6. Each of the binding domains of any of the CAR, the additional CAR, or the dual-targeting CAR can be, for example, independently selected from an scFv and a VHH.

[0160] As used herein, the term “chimeric antigen receptor"’ (CAR) refers to a recombinant polypeptide comprising at least an extracellular domain that binds specifically to an antigen or a target, a transmembrane domain and an intracellular signaling domain. Engagement of the extracellular domain of the CAR with the target antigen on the surface of a target cell results in clustering of the CAR and delivers an activation stimulus to the CAR-containing cell. CARs redirect the specificity of immune effector cells and trigger proliferation, cytokine production, phagocytosis and / or production of molecules that can mediate cell death of the target antigenexpressing cell in a major histocompatibility' (MHC)-independent manner.

[0161] As used herein, the term “signal peptide” refers to a leader sequence at the amino-terminus (N-terminus) of a nascent CAR protein, which co-translationally or post-translationally directs the nascent protein to the endoplasmic reticulum and subsequent surface expression.

[0162] As used herein, the term “extracellular antigen-binding domain,” “extracellular domain,"’ or “extracellular ligand binding domain” refers to the part of a CAR that is located outside of the cell membrane and is capable of binding to an antigen, target or ligand.

[0163] As used herein, the term “hinge region” or “hinge domain” refers to the part of a CAR that connects two adjacent domains of the CAR protein, i.e., the extracellular domain and the transmembrane domain of the CAR protein.

[0164] As used herein, the term “transmembrane domain” refers to the portion of a CAR that extends across the cell membrane and anchors the CAR to cell membrane. Docket No. CNTY-033-WO-01

[0165] As used herein, the term “intracellular signaling domain,’" “cytoplasmic signaling domain,” or “intracellular signaling domain” refers to the part of a CAR that is located inside of the cell membrane and is capable of transducing an effector signal.

[0166] As used herein, the term “stimulatory molecule” refers to a molecule expressed by an immune cell (e.g., NK cell or T cell) that provides the primary- cytoplasmic signaling sequence(s) that regulate primary activation of receptors in a stimulatory way for at least some aspect of the immune cell signaling pathway.

[0167] Stimulatory molecules comprise two distinct classes of cytoplasmic signaling sequence, those that initiate antigen-dependent primary activation (referred to as “primary signaling domains"’), and those that act in an antigen-independent manner to provide a secondary of co-stimulatory signal (referred to as “co-stimulatory signaling domains”).

[0168] In certain embodiments, the extracellular domain comprises an antigenbinding domain and / or an antigen-binding fragment. The antigen-binding fragment can, for example, be an antibody or antigen-binding fragment thereof that specifically binds a tumor antigen. The antigen-binding fragments of the application possess one or more desirable functional properties, including but not limited to high-affinity binding to a tumor antigen, high specificity to a tumor antigen, the ability to stimulate complement-dependent cytotoxicity (CDC), antibody-dependent phagocytosis (ADPC), and / or antibody -dependent cellular-mediated cytotoxicity (ADCC) against cells expressing a tumor antigen, and the ability to inhibit tumor grow th in subjects in need thereof and in animal models when administered alone or in combination with other anti-cancer therapies.

[0169] As used herein, the term “antibody” is used in a broad sense and includes immunoglobulin or antibody molecules including human, humanized, composite and chimeric antibodies and antibody fragments that are monoclonal or polyclonal. In general, antibodies are proteins or peptide chains that exhibit binding specificity to a specific antigen. Antibody structures are well known. Immunoglobulins can be assigned to five major classes (i.e., IgA, IgD, IgE, IgG and IgM), depending on the heavy chain constant domain amino acid sequence. IgA and IgG are further subclassified as the isotypes IgAl, IgA2, IgGl, IgG2, IgG3 and IgG4. Accordingly, the antibodies of the application can be of any of the five major classes or corresponding sub-classes. Preferably, the antibodies of the application are IgGl, IgG2. IgG3 or IgG4. Antibody light chains of vertebrate species can be assigned to one of two Docket No. CNTY-033-WO-01

[0170] clearly distinct types, namely kappa and lambda, based on the amino acid sequences of their constant domains. Accordingly, the antibodies of the application can contain a kappa or lambda light chain constant domain. According to particular embodiments, the antibodies of the application include heavy and / or light chain constant regions from rat or human antibodies. In addition to the heavy and light constant domains, antibodies contain an antigen-binding region that is made up of a light chain variable region and a heavy chain variable region, each of which contains three domains (i.e., complementarity determining regions 1-3; CDR1, CDR2, and CDR3). The light chain variable region domains are alternatively referred to as LCDR1, LCDR2, and LCDR3, and the heavy chain variable region domains are alternatively referred to as HCDR1, HCDR2, and HCDR3.

[0171] As used herein, the term an “isolated antibody’’ refers to an antibody which is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to the specific tumor antigen is substantially free of antibodies that do not bind to the tumor antigen). In addition, an isolated antibody is substantially free of other cellular material and / or chemicals.

[0172] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that can be present in minor amounts. The monoclonal antibodies of the application can be made by the hybridoma method, phage display technology, single lymphocyte gene cloning technology, or by recombinant DNA methods. For example, the monoclonal antibodies can be produced by a hybridoma which includes a B cell obtained from a transgenic nonhuman animal, such as a transgenic mouse or rat, having a genome comprising a human heavy chain transgene and a light chain transgene.

[0173] As used herein, the term “antigen-binding fragment” refers to an antibody fragment such as, for example, a diabody, a Fab, a Fab', a F(ab')2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide stabilized diabody (ds diabody), a single-chain antibody molecule (scFv), a single domain antibody (sdAb), a scFv dimer (bivalent diabody), a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a camelized single domain antibody, a minibody, a nanobody, a domain antibody, a bivalent domain antibody, a light chain variable domain (VL), a variable domain Docket No. CNTY-033-WO-01

[0174] (VHH) of a camelid antibody, or any other antibody fragment that binds to an antigen but does not comprise a complete antibody structure. An antigen-binding fragment is capable of binding to the same antigen to which the parent antibody or a parent antibody fragment binds.

[0175] As used herein, the term “single-chain antibody” refers to a conventional single-chain antibody in the field, which comprises a heavy chain variable region and a light chain variable region connected by a short peptide of about 15 to about 20 amino acids (e.g., a linker peptide).

[0176] As used herein, the term “single domain antibody” refers to a conventional single domain antibody in the field, which comprises a heavy chain variable region and a heavy chain constant region or which comprises only a heavy chain variable region.

[0177] As used herein, the term “human antibody” refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody- produced by a human made using any technique known in the art. This definition of a human antibody includes intact or full-length antibodies, fragments thereof, and / or antibodies comprising at least one human heavy7and / or light chain polypeptide.

[0178] As used herein, the term “humanized antibody” refers to a non-human antibody that is modified to increase the sequence homology to that of a human antibody, such that the antigen-binding properties of the antibody are retained, but its antigenicity' in the human body is reduced.

[0179] As used herein, the term “chimeric antibody” refers to an antibody wherein the amino acid sequence of the immunoglobulin molecule is derived from two or more species. The variable region of both the light and heavy chains often corresponds to the variable region of an antibody derived from one species of mammal (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity7, and capability7, while the constant regions correspond to the sequences of an antibody derived from another species of mammal (e.g.. human) to avoid eliciting an immune response in that species.

[0180] As used herein, the term “multispecific antibody” refers to an antibody that comprises a plurality7of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality7has binding specificity7for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In an embodiment, the first and Docket No. CNTY-033-WO-01

[0181] second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In an embodiment, the first and second epitopes overlap or substantially overlap. In an embodiment, the first and second epitopes do not overlap or do not substantially overlap. In an embodiment, the first and second epitopes are on different antigens, e.g., the different proteins (or different subunits of a multimeric protein). In an embodiment, a multispecific antibody comprises a third, fourth, or fifth immunoglobulin variable domain. In an embodiment, a multispecific antibody is a bispecific antibody molecule, a trispecific antibody molecule, or a tetraspecific antibody molecule.

[0182] As used herein, the term “bispecific antibody’" refers to a multispecific antibody that binds no more than two epitopes or two antigens. A bispecific antibody is characterized by a first immunoglobulin variable domain sequence which has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope. In an embodiment, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In an embodiment, the first and second epitopes overlap or substantially overlap. In an embodiment, the first and second epitopes are on different antigens, e.g., the different proteins (or different subunits of a multimeric protein). In an embodiment, a bispecific antibody comprises a heavy chain variable domain sequence and a light chain variable domain sequence which have binding specificity for a first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence which have binding specificity for a second epitope. In an embodiment, a bispecific antibody comprises a half antibody, or fragment thereof, having binding specificity for a first epitope and a half antibody, or fragment thereof, having binding specificity for a second epitope. In an embodiment, a bispecific antibody comprises a scFv, or fragment thereof, having binding specificity' for a first epitope, and a scFv, or fragment thereof, having binding specificity for a second epitope. In an embodiment, a bispecific antibody comprises a VHH having binding specificity for a first epitope, and a VHH having binding specificity for a second epitope. In an embodiment, the term XTY loop (wherein ‘X’ and ‘Y’ are antigens such as Nectin4 and an antigen selected from the group consisting of CD70, Folate Receptor alpha, FSHR, mesothelin, and SLITRK6 refers to an extracellular region in which one scFv is nested in between the VL and VH of the other scFv. In Docket No. CNTY-033-WO-01

[0183] some embodiments, X and Y may be the same antigen. In some embodiments, X and Y may be different antigens. In some embodiments. X and Y are tumor antigens.

[0184] As used herein, an antigen-binding domain or antigen-binding fragment that “specifically binds to a tumor antigen” refers to an antigen-binding domain or antigen-binding fragment that binds a tumor antigen, with a KD of 1 x 10-7M or less, preferably 1 x 10-8M or less, more preferably 5 x 10-9M or less, 1 x 10-10M or less, 5 x 10-11M or less, or 1x10-10M or less. The term “KD” refers to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and is expressed as a molar concentration (M). KD values for antibodies can be determined using methods in the art in view of the present disclosure. For example, the KD of an antigen-binding domain or antigen-binding fragment can be determined by using surface plasmon resonance, such as by using a biosensor system, e.g., a Biacore® system, or by using bio-layer interferometry technology, such as an Octet RED96 system.

[0185] The smaller the value of the KD of an antigen-binding domain or antigenbinding fragment, the higher affinity that the antigen-binding domain or antigenbinding fragment binds to a target antigen.

[0186] In various embodiments, antibodies or antibody fragments suitable for use in the CAR of the present disclosure include, but are not limited to, monoclonal antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies. polypeptide-Fc fusions, single-chain Fvs (scFv), single chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv), masked antibodies (e.g., Probodies®), Small Modular ImmunoPharmaceuticals (" SMIPsTM"), intrabodies, minibodies, single domain antibody variable domains, nanobodies, VHHs, diabodies, tandem diabodies (TandAb®), anti-idiotypic (anti-Id) antibodies (including, e.g., anti-id antibodies to antigen-specific TCR), and epitope-binding fragments of any of the above. Antibodies and / or antibody fragments may be derived from murine antibodies, rabbit antibodies, human antibodies, fully humanized antibodies, camelid antibody variable domains and humanized versions, shark antibody variable domains and humanized versions, and camelized antibody variable domains.

[0187] In some embodiments, the antigen-binding fragment is an Fab fragment, an Fab1fragment, an F(ab')2 fragment, an scFv fragment, an Fv fragment, a dsFv diabody, a VHH. a VNAR, a single-domain antibody (sdAb) or nanobody, a dAb fragment, a Fd' fragment, a Fd fragment, a heavy chain variable region, an isolated Docket No. CNTY-033-WO-01

[0188] complementarity determining region (CDR), a diabody, a triabody, or a decabody. In some embodiments, the antigen-binding fragment is an scFv fragment. In some embodiments, the antigen-binding fragment is a VHH.

[0189] In some embodiments, at least one of the extracellular tag-binding domain, the antigen-binding domain, or the tag comprises a single-domain antibody or nanobody. In some embodiments, at least one of the extracellular tag-binding domain, the antigen-binding domain, or the tag comprises a VHH.

[0190] In some embodiments, the extracellular tag-binding domain and the tag each comprise a VHH.

[0191] In some embodiments, the extracellular tag-binding domain, the tag, and the antigen-binding domain each comprise a VHH.

[0192] In some embodiments, at least one of the extracellular tag-binding domain, the antigen-binding domain, or the tag comprises an scFv.

[0193] In some embodiments, the extracellular tag-binding domain and the tag each comprise an scFv.

[0194] In some embodiments, the extracellular tag-binding domain, the tag, and the antigen-binding domain each comprise a scFv.

[0195] Alternative scaffolds to immunoglobulin domains that exhibit similar functional characteristics, such as high-affinity and specific binding of target biomolecules, may also be used in the CARs of the present disclosure. Such scaffolds have been shown to yield molecules with improved characteristics, such as greater stability or reduced immunogenicity. Non-limiting examples of alternative scaffolds that may be used in the CAR of the present disclosure include engineered, tenascin-derived, tenascin type III domain (e.g., Centyrin™); engineered, gamma-B crystallinderived scaffold or engineered, ubiquitin-derived scaffold (e.g., Affilins); engineered, fibronectin-derived, 10th fibronectin type III (10Fn3) domain (e.g., monobodies, AdNectins™, or AdNexins™);; engineered, ankyrin repeat motif containing polypeptide (e.g., DARPins™); engineered, low-density-lipoprotein-receptor-derived, A domain (LDLR-A) (e.g., Avimers™); lipocalin (e.g., anticalins); engineered, protease inhibitor-derived, Kunitz domain (e.g., EETI-II / AGRP, BPTI / LACI-D1 / ITI-D2); engineered, Protein-A-derived, Z domain (Affibodies™); Sac7d-derived polypeptides (e.g., Nanoffitins® or affitins); engineered, Fyn-derived, SH2 domain (e.g., Fynomers®); CTLDs (e.g.. Tetranectin); thioredoxin (e.g., peptide aptamer); KALBITOR®; the -sandwich (e.g., iMab); miniproteins; C-type lectin-like domain Docket No. CNTY-033-WO-01

[0196] scaffolds; engineered antibody mimics; and any genetically manipulated counterparts of the foregoing that retains its binding functionality’ (Worn A, Pluckthun A, J Mol Biol 305: 989-1010 (2001); Xu L et al., Chem Biol 9: 933-42 (2002); Wikman M et al., Protein Eng Des Sei 17: 455-62 (2004); Binz H et al., Nat Biolechnol 23: 1257-68 (2005); Hey T et al., Trends Biotechnol 23:514-522 (2005); Holliger P, Hudson P, Nat Biotechnol 23: 1126-36 (2005); Gill D, Damle N, Curr Opin Biotech 17: 653-8 (2006); Koide A, Koide S, Methods Mol Biol 352: 95-109 (2007); Skerra, Current Opin. in Biotech., 2007 18: 295-304; Byla P et al., J Biol Chem 285: 12096 (2010); Zoller F et al., Molecules 16: 2467-85 (2011), each of which is incorporated by reference in its entirety).

[0197] In some embodiments, the alternative scaffold is Affilin or Centyrin.

[0198] In some embodiments, the first polypeptide of the CARs of the present disclosure comprises a leader sequence. The leader sequence may be positioned at the N-terminus the extracellular tag-binding domain. The leader sequence may be optionally cleaved from the extracellular tag-binding domain during cellular processing and localization of the CAR to the cellular membrane. Any of various leader sequences known to one of skill in the art may be used as the leader sequence. Non-limiting examples of peptides from which the leader sequence may be derived include granulocyte-macrophage colony-stimulating factor receptor (GMCSFR), FceR, human immunoglobulin (IgG) heavy chain (HC) variable region. CD8a. or any of various other proteins secreted by T cells. In various embodiments, the leader sequence is compatible with the secretory’ pathw ay of a T cell. In certain embodiments, the leader sequence is derived from human immunoglobulin heavy chain (HC).

[0199] In some embodiments, the leader sequence is derived from GMCSFR. In one embodiment, the GMCSFR leader sequence comprises the amino acid sequence set forth in SEQ ID NO: 1, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90. at least 95. at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 1.

[0200] In some embodiments, the first polypeptide of the CARs of the present disclosure comprise a transmembrane domain, fused in frame betw een the extracellular tag-binding domain and the cytoplasmic domain.

[0201] The transmembrane domain may be derived from the protein contributing to the extracellular tag-binding domain, the protein contributing the signaling or co- Docket No. CNTY-033-WO-01

[0202] signaling domain, or by a totally different protein. In some instances, the transmembrane domain can be selected or modified by amino acid substitution, deletions, or insertions to minimize interactions with other members of the CAR complex. In some instances, the transmembrane domain can be selected or modified by amino acid substitution, deletions, or insertions to avoid binding of proteins naturally associated with the transmembrane domain. In certain embodiments, the transmembrane domain includes additional amino acids to allow for flexibility and / or optimal distance between the domains connected to the transmembrane domain.

[0203] The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Non-limiting examples of transmembrane domains of particular use in this disclosure may be derived from (i.e. comprise at least the transmembrane region(s) of) the a, P or chain of the T-cell receptor (TCR), CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD8a, CD9, CD16, CD22, CD28, CD33. CD37, CD40, CD64, CD80, CD86, CD134. CD137, or CD154. Alternatively, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. For example, a triplet of phenylalanine, tryptophan and / or valine can be found at each end of a synthetic transmembrane domain.

[0204] In some embodiments, it will be desirable to utilize the transmembrane domain of the r| or FceRly chains which contain a cysteine residue capable of disulfide bonding, so that the resulting chimeric protein will be able to form disulfide linked dimers with itself, or with unmodified versions of the r| or FceRly chains or related proteins. In some instances, the transmembrane domain will be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex. In other cases, it will be desirable to employ the transmembrane domain of p or FceRly and -, MB1

[0205]

[0206] (Iga.), B29 or CD3- y, or p, in order to retain physical association with other members of the receptor complex.

[0207] In some embodiments, the transmembrane domain is derived from CD8 or CD28. In one embodiment, the CD8 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 23, or a variant thereof having at least 50. at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least Docket No. CNTY-033-WO-01

[0208] 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 23. In one embodiment, the CD28 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 24, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 24.

[0209] In some embodiments, the first polypeptide of the CAR of the present disclosure comprises a spacer region betw een the extracellular tag-binding domain and the transmembrane domain, wherein the tag-binding domain, linker, and the transmembrane domain are in frame with each other.

[0210] The term “spacer region" as used herein generally means any oligo- or polypeptide that functions to link the tag-binding domain to the transmembrane domain. A spacer region can be used to provide more flexibility and accessibility for the tag-binding domain. A spacer region may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. A spacer region may be derived from all or part of naturally occurring molecules, such as from all or part of the extracellular region of CD8, CD4 or CD28, or from all or part of an antibody constant region. Alternatively, the spacer region may be a synthetic sequence that corresponds to a naturally occurring spacer region sequence, or may be an entirely synthetic spacer region sequence. Non-limiting examples of spacer regions w hich may be used in accordance to the disclosure include a part of human CD8a chain, partial extracellular domain of CD28, FcyRllla receptor, IgG, IgM, IgA, IgD, IgE, an Ig hinge, or functional fragment thereof. In some embodiments, additional linking amino acids are added to the spacer region to ensure that the antigen-binding domain is an optimal distance from the transmembrane domain. In some embodiments, when the spacer is derived from an Ig, the spacer may be mutated to prevent Fc receptor binding.

[0211] In some embodiments, the spacer region comprises a hinge domain. The hinge domain may be derived from CD8, CD8a, CD28, or an immunoglobulin (IgG). For example, the IgG hinge may be from IgGl, IgG2, IgG3, IgG4, IgG4 CH3, IgMl, IgM2, IgAl, IgA2, IgD, IgE, or a chimera thereof.

[0212] In certain embodiments, the hinge domain comprises an immunoglobulin IgG hinge or functional fragment thereof. In certain embodiments, the IgG hinge is from IgGl, IgG2, IgG3, IgG4, IgG4 CH3, IgMl, IgM2, IgAl, IgA2, IgD, IgE, or a chimera Docket No. CNTY-033-WO-01

[0213] thereof. In certain embodiments, the hinge domain comprises the CHI, CH2, CH3 and / or hinge region of the immunoglobulin. In certain embodiments, the hinge domain comprises the core hinge region of the immunoglobulin. The term '‘core hinge” can be used interchangeably with the term “short hinge” (a.k.a “SH”). Nonlimiting examples of suitable hinge domains are the core immunoglobulin hinge regions include EPKSCDKTHTCPPCP (SEQ ID NO: 57) from IgGl, ERKCCVECPPCP (SEQ ID NO: 58) from IgG2, ELKTPLGDTTHTCPRCP(EPKSCDTPPPCPRCP)3 (SEQ ID NO: 59) from IgG3, ESKYGPPCPSCP (SEQ ID NO: 60) from IgG4 (see also Wypych et al., JBC 2008 283(23): 16194-16205, which is incorporated herein by reference in its entirety for all purposes), and ESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK (SEQ ID NO: 96), or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70. at least 75. at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity. In certain embodiments, the hinge domain is a fragment of the immunoglobulin hinge.

[0214] In some embodiments, the hinge domain is derived from CD8 or CD28. In one embodiment, the CD8 hinge domain comprises the amino acid sequence set forth in SEQ ID NO: 21, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 21. In one embodiment, the CD28 hinge domain comprises the amino acid sequence set forth in SEQ ID NO: 22, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 22.

[0215] In some embodiments, the transmembrane domain and / or hinge domain is derived from CD8 or CD28. In some embodiments, both the transmembrane domain and hinge domain are derived from CD8. In some embodiments, both the transmembrane domain and hinge domain are derived from CD28.

[0216] In certain aspects, the first polypeptide of CARs of the present disclosure comprise a cytoplasmic domain, which comprises at least one intracellular signaling domain. In some embodiments, cytoplasmic domain also comprises one or more costimulatory signaling domains. Docket No. CNTY-033-WO-01

[0217] The cytoplasmic domain is responsible for activation of at least one of the normal effector functions of the host cell (e.g., T cell) in which the CAR has been placed in. The term “effector function” refers to a specialized function of a cell. Effector function of a T-cell, for example, may be cytolytic activity or helper activity' including the secretion of cytokines. Thus, the term “signaling domain” refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire signaling domain is present, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The term intracellular signaling domain is thus meant to include any truncated portion of the signaling domain sufficient to transduce the effector function signal.

[0218] Non-limiting examples of signaling domains which can be used in the CARs of the present disclosure include, e.g., signaling domains derived from DAP10, DAP12, Fc epsilon receptor I y chain (FCER1G), FcR 0, CD35. CD3s, CD3y, CD3g. CD5. CD22, CD226. CD66d, CD79a. and CD79b.

[0219] In some embodiments, the cytoplasmic domain comprises a CD3(^ signaling domain. In one embodiment, the CD3^ signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 6. or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85. at least 90. at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 6.

[0220] In some embodiments, the cytoplasmic domain further comprises one or more co-stimulatory signaling domains. In some embodiments, the one or more costimulatory signaling domains are derived from CD28, 4 IBB, IL2Rb, CD40, 0X40 (CD 134), CD80, CD86, CD27, ICOS, NKG2D, DAP 10, DAP 12, 2B4 (CD244), BTLA, CD30, GITR, CD226, CD79A, and HVEM.

[0221] In one embodiment, the co-stimulatory signaling domain is derived from 41BB. In one embodiment, the 41BB co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 8, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97. at least 98 or at least 99%, sequence identity with SEQ ID NO: 8. Docket No. CNTY-033-WO-01

[0222] In one embodiment, the co-stimulatory signaling domain is derived from IL2Rb. In one embodiment, the IL2Rb co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 9, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 9.

[0223] In one embodiment, the co-stimulatory signaling domain is derived from CD40. In one embodiment, the CD40 co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 10, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97. at least 98 or at least 99%. sequence identity with SEQ ID NO: 10.

[0224] In one embodiment, the co-stimulatory signaling domain is derived from 0X40. In one embodiment, the 0X40 co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 11, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity7with SEQ ID NO: 11.

[0225] In one embodiment, the co-stimulatory signaling domain is derived from CD80. In one embodiment, the CD80 co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 12, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 12.

[0226] In one embodiment, the co-stimulatory7signaling domain is derived from CD86. In one embodiment, the CD86 co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 13, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80. at least 85, at least 90, at least 95, at least 96, at least 97. at least 98 or at least 99%, sequence identity7with SEQ ID NO: 13.

[0227] In one embodiment, the co-stimulatory' signaling domain is derived from CD27. In one embodiment, the CD27 co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 14, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at Docket No. CNTY-033-WO-01

[0228] least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 14.

[0229] In one embodiment, the co-stimulatory signaling domain is derived from ICOS. In one embodiment, the ICOS co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 15, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80. at least 85, at least 90, at least 95, at least 96, at least 97. at least 98 or at least 99%, sequence identity with SEQ ID NO: 15.

[0230] In one embodiment, the co-stimulatory' signaling domain is derived from NKG2D. In one embodiment, the NKG2D co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 16, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 16.

[0231] In one embodiment, the co-stimulatory signaling domain is derived from DAP 10. In one embodiment, the DAP 10 co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 17, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 17.

[0232] In one embodiment, the co-stimulatory signaling domain is derived from DAP12. In one embodiment, the DAP 12 co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 18, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70. at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 18.

[0233] In one embodiment, the co-stimulatory signaling domain is derived from 2B4 (CD244). In one embodiment, the 2B4 (CD244) co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 19, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 19.

[0234] In another embodiment, the co-stimulatory signaling domain is derived from CD30. In one embodiment, the CD30 co-stimulatory signaling domain comprises the Docket No. CNTY-033-WO-01

[0235] amino acid sequence set forth in SEQ ID NO: 395, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80. at least 85. at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 395. In some embodiments, the CD30 co-stimulatory signaling domain is encoded by the polynucleotide set forth in SEQ ID NO: 396, or a variant thereof having at least 50, at least 55, at least 60, at least 65. at least 70, at least 75, at least 80, at least 85, at least 90. at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 396.

[0236] In some embodiments, the CAR of the present disclosure comprises one costimulatory signaling domains. In some embodiments, the CAR of the present disclosure comprises two or more costimulatory signaling domains. In certain embodiments, the CAR of the present disclosure comprises two, three, four, five, six or more costimulatory signaling domains.

[0237] In some embodiments, the signaling domain(s) and costimulatory signaling domain(s) can be placed in any order. In some embodiments, the signaling domain is upstream of the costimulatory signaling domains. In some embodiments, the signaling domain is downstream from the costimulatory signaling domains. In the cases where two or more costimulatory' domains are included, the order of the costimulatory signaling domains could be switched.

[0238] Non-limiting exemplary CAR regions and sequences are provided in Table 1. including amino acid and nucleic acid sequences for the various CAR constructs of the present disclosure.

[0239] Table 1.

[0240] CAR regions Sequence UniProt Id SEQ ID NO

[0241] Nectin4 Binding Domains:

[0242] EVQLLESGGGLVQAGGSLRLSCAASGSF DB01 C01 FSIYAMGWFRQAPGKEREFVAAY1SSGG

[0243] P2112 LTSYADSVKGRFTISRDNAKNTVYLQMN 105 (amino acid) SLKPEDTAVYYCAADLGAQTGYVQYDY WGQGTQVTVSS DB01 B01 EVQLLESGGGLVQPGGSLRLSCAASGFVS

[0244] P2106 SIYFMGWFRQ APGKEREFV S S SIGKGGST 106 (amino acid) RYADSVKGRFTISRDNSKNTLYLQMNSL

[0245]

[0246] Docket No. CNTY-033-WO-01

[0247] KPEDTAVYYCAGDEGLGTAHAEYDYWG QGTQVTVSS EVQLLESGGGLVQAGGSLRLSCAASGGIS DB01 BIO EFYFMGWFRQAPGKEREFVAAEISPGSY

[0248] P2110 TNYADSVKGRFTISRDNAKNTVYLQMNS 107 (amino acid) LKPEDTAVYYCAADRDGDTYTAEYDYW

[0249] GQGTQVTVSS EVQLLESGGGLVQPGGSLRLSCAASGSIS DB01 F03 SFYYIGWFRQAPGKEREFVS SRITSGGST

[0250] P2121 YYRDSVKGRFTISRDNSKNTLYLQMNSL 108 (amino acid) KPEDTAVYYCAAGTSRDYYYWGQGTQ

[0251] VTVSS EVQLLESGGGLVQPGGSLRLSCAASGST DB01 A11 SSIGIMGWFRQAPGKERELVSSITAGGST

[0252] P2105 YYADSVKGRFTISRDNSKNTLYLQMNSL 109 (amino acid) KPEDTAVYYCNAHVGYGRVHDVDYWG

[0253] QGTQVTVSS EVQLLESGGGLVQPGGSLRLSCAASGFV DB01 E03 SPSYIMGWFRQAPGKEREFVSSVIEYRGS

[0254] P2120 TYYLDSVKGRFTISRDNSKNTLYLQMNS 110 (amino acid) LKPEDTAVYYCAAGTPGGYDYWGQGT

[0255] QVTVSS EVQLLESGGGLVQPGGSLRLSCAASGST FSSNAMGWYRQAPGKERELVSSISGSGG NEC_S_2

[0256] STRYADSVKGRFTISRDNSKNTLYLQMN 111 (amino acid)

[0257] SLKPEDTAVYYCASYVLYLREYWGQGT QVTVSS EVQLLESGGGLVQPGGSLRLSCAASGLT FRYNAMGWYRQAPGKEREFVSAISGSG NEC_S_5

[0258] GGTYYADSVKGRFTISRDNSKNTLYLQM 112 (amino acid)

[0259] NSLKPEDTAVYYCAAEGLYDYWGQGTQ VTVSS EVQLLESGGGLVQPGGSLRLSCAASGLT NEC S l 1 SSGYAMGWYRQAPGKERELVSSISSSGG

[0260] P3112 LTHYADSVKGRFTISRDNSKNTLYLQMN 113 (amino acid) SLKPEDTAVYYCDADIAYTGADYWGQG

[0261] TQVTVSS

[0262]

[0263] Docket No. CNTY-033-WO-01

[0264] EVQLLESGGGLVQPGGSLRLSCAASGFT FSDYYMGWYRQAPGKEREFVSAISSTGG NEC_S_16

[0265] SPYYADSVKGRFTISRDNSKNTLYLQMN 114 (amino acid)

[0266] SLKPEDTAVYYCAVEVPYDYWGQGTQV TVSS EVQLLESGGGLVQPGGSLRLSCAASGFT NEC_S_31 YSSYAMGWYRQAPGKERELVSSISGSGG

[0267] P3113 STRYADSVKGRFTISRDNSKNTLYLQMN 115 (amino acid) SLKPEDTAVYYCAVAIGVGDYWGQGTQ

[0268] VTVSS EVQLLESGGGLVQPGGSLRLSCAASGFT NEC_S_55 LSSYAMGWYRQAPGKERELVSSISGSGG

[0269] P3114 STRYADSVKGRFTISRDNSKNTLYLQMN 116 (amino acid) SLKPEDTAVYYCAAYIGGDYLGQGTQV

[0270] TVSS EVQLLESGGGLVQPGGSLRLSCAASGLT LSSYAMGWFRQAPGKERELVSSISGSGG NEC_S_56

[0271] STRYADSVKGRFTISRDNSKNTLYLQMN 117 (amino acid)

[0272] SLKPEDTAVYYCAVDIRLTDYWGQGTQ VTVSS EVQLLESGGGLVQPGGSLRLSCAASGFA NEC_S_64 YSSYYMGWYRQAPGKEREFVSAISSSGG

[0273] (amino acid) GTYYADSVKGRFTISRDNSKNTLYLQMN 118

[0274] SLKPEDTAVYYCAVELTYNYWGQGTQV TVSS EVQLLESGGGLVQPGGSLRLSCAASGFT FRSYAMGWFRQAPGKEREGVSVITGTG NEC_S_81

[0275] GSTYYADSVKGRFTISRDNSKNTLYLQM 119 (amino acid)

[0276] NSLKPEDTAVYYCARAYLSGYLYEYWG QGTQVTVSS EVQLLESGGGLVQPGGSLRLSCAASGLT LSSYAMGWFRQAPGKERELVSSISGSGG NEC_S_85

[0277] STRYADSVKGRFTISRDNSKNTLYLQMN 120 (amino acid)

[0278] SLKPEDTAVYYCAVDIRLTDYWGQGTQ VTVSS NEC_M_5 EVQLLESGGGLVQPGGSLRLSCAASGLS

[0279] P3106 FSSYSMGWYRQAPGKEREFVSAISGSSG 121 (amino acid) STNYADSVKGRFTISRDNSKNTLYLQMN

[0280]

[0281] Docket No. CNTY-033-WO-01

[0282] SLKPEDTAVYYCAAEHRVTTSGVFYDY WGQGTQVTVSS EVQLLESGGGLVQPGGSLRLSCAASGFT NEC_M_8 FSSYAMGWYRQAPGKEREFVSSISGSGG

[0283] P3107 LTRYADSVKGRFTISRDNSKNTLYLQMN 122 (amino acid) SLKPEDTAVYYCAVTTGYQGGVYDYW

[0284] GQGTQVTVSS EVQLLESGGGLVQPGGSLRLSCAASGFT NEC_M_17 YSGYAMGWYRQAPGKERELVSSISGSGT

[0285] P3108 LTSYADSVKGRFTISRDNSKNTLYLQMN 123 (amino acid) SLKPEDTAVYYCDVDIPVGDATTVGDY WGQGTQVTVSS EVQLLESGGGLVQPGGSLRLSCAASGLT FSSNAMGWYRQAPGKERELVSSISGSGG NEC_M_21

[0286] STNYADSVKGRFTISRDNSKNTLYLQMN 124 (amino acid)

[0287] SLKPEDTAVYYCAAEIATYYDFADYWG QGTQVTVSS EVQLLESGGGLVQPGGSLRLSCAASGFT YTSYVMGWYRQAPGKERELVSAIYGSA NEC_M_29

[0288] GSGYYADSVKGRFTISRDNSKNTLYLQM 125 (amino acid)

[0289] NSLKPEDTAVYYCARVTTTTHLWSAEY WGQGTQVTVSS EVQLLESGGGLVQPGGSLRLSCAASGRT NEC_M_44 LSSYAMGWYRQAPGKERELVSSISGSGG

[0290] P3109 STRYADSVKGRFTISRDNSKNTLYLQMN 126 (amino acid) SLKPEDTAVYYCAVYILELAPGAEYWGQ

[0291] GTQVTVSS EVQLLESGGGLVQPGGSLRLSCAASGYT NEC_M_46 FSDYAMGWYRQAPGKERELVSSISGSGG

[0292] P3110 STRYADSVKGRFTISRDNSKNTLYLQMN 127 (amino acid) SLKPEDTAVYYCAAVIRQPSTGFYEYWG

[0293] QGTQVTVSS EVQLLESGGGLVQPGGSLRLSCAASGFA YSSYSIGWYRQAPGKERELVSAISGSGGS NEC_M_66

[0294] SRYADSVKGRFTISRDNSKNTLYLQMNS 128 (amino acid)

[0295] LKPEDTAVYYCAAIAYVYTSTARNYWG QGTQVTVSS

[0296]

[0297] Docket No. CNTY-033-WO-01

[0298] EVQLLESGGGLVQPGGSLRLSCAASGFT YSDYAMGWYRQAPGKERELVSAISSYG NEC_M_82

[0299] GVTNYADSVKGRFTISRDNSKNTLYLQM 129 (amino acid)

[0300] NSLKPEDTAVYYCAAYGALSIRGYDYW GQGTQVTVSS EVQLLESGGGLVQPGGSLRLSCAASGFA FSIDAMGWYRQAPGKEREGVSSISGSGG NEC M 84

[0301] LTRYADSVKGRFTISRDNSKNTLYLQMN 130 (amino acid)

[0302] SLKPEDTAVYYCDTAVLTGYSEAFDYW GQGTQVTVSS GAGGTACAACTTTTGGAGTCAGGCGGC GGGTTGGTCCAGGCGGGTGGCTCACTC CGCCTTAGTTGTGCCGCCTCAGGGTCA TTCTTTAGTATCTACGCTATGGGCTGGT TTCGACAGGCCCCTGGTAAGGAACGTG AGTTTGTGGCCGCCTACATTTCCTCAG DB01 C01

[0303] GGGGGCTCACCAGCTACGCGGATAGTG

[0304] P2112 131

[0305] TTAAGGGTAGATTCACCATCTCCAGAG

[0306] (nucleotide)

[0307] ACAATGCAAAGAATACGGTATACCTCC AAATGAACAGCCTGAAGCCTGAAGAC ACGGCTGTCTACTATTGCGCAGCAGAC TTGGGAGCCCAGACCGGATACGTTCAG TACGACTACTGGGGGCAGGGAACCCA GGTGACGGTCTCGAGC GAGGTACAACTTTTGGAGTCAGGCGGC GGCCTGGTGCAGCCCGGAGGGAGTCTC CGACTGTCTTGCGCTGCATCTGGATTC GTGAGCTCTATATACTTTATGGGATGG TTCAGGCAGGCTCCTGGGAAGGAGCGC GAGTTTGTGTCTAGTAGTATTGGCAAG DB01 B01

[0308] GGTGGCTCAACACGCTATGCGGATTCT

[0309] P2106 132

[0310] GTGAAAGGGAGGTTCACAATAAGCAG

[0311] (nucleotide)

[0312] GGACAACTCAAAGAATACACTGTACCT CCAGATGAACTCCTTAAAACCAGAGGA TACTGCAGTCTATTACTGTGCTGGAGA TGAGGGATTGGGAACTGCACATGCTGA ATACGACTACTGGGGCCAGGGGACCCA GGTGACGGTCTCGAGC

[0313]

[0314] Docket No. CNTY-033-WO-01

[0315] GAGGTACAACTTTTGGAGTCAGGCGGA GGACTCGTGCAGGCTGGAGGGTCCCTT AGGCTCAGCTGCGCCGCTTCCGGAGGG ATCTCCGAATTCTATTTCATGGGGTGGT TTAGACAAGCGCCCGGAAAGGAAAGG GAATTCGTCGCAGCAGAGATCTCACCT DB01 B10

[0316] GGAAGCTACACCAACTACGCAGATAGC

[0317] P2110 133

[0318] GTGAAGGGGCGCTTTACCATCTCTAGG

[0319] (nucleotide)

[0320] GATAACGCGAAAAATACAGTCTACCTC CAAATGAACTCTTTGAAGCCAGAGGAT ACCGCCGTGTATTATTGCGCTGCCGAC AGGGACGGAGACACATATACAGCAGA ATATGATTATTGGGGCCAGGGGACCCA GGTGACGGTCTCGAGC GAGGTACAACTTTTGGAGTCAGGCGGC GGGTTAGTGCAGCCTGGAGGATCACTG AGGCTGAGCTGCGCCGCCTCTGGCTCA ATTAGCAGTTTCTATTATATCGGATGGT TCCGCCAGGCTCCGGGAAAAGAGAGA DB01 F03 GAGTTTGTTTCCTCTCGCATTACCTCAG

[0321] P2121 GAGGAAGCACTTACTACAGGGACTCTG 134 (nucleotide) TTAAAGGACGCTTTACAATCTCCAGAG ATAATTCCAAGAACACCTTATATCTGC AAATGAATAGTTTGAAGCCCGAGGACA CTGCCGTGTATTATTGCGCAGCCGGGA CATCCCGCGATTACTACTACTGGGGAC AAGGAACCCAGGTGACGGTCTCGAGC GAGGTACAACTTTTGGAGTCAGGCGGG GGGCTGGTGCAGCCTGGGGGATCACTG CGCCTTTCATGCGCAGCGAGTGGTTCC ACCTCTAGTATCGGAATTATGGGCTGG TTTCGCCAGGCTCCTGGAAAGGAAAGG GAGCTGGTCTCCAGCATCACAGCCGGC DB01 A11

[0322] GGATCTACCTACTACGCCGACTCCGTT

[0323] P2105 135

[0324] AAGGGGCGATTCACTATCTCCCGCGAC

[0325] (nucleotide)

[0326] AATAGCAAGAACACCTTGTATCTGCAG ATGAACTCCCTCAAACCCGAGGATACT GCCGTGTACTATTGCAACGCACATGTG GGCTACGGGAGGGTGCACGATGTGGAT TACTGGGGGCAGGGGACCCAGGTGAC GGTCTCGAGC

[0327]

[0328] Docket No. CNTY-033-WO-01

[0329] GAGGTACAACTTTTGGAGTCAGGCGGT GGTCTCGTCCAGCCGGGGGGGAGCCTC CGCTTATCATGTGCCGCCTCCGGATTTG TCTCACCCTCTTATATAATGGGGTGGTT CCGGCAAGCACCAGGCAAAGAACGGG DB01 E03 AGTTTGTTTCCTCCGTCATAGAGTACCG

[0330] P2120 AGGGTCCACCTATTACCTGGATAGCGT 136 (nucleotide) GAAGGGGCGGTTCACCATCTCCCGCGA TAATTCCAAGAATACCCTGTACCTGCA GATGAATAGTCTGAAACCTGAGGACAC GGCCGTGTACTATTGCGCAGCGGGCAC CCCCGGAGGCTACGATTACTGGGGCCA AGGGACCCAGGTGACGGTCTCGAGC GAGGTACAACTTTTGGAGTCAGGCGGT GGACTGGTACAACCGGGTGGTTCATTG CGTTTGAGCTGCGCTGCCTCTGGTTCTA CCTTCTCAAGCAATGCAATGGGTTGGT ATCGCCAAGCGCCGGGCAAAGAACGC GAGTTGGTCTCCTCCATATCCGGTTCCG NEC_S_2

[0331] GTGGTAGCACCCGCTACGCGGACTCGG 137 (nucleotide)

[0332] TAAAAGGCCGTTTTACGATCAGTCGTG ATAATTCCAAGAATACCTTGTACCTGC AAATGAATAGCCTTAAGCCCGAAGACA CAGCGGTGTATTATTGTGCCTCTTATGT CCTGTATCTCCGAGAATACTGGGGCCA GGGTAC CC AGGTGAC GGTCTC GAGC GAGGTACAACTTTTGGAGTCAGGCGGT GGACTGGTACAACCGGGTGGTTCATTG CGTTTGAGCTGCGCTGCCTCTGGTCTG ACTTTT AGGTAT A AC GC AATGGGATGG TACCGCCAAGCGCCGGGCAAAGAACG CGAGTTTGTCTCCGCGATCAGCGGCTC NEC_S_5

[0333] TGGGGGGGGTACCTATTACGCGGACTC 138 (nucleotide)

[0334] GGTAAAAGGCCGTTTTACGATCAGTCG TGATAATTCCAAGAATACCTTGTACCT GCAAATGAATAGCCTTAAGCCCGAAGA CACAGCGGTGTATTATTGTGCCGCGGA AGGCCTGTATGATTATTGGGGCCAGGG TACCCAGGTGACGGTCTCGAGC

[0335]

[0336] Docket No. CNTY-033-WO-01

[0337] GAGGTACAACTTTTGGAGTCAGGCGGT GGACTGGTACAACCGGGTGGTTCATTG CGTTTGAGCTGCGCTGCCTCTGGTTTAA CAAGCTCTGGTTACGCTATGGGCTGGT ATCGCCAAGCGCCGGGCAAAGAACGC GAGCTGGTGAGCAGTATTTCTTCCTCA NEC_S_11

[0338] GGCGGACTGACCCATTACGCGGACTCG

[0339] P3112 139

[0340] GTAAAAGGCCGTTTTACGATCAGTCGT

[0341] (nucleotide)

[0342] GATAATTCCAAGAATACCTTGTACCTG CAAATGAATAGCCTTAAGCCCGAAGAC ACAGCGGTGTATTATTGTGACGCAGAT ATTGCTTACACTGGCGCCGATTATTGG GGCCAGGGTACCCAGGTGACGGTCTCG AGC GAGGTACAACTTTTGGAGTCAGGCGGT GGACTGGTACAACCGGGTGGTTCATTG CGTTTGAGCTGCGCTGCCTCTGGTTTTA CGTTTAGCGACTATTATATGGGCTGGT ACCGCCAAGCGCCGGGCAAAGAACGC GAGTTCGTATCAGCTATTTCGTCCACG NEC_S_16

[0343] GGCGGCTCCCCCTACTACGCGGACTCG 140 (nucleotide)

[0344] GTAAAAGGCCGTTTTACGATCAGTCGT GATAATTCCAAGAATACCTTGTACCTG CAAATGAATAGCCTTAAGCCCGAAGAC ACAGCGGTGTATTATTGTGCCGTGGAA GTCCCTTACGATTATTGGGGCCAGGGT ACCCAGGTGACGGTCTCGAGC GAGGTACAACTTTTGGAGTCAGGCGGT GGACTGGTACAACCGGGTGGTTCATTG CGTTTGAGCTGCGCTGCCTCTGGTTTTA CGTACTCTTCTTATGCAATGGGCTGGT ATCGCCAAGCGCCGGGCAAAGAACGC NEC_S_31 GAGTTGGTCTCCTCCATATCCGGTTCCG

[0345] P3113 GTGGTAGCACCCGCTACGCGGACTCGG 141 (nucleotide) TAAAAGGCCGTTTTACGATCAGTCGTG ATAATTCCAAGAATACCTTGTACCTGC AAATGAATAGCCTTAAGCCCGAAGACA CAGCGGTGTATTATTGTGCCGTGGCGA TTGGCGTAGGAGACTATTGGGGCCAGG GTACCCAGGTGACGGTCTCGAGC

[0346]

[0347] Docket No. CNTY-033-WO-01

[0348] GAGGTACAACTTTTGGAGTCAGGCGGT GGACTGGTACAACCGGGTGGTTCATTG CGTTTGAGCTGCGCTGCCTCTGGTTTTA CCCTGTCTTCGTATGCAATGGGCTGGT ACCGCCAAGCGCCGGGCAAAGAACGC NEC_S_55 GAGCTCGTTTCCAGCATCAGTGGGTCG

[0349] P3114 GGTGGCTCTACCCGGTACGCGGACTCG 142 (nucleotide) GTAAAAGGCCGTTTTACGATCAGTCGT GATAATTCCAAGAATACCTTGTACCTG CAAATGAATAGCCTTAAGCCCGAAGAC ACAGCGGTGTATTATTGTGCCGCTTAC ATCGGCGGAGATTACTTGGGCCAGGGT ACCCAGGTGACGGTCTCGAGC GAGGTACAACTTTTGGAGTCAGGCGGT GGACTGGTACAACCGGGTGGTTCATTG CGTTTGAGCTGCGCTGCCTCTGGTCTG ACACTGTCATCGTACGCAATGGGTTGG TTCCGCCAAGCGCCGGGCAAAGAACGC GAGCTGGTGTCCTCAATAAGCGGTAGT NEC_S_56

[0350] GGCGGCAGCACGCGCTACGCGGACTCG 143 (nucleotide)

[0351] GTAAAAGGCCGTTTTACGATCAGTCGT GATAATTCCAAGAATACCTTGTACCTG CAAATGAATAGCCTTAAGCCCGAAGAC ACAGCGGTGTATTATTGTGCCGTTGAT ATCCGGCTGACGGATTATTGGGGCCAG GGTACCCAGGTGACGGTCTCGAGC GAGGTACAACTTTTGGAGTCAGGCGGT GGACTGGTACAACCGGGTGGTTCATTG CGTTTGAGCTGCGCTGCCTCTGGTTTTG CCTACTCTTCGTATTACATGGGTTGGTA TCGCCAAGCGCCGGGCAAAGAACGCG NEC_S_64 AGTTTGTGAGTGCAATATCATCTAGTG

[0352] (nucleotide) GCGGCGGTACCTACTACGCGGACTCGG 144

[0353] TAAAAGGCCGTTTTACGATCAGTCGTG ATAATTCCAAGAATACCTTGTACCTGC AAATGAATAGCCTTAAGCCCGAAGACA C AGC GGTGT ATT ATTGTGC C GTTGAAC TTACCTATAACTACTGGGGCCAGGGTA CCCAGGTGACGGTCTCGAGC NEC_S_81

[0354] GAGGTACAACTTTTGGAGTCAGGCGGT 145 (nucleotide)

[0355]

[0356] GGACTGGTACAACCGGGTGGTTCATTG Docket No. CNTY-033-WO-01

[0357] CGTTTGAGCTGCGCTGCCTCTGGTTTCA TATACTCTAGCTACTATATGGGATGGT ACCGCCAAGCGCCGGGCAAAGAACGC GAGTTTGTTAGCGCTATTACCTCATCA GGCGGTTCTACATATTACGCGGACTCG GTAAAAGGCCGTTTTACGATCAGTCGT GATAATTCCAAGAATACCTTGTACCTG CAAATGAATAGCCTTAAGCCCGAAGAC ACAGCGGTGTATTATTGTGCCGTGGAA GTCCCTGAATTTGATTATTGGGGCCAG GGTACCCAGGTGACGGTCTCGAGC GAGGTACAACTTTTGGAGTCAGGCGGT GGACTGGTACAACCGGGTGGTTCATTG CGTTTGAGCTGCGCTGCCTCTGGTTTCA CATTTCGTTCTTATGCGATGGGGTGGTT TCGCCAAGCGCCGGGCAAAGAACGCG AGGGTGTTTCTGTGATTACAGGTACTG NEC_S_85 GCGGTTCAACCTATTACGCGGACTCGG

[0358] 146 (nucleotide) TAAAAGGCCGTTTTACGATCAGTCGTG

[0359] ATAATTC C AAGAAT ACCTTGT AC CTGC AAATGAATAGCCTTAAGCCCGAAGACA CAGCGGTGTATTATTGTGCCCGCGCAT ATCTGAGTGGCTATCTGTACGAGTATT GGGGCCAGGGTACCCAGGTGACGGTCT CGAGC GAGGTACAACTTTTGGAGTCAGGCGGT GGACTGGTACAACCGGGTGGTTCATTG CGTTTGAGCTGCGCTGCCTCTGGTCTG AGCTTCTCATCTTATTCAATGGGGTGGT ATCGCCAAGCGCCGGGCAAAGAACGC GAGTTTGTTTCTGCAATCTCAGGCTCAT NEC_M_5

[0360] CAGGTTCAACGAATTACGCGGACTCGG

[0361] P3106 147

[0362] TAAAAGGCCGTTTTACGATCAGTCGTG

[0363] (nucleotide)

[0364] ATAATTC C AAGAAT ACCTTGT AC CTGC AAATGAATAGCCTTAAGCCCGAAGACA CAGCGGTGTATTATTGTGCCGCCGAAC ACCGGGTGACCACTTCCGGAGTTTTCT ACGACTATTGGGGCCAGGGTACCCAGG TGACGGTCTCGAGC

[0365]

[0366] Docket No. CNTY-033-WO-01

[0367] GAGGTACAACTTTTGGAGTCAGGCGGT GGACTGGTACAACCGGGTGGTTCATTG CGTTTGAGCTGCGCTGCCTCTGGTTTCA CTTTCAGCAGTTATGCGATGGGATGGT ATCGCCAAGCGCCGGGCAAAGAACGC GAGTTTGTTAGCAGCATAAGCGGTTCA NEC_M_8

[0368] GGGGGGTTAACCCGGTACGCGGACTCG

[0369] P3107 148

[0370] GTAAAAGGCCGTTTTACGATCAGTCGT

[0371] (nucleotide)

[0372] GATAATTCCAAGAATACCTTGTACCTG CAAATGAATAGCCTTAAGCCCGAAGAC ACAGCGGTGTATTATTGTGCCGTAACC ACCGGCTACCAAGGCGGCGTATATGAC TACTGGGGCCAGGGTACCCAGGTGACG GTCTCGAGC GAGGTACAACTTTTGGAGTCAGGCGGT GGACTGGTACAACCGGGTGGTTCATTG CGTTTGAGCTGCGCTGCCTCTGGTTTCA CTTACTCTGGGTATGCAATGGGGTGGT ATCGCCAAGCGCCGGGCAAAGAACGC GAGCTAGTCTCAAGCATCTCAGGTTCG NEC_M_17

[0373] GGTACCCTTACTTCGTACGCGGACTCG

[0374] P3108 149

[0375] GTAAAAGGCCGTTTTACGATCAGTCGT

[0376] (nucleotide)

[0377] GATAATTCCAAGAATACCTTGTACCTG CAAATGAATAGCCTTAAGCCCGAAGAC ACAGCGGTGTATTATTGTGACGTAGAT ATACCTGTGGGCGACGCTACAACCGTT GGTGATTACTGGGGCCAGGGTACCCAG GTGACGGTCTCGAGC GAGGTACAACTTTTGGAGTCAGGCGGT GGACTGGTACAACCGGGTGGTTCATTG CGTTTGAGCTGCGCTGCCTCTGGTCTTA CATTTTCTAGCAATGCTATGGGTTGGT ACCGCCAAGCGCCGGGCAAAGAACGC NEC_M_21 GAGCTGGTGAGTTCTATTTCGGGGAGC 150 (nucleotide) GGAGGTAGTACCAATTACGCGGACTCG GTAAAAGGCCGTTTTACGATCAGTCGT GATAATTCCAAGAATACCTTGTACCTG CAAATGAATAGCCTTAAGCCCGAAGAC ACAGCGGTGTATTATTGTGCCGCGGAG

[0378]

[0379] ATTGCTACCTACTACGATTTTGCAGATT Docket No. CNTY-033-WO-01

[0380] ACTGGGGCCAGGGTACCCAGGTGACG GTCTCGAGC GAGGTACAACTTTTGGAGTCAGGCGGT GGACTGGTACAACCGGGTGGTTCATTG CGTTTGAGCTGCGCTGCCTCTGGTTTTA CCTATACGTCTTACGTCATGGGCTGGT ATCGCCAAGCGCCGGGCAAAGAACGC GAGCTGGTATCTGCGATCTATGGCAGT NEC_M_29 GCAGGCAGTGGGTACTACGCGGACTCG

[0381] 151 (nucleotide) GTAAAAGGCCGTTTTACGATCAGTCGT GATAATTCCAAGAATACCTTGTACCTG CAAATGAATAGCCTTAAGCCCGAAGAC ACAGCGGTGTATTATTGTGCCCGCGTT ACTACCACAACCCACCTGTGGTCTGCC GAGTATTGGGGCCAGGGTACCCAGGTG ACGGTCTCGAGC GAGGTACAACTTTTGGAGTCAGGCGGT GGACTGGTACAACCGGGTGGTTCATTG CGTTTGAGCTGCGCTGCCTCTGGTCGT ACGCTAAGTAGTTACGCGATGGGGTGG TACCGCCAAGCGCCGGGCAAAGAACG CGAGCTCGTTAGTAGTATTTCCGGGAG NEC_M_44

[0382] TGGAGGCTCTACCCGTTACGCGGACTC

[0383] P3109 152

[0384] GGTAAAAGGCCGTTTTACGATCAGTCG

[0385] (nucleotide)

[0386] TGATAATTCCAAGAATACCTTGTACCT GCAAATGAATAGCCTTAAGCCCGAAGA CACAGCGGTGTATTATTGTGCCGTCTA CATTCTGGAGCTGGCTCCTGGCGCGGA ATACTGGGGCCAGGGTACCCAGGTGAC GGTCTCGAGC GAGGTACAACTTTTGGAGTCAGGCGGT GGACTGGTACAACCGGGTGGTTCATTG CGTTTGAGCTGCGCTGCCTCTGGTTAC ACATTTTCGGATTATGCAATGGGTTGG NEC_M_46 TATCGCCAAGCGCCGGGCAAAGAACG

[0387] P3110 CGAGCTCGTTAGTAGTATTTCCGGGAG 153 (nucleotide) TGGAGGCTCTACCCGTTACGCGGACTC GGTAAAAGGCCGTTTTACGATCAGTCG TGATAATTCCAAGAATACCTTGTACCT GCAAATGAATAGCCTTAAGCCCGAAGA

[0388]

[0389] CACAGCGGTGTATTATTGTGCCGCAGT Docket No. CNTY-033-WO-01

[0390] AATTCGGCAGCCTAGCACCGGTTTCTA TGAATACTGGGGCCAGGGTACCCAGGT GACGGTCTCGAGC GAGGTACAACTTTTGGAGTCAGGCGGT GGACTGGTACAACCGGGTGGTTCATTG CGTTTGAGCTGCGCTGCCTCTGGTTTTG CATATTCGAGCTACAGCATCGGCTGGT ACCGCCAAGCGCCGGGCAAAGAACGC GAGTTAGTCAGCGCTATCTCCGGCTCT NEC_M_66 GGAGGCTCCTCGCGCTACGCGGACTCG

[0391] 154 (nucleotide) GTAAAAGGCCGTTTTACGATCAGTCGT GATAATTCCAAGAATACCTTGTACCTG CAAATGAATAGCCTTAAGCCCGAAGAC ACAGCGGTGTATTATTGTGCCGCCATA GCGTACGTGTACACGTCCACGGCCCGT AATTACTGGGGCCAGGGTACCCAGGTG ACGGTCTCGAGC GAGGTACAACTTTTGGAGTCAGGCGGT GGACTGGTACAACCGGGTGGTTCATTG CGTTTGAGCTGCGCTGCCTCTGGTTTCA CCTATAGCGATTATGCGATGGGATGGT ACCGCCAAGCGCCGGGCAAAGAACGC GAGTTGGTTTCAGCGATCTCATCGTAC NEC_M_82 GGGGGCGTTACAAATTACGCTGACTCG

[0392] 155 (nucleotide) GTAAAAGGCCGTTTTACGATCAGTCGT GATAATTCCAAGAATACCTTGTACCTG CAAATGAATAGCCTTAAGCCCGAAGAC ACAGCGGTGTATTATTGTGCCGCCTAT GGCGCTCTGTCCATTCGGGGCTATGAT TACTGGGGCCAGGGTACCCAGGTGACG GTCTCGAGC GAGGTACAACTTTTGGAGTCAGGCGGT GGACTGGTACAACCGGGTGGTTCATTG CGTTTGAGCTGCGCTGCCTCTGGTTTTG CGTTCTCC ATC GACGCGATGGGTTGGT NEC_M_84 ATCGCCAAGCGCCGGGCAAAGAACGC 156 (nucleotide) GAGGGCGTTTCCTCCATATCGGGTAGC GGTGGTCTGACACGCTACGCGGACTCG GTAAAAGGCCGTTTTACGATCAGTCGT GATAATTCCAAGAATACCTTGTACCTG

[0393]

[0394] CAAATGAATAGCCTTAAGCCCGAAGAC Docket No. CNTY-033-WO-01

[0395] ACAGCGGTGTATTATTGTGACACAGCC GTACTGACTGGCTATTCCGAGGCTTTT GACTACTGGGGCCAGGGTACCCAGGTG ACGGTCTCGAGC CD 19 Binding Domains:

[0396] FMC63 VH EVKLQESGPGLVAPSQSLSVTCTVSGVSL

[0397] (amino acid PDYGVSWIRQPPRKGLEWLGVIWGSETT

[0398] sequence) YYNSALKSRLTIIKDNSKSQVFLKMNSLQ 379

[0399] TDDTAIYYCAKHYYYGGSYAMDYWGQ GTSVTVSS FMC63 VH GAAGTGAAACTGCAAGAGTCTGGCCCT

[0400] (nucleotide GGACTGGTGGCCCCATCTCAGTCTCTG

[0401] sequence) AGCGTGACCTGTACAGTCAGCGGAGTG TCCCTGCCTGATTACGGCGTGTCCTGG ATCAGACAGCCTCCTCGGAAAGGCCTG GAATGGCTGGGAGTGATCTGGGGCAGC GAGACAACCTACTACAACAGCGCCCTG

[0402] 382 AAGTCCCGGCTGACCATCATCAAGGAC AACTCCAAGAGCCAGGTGTTCCTGAAG ATGAACAGCCTGCAGACCGACGACACC GCCATCTACTATTGCGCCAAGCACTAC TACTACGGCGGCAGCTACGCCATGGAT TATTGGGGCCAGGGCACCAGCGTGACC GTGTCTAGC FMC63 VL DIQMTQTTSSLSASLGDRVTTSCRASQDIS

[0403] (amino acid KYLNWYQQKPDGTVKLLIYHTSRLHSG

[0404] 380 sequence) VPSRFSGSGSGTDYSLTISNLEQEDIATYF CQQGNTLPYTFGGGTKLEIT FMC63 VL GACATCCAGATGACCCAGACCACAAGC

[0405] (nucleotide AGCCTGTCTGCCAGCCTGGGCGATAGA

[0406] sequence) GTGACCATCAGCTGTAGAGCCAGCCAG GACATCAGCAAGTACCTGAACTGGTAT CAGCAAAAGCCCGACGGCACCGTGAA 383 GCTGCTGATCTACCACACCAGCAGACT GCACAGCGGCGTGCCAAGCAGATTTTC TGGCAGCGGCTCTGGCACCGACTACAG CCTGACAATCAGCAACCTGGAACAAGA

[0407]

[0408] GGATATCGCTACCTACTTCTGCCAGCA Docket No. CNTY-033-WO-01

[0409] AGGCAACACCCTGCCTTACACCTTTGG CGGAGGCACCAAGCTGGAAATCACC FMC63 scFV EVKLQESGPGLVAPSQSLSVTCTVSGVSL PDYGVSWIRQPPRKGLEWLGVIWGSETT YYNSALKSRLTIIKDNSKSQVFLKMNSLQ TDDTAIYYCAKHYYYGGSYAMDYWGQ GTSVTVSSGSTSGSGKPGSGEGSTKGDIQ 381 MTQTTSSLSASLGDRVTISCRASQDISKY LNWYQQKPDGTVKLLIYHTSRLHS GVPS RFSGSGSGTDYSLTTSNLEQEDIATYFCQ QGNTLPYTFGGGTKLEIT CD22 Binding Domains:

[0410] CD22 D04 QVQLVESGGGLVQAGGSLRLSCAASGSE FTGYPMGWFRQAPGKEREFVAGSVGIG GSTNYADSVKGRFTISRDNAKNTVYLQ 384 MNSLKPEDTAVYYCAADKDYYKPYSRY RTVIRYETWGQGTQVTVSS CD22 CNTY V EVQLLESGGGLVQPGGSLRLSCAASGLT

[0411] HH1 A01 SYSYAMGWYRQAPGKEREFVSAISSGGS

[0412] (amino acid AYYADSVKGRFTISRDNSKNTLYLQMNS 385 sequence) LRAEDTAVYYCAVGPYYGFRAVTEADY WGQGTQVTVSS CD22 CNTY V GAGGTACAACTTTTGGAGTCAGGCGGT

[0413] HH1 A01 GGACTGGTACAACCGGGTGGTTCATTG

[0414] (nucleotide CGTTTGAGCTGCGCTGCCTCTGGTTTGA

[0415] sequence) CCTCTTATTCCTACGCGATGGGCTGGT ATCGCCAAGCGCCGGGCAAAGAACGC GAGTTTGTCAGCGCAATCAGCTCGGGT GGTAGCGCGTACTACGCGGACTCGGTA

[0416] 389 AAAGGCCGTTTTACGATCAGTCGTGAT AATTCCAAGAATACCTTGTACCTGCAA ATGAATAGCCTTCGCGCAGAAGACACA GCGGTGTATTATTGTGCCGTTGGACCG TACTACGGATTTAGAGCGGTTACCGAA GCAGATTATTGGGGCCAGGGTACCCAG GTGACGGTCTCGAGC CD22 CNTY V EVQLLESGGGLVQPGGTLRLSCAASGLT

[0417] HH1 A01 CYSY AMGWYRQ APGKEREF VS AIS S GGS 386

[0418]

[0419] AYYADSVKGRFTICRDNSKNTLYLQMNS Docket No. CNTY-033-WO-01

[0420] (amino acid LRAEDTAVYYCAVGPYYGFRAVTEADY

[0421] sequence) WGQGTQVTVSS

[0422] CD22 CNTY V GAAGTCCAGCTGCTGGAAAGCGGTGGC

[0423] HH1 A01 GGTCTGGTCCAGCCTGGCGGCACCCTG

[0424] (nucleotide CGCCTGTCCTGTGCCGCTAGCGGCCTG

[0425] sequence) ACCTGCTATAGCTATGCCATGGGTTGG TACCGCCAGGCCCCTGGTAAGGAGCGC GAATTCGTGTCCGCTATTTCCAGCGGC GGCTCCGCCTATTATGCTGATAGCGTC

[0426] 390 AAGGGTCGCTTCACCATTTGCCGCGAC AACAGCAAAAACACTCTGTATCTGCAG ATGAACTCCCTGCGCGCTGAGGATACC GCCGTCTACTACTGCGCTGTGGGCCCT TATTATGGCTTCCGCGCTGTGACTGAG GCTGACTACTGGGGTCAGGGCACTCAG GTGACTGTGAGCAGC CD22 CNTY V EVQLLESGGGLVQPGGSLRLSCAASGFT

[0427] HH1 E04 S S SYVMGWYRQAPGKEREFVS SISTGGD

[0428] (amino acid AYYADSVKGRFTISRDNSKNTLYLQMNS 387 sequence) LRAEDTAVYYCAADVWYYHGGAYDY WGQGTQVTVSS CD22 CNTY V GAGGTACAACTTTTGGAGTCAGGCGGT

[0429] HH1 E04 GGACTGGTACAACCGGGTGGTTCATTG

[0430] (nucleotide CGTTTGAGCTGCGCTGCCTCTGGTTTTA

[0431] sequence) CCAGCTCCTCCTACGTGATGGGCTGGT ATCGCCAAGCGCCGGGCAAAGAACGC GAGTTTGTCAGCTCGATCAGCACCGGT GGTGATGCCTACTACGCGGACTCGGTA

[0432] 391 AAAGGCCGTTTTACGATCAGTCGTGAT AATTCCAAGAATACCTTGTACCTGCAA ATGAATAGCCTTCGCGCAGAAGACACA GCGGTGTATTATTGTGCCGCTGACGTTT GGTACTACCACGGCGGCGCGTACGATT ATTGGGGCCAGGGTACCCAGGTGACGG TCTCGAGC DO4-D11-CD22 QVQLVESGGGLVQAGGSLRLSCAASGSE

[0433] VHH (amino FTGYPMGWFRQAPGKEREFVAGSVGIG

[0434] acid sequence) GSTNYADSVKGRFTISRDNAKNTVYLQ 388

[0435] MNSLKPEDTAVYYCAADKDYYKPYSRY RTAIRYDTWGQGTQVTVSS

[0436]

[0437] Docket No. CNTY-033-WO-01

[0438] DO4-D11-CD22 CAGGTGCAGCTGGTTGAGTCTGGGGGA

[0439] VHH GGCCTTGTCCAGGCTGGGGGGTCCCTG

[0440] (nucleotide AGACTCTCCTGTGCAGCGTCTGGAAGC

[0441] sequence) GAATTCACCGGTTATCCCATGGGCTGG TTTCGCCAGGCTCCAGGCAAGGAAAGG GAGTTTGTCGCTGGCTCCGTAGGTATC GGTGGTAGTACAAACTATGCAGACTCC GTGAAGGGCCGATTCACCATCTCCAGA 392 GACAATGCGAAGAACACGGTCTATCTG CAAATGAACAGCCTGAAGCCAGAGGA CACGGCTGTGTATTACTGTGCGGCCGA CAAAGACTACTACAAACCTTATAGTCG ATATAGGACCGCTATCAGGTACGATAC CTGGGGCCAAGGGACCCAGGTCACCGT CTCGAGC

[0442] Signaling / Co-stimulatory Domains:

[0443] CD30 CHRRACRKRIRQKLHLCYPVQTSQPKLE LVDSRPRRSSTQLRSGASVTEPVAEERGL

[0444] (amino acid) MSQPLMETCHSVGAAYLESLPLQDASPA GGPSSPRDLPEPRVSTEHTNNKIEKIYIM 395 KADTVIVGTVKAELPEGRGLAGPAEPEL EEELEADHTPHYPEQETEPPLGSCSDVM LSVEEEGKEDPLPTAASGK CD30 TGCCATAGGCGGGCTTGCAGAAAAAG

[0445] (nucleic acid) AATAAGGCAAAAATTGCATTTGTGCTA TCCAGTCCAAACGTCCCAGCCGAAGCT GGAGCTGGTAGACAGCAGACCACGAC GATCCTCCACGCAACTGAGATCAGGGG CCTCAGTTACGGAGCCTGTCGCGGAGG AAAGGGGCCTCATGTCTCAGCCTCTGA TGGAGACTTGCCATAGCGTAGGTGCTG CCTACTTGGAGTCACTGCCACTCCAGG 396 ATGCTAGTCCAGCAGGAGGCCCCTCCT CACCTAGGGATTTGCCAGAGCCTCGCG TGAGTACCGAGCACACCAATAACAAA ATTGAAAAAATATATATTATGAAGGCG GACACCGTCATCGTGGGAACGGTGAA GGCCGAGCTCCCGGAGGGCCGGGGGT TGGCCGGACCTGCGGAACCAGAACTCG AGGAAGAACTTGAAGCAGATCATACG

[0446]

[0447] CCTCACTACCCAGAACAAGAGACGGA Docket No. CNTY-033-WO-01

[0448] ACCGCCTTTGGGTTCTTGCAGTGACGT GATGTTGTCTGTGGAAGAGGAAGGAA AGGAGGATCCTCTTCCGACCGCTGCGA GCGGGAAA CD3-zeta RVKFSRSADAPAYQQGQNQLYNELNLG P20963-3 isoform 3 RREEYDVLDKRRGRDPEMGGKPRRKNP QEGLYNELQKDKMAEAYSEIGMKGERR 6 (AA 52-163)

[0449] RGKGHDGLYQGLSTATKDTYDALHMQ ALPPR CD3-zeta AGAGTGA AGTTC AGC AGATC C GC C GAT

[0450] isoform 3 GCTCCCGCCTATCAGCAGGGCCAAAAC CAGCTGTACAACGAGCTGAACCTGGGG

[0451] (nucleotide AGAAGAGAAGAGTACGACGTGCTGGA

[0452] sequence)

[0453] CAAGCGGAGAGGCAGAGATCCTGAAA TGGGCGGCAAGCCCAGACGGAAGAAT CCTCAAGAGGGCCTGTATAATGAGCTG 101 C AGAAAGAC AAGATGGCCGAGGC CTA CAGCGAGATCGGAATGAAGGGCGAGC GC AGAAGAGGC AAGGGAC AC GATGGA CTGTACCAGGGCCTGAGCACCGCCACC AAGGATACCTATGATGCCCTGCACATG CAGGCCCTGCCTCCAAGA

[0454] 41BB KRGRKKLLYIFKQPFMRPVQTTQEEDGC Q07011

[0455] SCRFPEEEEGGCEL 8 (AA 214-255)

[0456] IL2Rb NCRNTGPWLKKVLKCNTPDPSKFFSQLS P14784

[0457] SEHGGDVQKWLSSPFPSSSFSPGGLAPEI

[0458] (AA 266-551) 9

[0459] SPLEVLERDKVTQLLPLNTDAYLSLQEL QGQDPTHLV CD40 KKVAKKPTNKAPHPKQEPQEINFPDDLP P25942

[0460] GSNTAAPVQETLHGCQPVTQEDGKESRI 10 (AA 216-277) SVQERQ

[0461] 0X40 ALYLLRRDQRLPPDAHKPPGGGSFRTPIQ P43489

[0462] EEQADAHSTLAKI 11 (AA 236-277)

[0463] CD80 TYCFAPRCRERRRNERLRRESVRPV P33681

[0464] 12 (AA 264-288)

[0465]

[0466] Docket No. CNTY-033-WO-01

[0467] CD86 (AA269- KWKKKKRPRNSYKCGTNTMEREESEQT P42081

[0468] 329) KKREKIHIPERSDEAQRVFKSSKTSSCDK 13

[0469] SDTCF CD27 QRRKYRSNKGESPVEPAEPCHYSCPREE P26842

[0470] EGSTIPIQEDYRKPEPACSP 14 (AA 213-260)

[0471] ICOS CWLTKKKYS S S VHDPNGEYMFMRAVNT Q9Y6W8

[0472] AKKSRLTDVTL 15 (AA 162-199)

[0473] NKG2D MGWIRGRRSRHSWEMSEFHNYNLDLKK P26718

[0474] SDFSTRWQKQRCPVVKSKCRENAS 16 (AA 1-51)

[0475] DAP 10 LCARPRRSPAQEDGKVYINMPGRG Q9UBK5

[0476] 17 (AA 70-93)

[0477] DAP 12 YFLGRLVPRGRGAAEAATRKQRITETES 054885

[0478] PYQELQGQRSDVYSDLNTQRPYYK 18 (AA 62-113)

[0479] 2B4 / CD244 WRRKRKEKQSETSPKEFLTIYEDVKDLK Q9BZW8

[0480] (AA 251-370) TRRNHEQEQTFPGGGSTIYSMIQSQSSAP TSQEPAYTLYSLIQPSRKSGSRKRNHSPS 19 FNSTIYEVIGKSQPKAQNPARLSRKELEN FDVYS CD3-zeta RVKFSRSADAPAYQQGQNQLYNELNLG P20963-3 isoform 3 RREEYDVLDKRRGRDPEMGGKPRRKNP QEGLYNELQKDKMAEAYSEIGMKGERR 6 (AA 52-163)

[0481] RGKGHDGLYQGLSTATKDTYDALHMQ ALPPR CD28 RSKRSRLLHSDYMNMTPRRPGPTRKHY P10747-1

[0482] QPYAPPRDFAAYRS 20 (AA 180-220)

[0483] Spacer / H inge:

[0484] CD8 TTTPAPRPPTPAPTIASQPLSLRPEACRPA P01732

[0485] AGGAVHTRGLDFACD 21 (AA 136-182)

[0486] CD28 IEVMYPPPYLDNEKSNGTIIHVKGKHLCP P10747-1

[0487] SPLFPGPSKP 22 (AA 114-151)

[0488]

[0489] Docket No. CNTY-033-WO-01

[0490] IgG4 CH3 ESKYGPPCPPCPGQPREPQVYTLPPSQEE MTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSRLTVD 96 KSRWQEGNVFSCSVMHEALHNHYTQKS LSLSLGK

[0491] Transmembrane:

[0492] CD28 IEVMYPPPYLDNEKSNGTI1HVKGKHLCP Pl 0747-1

[0493] SPLFPGPSKPFWVLVVVGGVLACYSLLV

[0494] (AA 114-220) 5

[0495] TVAFIIFWVRSKRSRLLHSDYMNMTPRR PGPTRKHYQPYAPPRDFAAYRS CD28 ATCGAAGTGATGTACCCTCCACCTTAC CTGGACAACGAGAAGTCCAACGGCAC

[0496] (nucleotide CATCATCCACGTGAAGGGCAAGCACCT

[0497] sequence) GTGTCCTTCTCCACTGTTCCCCGGACCT AGCAAGCCTTTCTGGGTGCTCGTTGTT GTTGGCGGCGTGCTGGCCTGTTATAGC

[0498] 100 CTGCTTGTGACCGTGGCCTTCATCATCT TTTGGGTCCGAAGCAAGCGGAGCCGGC TGCTGCACTCCGACTACATGAACATGA CCCCTAGACGGCCCGGACCAACCAGA AAGCACTACCAGCCTTACGCTCCTCCT AGAGACTTCGCCGCCTACCGGTCC CD8 IYIWAPLAGTCGVLLLSLVIT P01732

[0499] 23 (AA 183-203)

[0500] CD28 FWVLVVVGGVLACYSLLVTVAFIIFWV Pl 0747-1

[0501] 24 (AA 153-179)

[0502] Linkers:

[0503] Whitlow Linker GSTSGSGKPGSGEGSTKG 3 (G4S)3 GGGGSGGGGSGGGGS 25 Linker 3 GGSEGKSSGSGSESKSTGGS 26 Linker 4 GGGSGGGS 27 Linker 5 GGGSGGGSGGGS 28 Linker 6 GGGSGGGSGGGSGGGS 29

[0504]

[0505] Docket No. CNTY-033-WO-01

[0506] Linker 7 GGGSGGGSGGGSGGGSGGGS 30 Linker 8 GGGGSGGGGSGGGGSGGGGS 31 Linker 9 GGGGSGGGGSGGGGSGGGGSGGGGS 32 Linker 10 IRPRAIGGSKPRVA 33 Linker 11 GKGGS GKGGS GKGGS 34 Linker 12 GGKGS GGKGS GGKGS 35 Linker 13 GGGKS GGGKS GGGKS 36 Linker 14 GKGKS GKGKS GKGKS 37 Linker 15 GGGKS GGKGS GKGGS 38 Linker 16 GKPGSGKPGSGKPGS 39 Linker 17 GKPGSGKPGSGKPGSGKPGS 40 Linker 18 GKGKS GKGKS GKGKS GKGKS 41 Linker 19 STAGDTHLGGEDFD 42 Linker 20 GEGGSGEGGSGEGGS 43 Linker 21 GGEGSGGEGSGGEGS 44 Linker 22 GEGESGEGESGEGES 45 Linker 23 GGGESGGEGSGEGGS 46 Linker 24 GEGESGEGESGEGESGEGES 47 Linker 25 GSTSGSGKPGSGEGSTKG 48 Linker 26 PRGASKSGSASQTGSAPGS 49 Linker 27 GTAAAGAGAAGGAAAGAAG 50 Linker 28 GTSGSSGSGSGGSGSGGGG 51 Linker 29 GKPGSGKPGSGKPGSGKPGS 52 Linker 30 GSGS 53 Linker 31 APAPAPAPAP 54 Linker 32 APAPAPAPAPAPAPAPAPAP 55

[0507]

[0508] Docket No. CNTY-033-WO-01

[0509] AEAAAKEAAAKEAAAAKEAAAAKEAA

[0510] Linker 33 56

[0511] AAKAAA

[0512] Linker 34 GGGGS 104 Linker 35 GGGGSGGGGS 103 Whitlow Linker GGCTCTACAAGCGGCAGCGGCAAACCT

[0513] (nucleotide GGATCTGGCGAGGGATCTACCAAGGG 99 sequence) C

[0514] Whitlow Linker GGCAGTACTTCTGGTAGCGGAAAACCC

[0515] (nucleotide GGTAGCGGCGAGGGGTCAACTAAAGG 102 sequence) A

[0516] Signal Peptide:

[0517] GMCSFR Signal

[0518] MLLLVTSLLLCELPHPAFLLIP 1 Peptide

[0519] IgK Signal

[0520] Peptide Variant

[0521] MARSPAQLLGLLLLWLSGARC 97 (amino acid

[0522] sequence)

[0523] IgK Signal

[0524] Peptide Variant ATGGCCAGATCTCCTGCTCAACTGCTGGGACTGCT

[0525] 98 (nucleotide GCTGCTGTGGCTTAGCGGAGCCAGATGC

[0526] sequence)

[0527] MARS SP ATGGCCAGATCCCCGGCACAACTGCTCGGACTCCT

[0528] 378 (nucleic acid) CCTGTTGTGGTTGAGCGGGGCCCGCTGT

[0529] Nectin4 Targeting CARs:

[0530] MARS MARSPAQLLGLLLLWLSGARCEVQLLESGGGLVQAG SP_DB01_C01_ GSLRLSCAASGSFFSIYAMGWFRQAPGKEREFVAAYI P2112_CD8 SSGGLTSYADSVKGRFTISRDNAKNTVYLQMNSLKPE Hinge_CD28 DTAVYYCAADLGAQTGYVQYDYWGQGTQVTVSST tm_41BB_CD3 TTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRG

[0531] Z (amino acid) LDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVKRG 157

[0532] RKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGC ELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV LDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAE AYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL HMQALPPR

[0533]

[0534] Docket No. CNTY-033-WO-01

[0535] MARS MARSPAQLLGLLLLWLSGARCEVQLLESGGGLVQPG SP_DB01_B01_ GSLRLSCAASGFVSSIYFMGWFRQAPGKEREFVSSSIG P2106_CD8 KGGSTRYADSVKGRFTISRDNSKNTLYLQMNSLKPE Hinge_CD28 DTAVYYC AGDEGLGTAH AEYDYWGQGTQVTV S SET tm_41BB_CD3 TPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGL

[0536] Z (amino acid) DFACDFWVLVVVGGVLACYSLLVTVAFIIFWVKRGR 158

[0537] KKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEA YSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH MQALPPR MARS MARSPAQLLGLLLLWLSGARCEVQLLESGGGLVQAG SP_DB01_B10_ GSLRLSCAASGGISEFYFMGWFRQAPGKEREFVAAEI P2110 CD8 SPGSYTNYADSVKGRFTISRDNAKNTVYLQMNSLKP Hinge_CD28 EDTAVYYCAADRDGDTYTAEYDYWGQGTQVTVSST tm_41BB_CD3 TTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRG

[0538] Z (amino acid) LDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVKRG 159

[0539] RKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGC ELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV LDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAE AYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL HMQALPPR MARS MARSPAQLLGLLLLWLSGARCEVQLLESGGGLVQPG SP_DBO1_FO3_ GSLRLSCAASGSISSFYYIGWFRQAPGKEREFVSSRITS P2121 CD8 GGSTYYRDSVKGRFTISRDNSKNTLYLQMNSLKPEDT Hinge_CD28 AVYYCAAGTSRDYYYWGQGTQVTVSSTTTPAPRPPT tm_41BB_CD3 PAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFW

[0540] 160 Z (amino acid) VLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFK QPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRS ADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRD PEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMK GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR MARS MARSPAQLLGLLLLWLSGARCEVQLLESGGGLVQPG SP_DBO1_A11_ GSLRLSCAASGSTSSIGIMGWFRQAPGKERELVSSITA P2105_CD8 GGSTYYADSVKGRFTISRDNSKNTLYLQMNSLKPED Hinge_CD28 TAVYYCNAHV GYGRVHDVDYWGQGTQVTVS STTTP tm_41BB_CD3 APRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDF 161 Z (amino acid) ACDFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKK LLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELR VKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD

[0541]

[0542] KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAY Docket No. CNTY-033-WO-01

[0543] SEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPR MARS MARSPAQLLGLLLLWLSGARCEVQLLESGGGLVQPG SP DB01 E03 GSLRLSCAASGFVSPSYIMGWFRQAPGKEREFVSSVIE P2120_CD8 YRGSTYYLDSVKGRFTISRDNSKNTLYLQMNSLKPED Hinge_CD28 TAVYYC AAGTPGGYDYWGQGTQVTVS STTTP APRPP tm_41BB_CD3 TPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDF

[0544] Z (amino acid) WVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYI 162

[0545] FKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFS RSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRG RDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP PR MARSPAQLLGLLLLWLSGARCEVQLLESGGGLVQPG GSLRLSCAASGLTSSGYAMGWYRQAPGKERELVSSIS SSGGLTHYADSVKGRFTISRDNSKNTLYLQMNSLKPE MARS DTAVYYCDADIAYTGADYWGQGTQVTVSSTTTPAPR SP_NEC_S_11_

[0546] PPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC

[0547] P3112 CD8

[0548] DFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLL

[0549] Hinge_CD28

[0550] YIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVK 163 tm_41BB_CD3

[0551] FSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRR

[0552] Z (amino acid)

[0553] GRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEI GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQA LPPR

[0554] MARSPAQLLGLLLLWLSGARCEVQLLESGGGLVQPG GSLRLSCAASGFTYSSYAMGWYRQAPGKERELVSSIS GSGGSTRYADSVKGRFTISRDNSKNTLYLQMNSLKPE MARS DTAVYYCAVAIGVGDYWGQGTQVTVSSTTTP APRPP SP_NEC_S_31_

[0555] TPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDF

[0556] P3113 CD8

[0557] WVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYI 164 Hinge_CD28

[0558] FKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFS

[0559] tm 4 IBB CD3

[0560] RSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRG

[0561] Z (amino acid)

[0562] RDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP PR MARS MARSPAQLLGLLLLWLSGARCEVQLLESGGGLVQPG SP_NEC_S_55_ GSLRLSCAASGFTYSSYAMGWYRQAPGKERELVSSIS 165 P3114 CD8 GSGGSTRYADSVKGRFTISRDNSKNTLYLQMNSLKPE

[0563]

[0564] Hinge_CD28 DTAVYYCAVAIGVGDYWGQGTQVTVSSTTTP APRPP Docket No. CNTY-033-WO-01

[0565] tm_41BB_CD3 TPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDF

[0566] Z (amino acid) WVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYI FKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFS RSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRG RDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP PR MARSPAQLLGLLLLWLSGARCEVQLLESGGGLVQPG GSLRLSCAASGLSFSSYSMGWYRQAPGKEREFVSAIS GSSGSTNYADSVKGRFTISRDNSKNTLYLQMNSLKPE MARS DTAVYYC AAEHRVTTSGVFYDYWGQGTQVTV S STT SP_NEC_M_5_

[0567] TPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGL

[0568] P3106 CD8

[0569] DFACDFWVLVVVGGVLACYSLLVTVAFIIFWVKRGR 166 Hinge_CD28

[0570] KKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE

[0571] tm_41BB_CD3

[0572] LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL

[0573] Z (amino acid)

[0574] DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEA YSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH MQALPPR MARSPAQLLGLLLLWLSGARCEVQLLESGGGLVQPG GSLRLSCAASGFTFSSYAMGWYRQAPGKEREFVSSIS GSGGLTRYADSVKGRFTISRDNSKNTLYLQMNSLKPE MARS DTAVYYC AVTTGYQGGVYDYWGQGTQVTVSSTTTP SP_NEC_M_8_

[0575] APRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDF

[0576] P3107 CD8

[0577] ACDFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKK 167 Hinge_CD28

[0578] LLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELR

[0579] tm_41BB_CD3

[0580] VKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD

[0581] Z (amino acid)

[0582] KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAY SEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPR MARSPAQLLGLLLLWLSGARCEVQLLESGGGLVQPG GSLRLSCAASGFTYSGYAMGWYRQAPGKERELVSSI SGSGTLTSYADSVKGRFTISRDNSKNTLYLQMNSLKP MARS EDTAVYYCDVDIPVGDATTVGDYWGQGTQVTVSST SP_NEC_M_17

[0583] TTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRG

[0584] P3108 CD8

[0585] LDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVKRG 168 Hinge_CD28

[0586] RKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGC

[0587] tm_41BB_CD3

[0588] ELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV

[0589] Z (amino acid)

[0590] LDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAE AYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL HMQALPPR

[0591]

[0592] Docket No. CNTY-033-WO-01

[0593] MARSPAQLLGLLLLWLSGARCEVQLLESGGGLVQPG GSLRLSCAASGRTLSSYAMGWYRQAPGKERELVSSIS GSGGSTRYADSVKGRFTISRDNSKNTLYLQMNSLKPE MARS DTAVYYCAVYILELAPGAEYWGQGTQVTVSSTTTPA SP_NEC_M_44

[0594] PRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFA

[0595] P3109 CD8

[0596] CDFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKL 169 Hinge_CD28

[0597] LYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRV

[0598] tm_41BB_CD3

[0599] KFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDK

[0600] Z (amino acid)

[0601] RRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYS EIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ ALPPR MARSPAQLLGLLLLWLSGARCEVQLLESGGGLVQPG GSLRLSCAASGYTFSDYAMGWYRQAPGKERELVSSI SGSGGSTRYADSVKGRFTISRDNSKNTLYLQMNSLKP MARS EDTAVYYCAAVIRQPSTGFYEYWGQGTQVTVSSTTT SP_NEC_M_46

[0602] PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD

[0603] P3110 CD8

[0604] FACDFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRK 170 Hinge_CD28

[0605] KLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0606] tm_41BB_CD3

[0607] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL

[0608] Z (amino acid)

[0609] DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEA YSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH MQALPPR EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMGW YRQAPGKEREFVSSISGSGGLTRYADSVKGRFTISRD NSKNTLYLQMNSLKPEDTAVYYCAVTTGYQGGVYD YWGQGTQVTV S STTTP APRPPTP APTI AS QPLSLRPE A

[0610] M8-CD8h- CRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSL CD28tmd- LVTVAFIIFWVCHRRACRKRIRQKLHLCYPVQTSQPK CD30-CD3z LELVDSRPRRSSTQLRSGASVTEPVAEERGLMSQPLM

[0611] 393 (amino acid) ETCHSVGAAYLESLPLQDASPAGGPSSPRDLPEPRVST EHTNNKIEKIYIMKADTVIVGTVKAELPEGRGLAGPA

[0612] #5034 EPELEEELEADHTPHYPEQETEPPLGSCSDVMLSVEEE GKEDPLPTAASGKRVKFSRSADAPAYQQGQNQLYNE LNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGL YNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGL STATKDTYDALHMQALPPR MARS ATGGCCAGATCTCCTGCTCAACTGCTGGGACTGCT SP_DB01_C01_ GCTGCTGTGGCTTAGCGGAGCCAGATGCGAGGTAC P2112 CD8 AACTTTTGGAGTCAGGCGGCGGGTTGGTCCAGGCG 171 Hinge_CD28 GGTGGCTCACTCCGCCTTAGTTGTGCCGCCTCAGG

[0613]

[0614] GTCATTCTTTAGTATCTACGCTATGGGCTGGTTTCG Docket No. CNTY-033-WO-01

[0615] tm_41BB_CD3 ACAGGCCCCTGGTAAGGAACGTGAGTTTGTGGCCG

[0616] Z (nucleotide) CCTACATTTCCTCAGGGGGGCTCACCAGCTACGCG GATAGTGTTAAGGGTAGATTCACCATCTCCAGAGA CAATGCAAAGAATACGGTATACCTCCAAATGAACA GCCTGAAGCCTGAAGACACGGCTGTCTACTATTGC GCAGCAGACTTGGGAGCCCAGACCGGATACGTTCA GTACGACTACTGGGGGCAGGGAACCCAGGTGACG GTCTCGAGCACAACAACTCCAGCCCCAAGACCACC TACGCCTGCACCTACTATCGCATCTCAACCACTGTC CCTGCGCCCTGAGGCATGCCGACCAGCAGCCGGTG GCGCGGTGCATACCCGCGGACTGGACTTTGCCTGC GATTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTG GCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATT ATTTTCTGGGTGAAACGGGGCAGAAAGAAACTCCT GTATATATTCAAACAACCATTTATGCGACCAGTAC AAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGA TTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGA GAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGC GTACCAGCAGGGCCAGAACCAGCTCTATAACGAGC TCAATCTAGGACGAAGAGAGGAGTACGATGTTTTG GACAAGCGACGTGGCCGGGACCCTGAGATGGGGG GAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCT GTACAATGAACTGCAGAAAGATAAGATGGCGGAG GCCTACAGTGAGATTGGGATGAAAGGCGAGCGCC GGAGGGGCAAGGGGCACGATGGCCTTTACCAGGG ACTCAGTACAGCCACCAAGGACACCTACGACGCCC TTCACATGCAGGCCCTGCCCCCTCGC MARS ATGGCCAGATCTCCTGCTCAACTGCTGGGACTGCT SP_DB01_B01_ GCTGCTGTGGCTTAGCGGAGCCAGATGCGAGGTAC P2106 CD8 AACTTTTGGAGTCAGGCGGCGGCCTGGTGCAGCCC Hinge_CD28 GGAGGGAGTCTCCGACTGTCTTGCGCTGCATCTGG tm_41BB_CD3 ATTCGTGAGCTCTATATACTTTATGGGATGGTTCAG

[0617] Z (nucleotide) GCAGGCTCCTGGGAAGGAGCGCGAGTTTGTGTCTA GTAGTATTGGCAAGGGTGGCTCAACACGCTATGCG GATTCTGTGAAAGGGAGGTTCACAATAAGCAGGGA 172 CAACTCAAAGAATACACTGTACCTCCAGATGAACT CCTTAAAACCAGAGGATACTGCAGTCTATTACTGT GCTGGAGATGAGGGATTGGGAACTGCACATGCTGA ATACGACTACTGGGGCCAGGGGACCCAGGTGACG GTCTCGAGCACAACAACTCCAGCCCCAAGACCACC TACGCCTGCACCTACTATCGCATCTCAACCACTGTC CCTGCGCCCTGAGGCATGCCGACCAGCAGCCGGTG

[0618]

[0619] GCGCGGTGCATACCCGCGGACTGGACTTTGCCTGC Docket No. CNTY-033-WO-01

[0620] GATTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTG GCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATT ATTTTCTGGGTGAAACGGGGCAGAAAGAAACTCCT GTATATATTCAAACAACCATTTATGCGACCAGTAC AAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGA TTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGA GAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGC GTACCAGCAGGGCCAGAACCAGCTCTATAACGAGC TCAATCTAGGACGAAGAGAGGAGTACGATGTTTTG GACAAGCGACGTGGCCGGGACCCTGAGATGGGGG GAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCT GTACAATGAACTGCAGAAAGATAAGATGGCGGAG GCCTACAGTGAGATTGGGATGAAAGGCGAGCGCC GGAGGGGCAAGGGGCACGATGGCCTTTACCAGGG ACTCAGTACAGCCACCAAGGACACCTACGACGCCC TTCACATGCAGGCCCTGCCCCCTCGC MARS ATGGCCAGATCTCCTGCTCAACTGCTGGGACTGCT SP_DB01_B10_ GCTGCTGTGGCTTAGCGGAGCCAGATGCGAGGTAC P2110_CD8 AACTTTTGGAGTCAGGCGGCGGGTTGGTCCAGGCG Hinge_CD28 GGTGGCTCACTCCGCCTTAGTTGTGCCGCCTCAGG tm 4 IBB CD3 GTCATTCTTTAGTATCTACGCTATGGGCTGGTTTCG

[0621] Z (nucleotide) ACAGGCCCCTGGTAAGGAACGTGAGTTTGTGGCCG CCTACATTTCCTCAGGGGGGCTCACCAGCTACGCG GATAGTGTTAAGGGTAGATTCACCATCTCCAGAGA CAATGCAAAGAATACGGTATACCTCCAAATGAACA GCCTGAAGCCTGAAGACACGGCTGTCTACTATTGC GCAGCAGACTTGGGAGCCCAGACCGGATACGTTCA GTACGACTACTGGGGGCAGGGAACCCAGGTGACG GTCTCGAGCACAACAACTCCAGCCCCAAGACCACC TACGCCTGCACCTACTATCGCATCTCAACCACTGTC 173 CCTGCGCCCTGAGGCATGCCGACCAGCAGCCGGTG GCGCGGTGCATACCCGCGGACTGGACTTTGCCTGC GATTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTG GCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATT ATTTTCTGGGTGAAACGGGGCAGAAAGAAACTCCT GTATATATTCAAACAACCATTTATGCGACCAGTAC AAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGA TTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGA GAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGC GTACCAGCAGGGCCAGAACCAGCTCTATAACGAGC TCAATCTAGGACGAAGAGAGGAGTACGATGTTTTG GACAAGCGACGTGGCCGGGACCCTGAGATGGGGG

[0622]

[0623] GAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCT Docket No. CNTY-033-WO-01

[0624] GTACAATGAACTGCAGAAAGATAAGATGGCGGAG GCCTACAGTGAGATTGGGATGAAAGGCGAGCGCC GGAGGGGCAAGGGGCACGATGGCCTTTACCAGGG ACTCAGTACAGCCACCAAGGACACCTACGACGCCC TTCACATGCAGGCCCTGCCCCCTCGC MARS ATGGCCAGATCTCCTGCTCAACTGCTGGGACTGCT SP_DB01_F03_ GCTGCTGTGGCTTAGCGGAGCCAGATGCGAGGTAC P2121_CD8 AACTTTTGGAGTCAGGCGGCGGGTTAGTGCAGCCT Hinge_CD28 GGAGGATCACTGAGGCTGAGCTGCGCCGCCTCTGG tm_41BB_CD3 CTCAATTAGCAGTTTCTATTATATCGGATGGTTCCG

[0625] Z (nucleotide) CCAGGCTCCGGGAAAAGAGAGAGAGTTTGTTTCCT CTCGCATTACCTCAGGAGGAAGCACTTACTACAGG GACTCTGTTAAAGGACGCTTTACAATCTCCAGAGA TAATTCCAAGAACACCTTATATCTGCAAATGAATA GTTTGAAGCCCGAGGACACTGCCGTGTATTATTGC GCAGCCGGGACATCCCGCGATTACTACTACTGGGG ACAAGGAACCCAGGTGACGGTCTCGAGCACAACA ACTCCAGCCCCAAGACCACCTACGCCTGCACCTAC TATCGCATCTCAACCACTGTCCCTGCGCCCTGAGGC ATGCCGACCAGCAGCCGGTGGCGCGGTGCATACCC GCGGACTGGACTTTGCCTGCGATTTTTGGGTGCTGG

[0626] 174 TGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGC TAGTAACAGTGGCCTTTATTATTTTCTGGGTGAAAC GGGGCAGAAAGAAACTCCTGTATATATTCAAACAA CCATTTATGCGACCAGTACAAACTACTCAAGAGGA AGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAG AAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAG GAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAG AACCAGCTCTATAACGAGCTCAATCTAGGACGAAG AGAGGAGTACGATGTTTTGGACAAGCGACGTGGCC GGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAA GAACCCTCAGGAAGGCCTGTACAATGAACTGCAGA AAGATAAGATGGCGGAGGCCTACAGTGAGATTGG GATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCAC GATGGCCTTTACCAGGGACTCAGTACAGCCACCAA GGACACCTACGACGCCCTTCACATGCAGGCCCTGC CCCCTCGC MARS ATGGCCAGATCTCCTGCTCAACTGCTGGGACTGCT SP_DBO1_A11_ GCTGCTGTGGCTTAGCGGAGCCAGATGCGAGGTAC P2105_CD8 AACTTTTGGAGTCAGGCGGGGGGCTGGTGCAGCCT 175 Hinge_CD28 GGGGGATCACTGCGCCTTTCATGCGCAGCGAGTGG

[0627]

[0628] TTCCACCTCTAGTATCGGAATTATGGGCTGGTTTCG Docket No. CNTY-033-WO-01

[0629] tm_41BB_CD3 CCAGGCTCCTGGAAAGGAAAGGGAGCTGGTCTCCA

[0630] Z (nucleotide) GCATCACAGCCGGCGGATCTACCTACTACGCCGAC TCCGTTAAGGGGCGATTCACTATCTCCCGCGACAA TAGCAAGAACACCTTGTATCTGCAGATGAACTCCC TCAAACCCGAGGATACTGCCGTGTACTATTGCAAC GCACATGTGGGCTACGGGAGGGTGCACGATGTGGA TTACTGGGGGCAGGGGACCCAGGTGACGGTCTCGA GCACAACAACTCCAGCCCCAAGACCACCTACGCCT GCACCTACTATCGCATCTCAACCACTGTCCCTGCGC CCTGAGGCATGCCGACCAGCAGCCGGTGGCGCGGT GCATACCCGCGGACTGGACTTTGCCTGCGATTTTTG GGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCT ATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCT GGGTGAAACGGGGCAGAAAGAAACTCCTGTATAT ATTCAAACAACCATTTATGCGACCAGTACAAACTA CTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCA GAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGA AGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAG CAGGGCCAGAACCAGCTCTATAACGAGCTCAATCT AGGACGAAGAGAGGAGTACGATGTTTTGGACAAG CGACGTGGCCGGGACCCTGAGATGGGGGGAAAGC CGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAA TGAACTGCAGAAAGATAAGATGGCGGAGGCCTAC AGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGG GCAAGGGGCACGATGGCCTTTACCAGGGACTCAGT ACAGCCACCAAGGACACCTACGACGCCCTTCACAT GCAGGCCCTGCCCCCTCGC MARS ATGGCCAGATCTCCTGCTCAACTGCTGGGACTGCT SP_DB01_E03_ GCTGCTGTGGCTTAGCGGAGCCAGATGCGAGGTAC P2120_CD8 AACTTTTGGAGTCAGGCGGTGGTCTCGTCCAGCCG Hinge_CD28 GGGGGGAGCCTCCGCTTATCATGTGCCGCCTCCGG tm_41BB_CD3 ATTTGTCTCACCCTCTTATATAATGGGGTGGTTCCG

[0631] Z (nucleotide) GCAAGCACCAGGCAAAGAACGGGAGTTTGTTTCCT CCGTCATAGAGTACCGAGGGTCCACCTATTACCTG GATAGCGTGAAGGGGCGGTTCACCATCTCCCGCGA 176 TAATTCCAAGAATACCCTGTACCTGCAGATGAATA GTCTGAAACCTGAGGACACGGCCGTGTACTATTGC GCAGCGGGCACCCCCGGAGGCTACGATTACTGGGG CCAAGGGACCCAGGTGACGGTCTCGAGCACAACA ACTCCAGCCCCAAGACCACCTACGCCTGCACCTAC TATCGCATCTCAACCACTGTCCCTGCGCCCTGAGGC ATGCCGACCAGCAGCCGGTGGCGCGGTGCATACCC

[0632]

[0633] GCGGACTGGACTTTGCCTGCGATTTTTGGGTGCTGG Docket No. CNTY-033-WO-01

[0634] TGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGC TAGTAACAGTGGCCTTTATTATTTTCTGGGTGAAAC GGGGCAGAAAGAAACTCCTGTATATATTCAAACAA CCATTTATGCGACCAGTACAAACTACTCAAGAGGA AGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAG AAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAG GAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAG AACCAGCTCTATAACGAGCTCAATCTAGGACGAAG AGAGGAGTACGATGTTTTGGACAAGCGACGTGGCC GGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAA GAACCCTCAGGAAGGCCTGTACAATGAACTGCAGA AAGATAAGATGGCGGAGGCCTACAGTGAGATTGG GATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCAC GATGGCCTTTACCAGGGACTCAGTACAGCCACCAA GGACACCTACGACGCCCTTCACATGCAGGCCCTGC CCCCTCGC ATGGCCAGATCTCCTGCTCAACTGCTGGGACTGCT GCTGCTGTGGCTTAGCGGAGCCAGATGCGAGGTAC AACTTTTGGAGTCAGGCGGTGGACTGGTACAACCG GGTGGTTCATTGCGTTTGAGCTGCGCTGCCTCTGGT TTAACAAGCTCTGGTTACGCTATGGGCTGGTATCG CCAAGCGCCGGGCAAAGAACGCGAGCTGGTGAGC AGTATTTCTTCCTCAGGCGGACTGACCCATTACGCG GACTCGGTAAAAGGCCGTTTTACGATCAGTCGTGA TAATTCCAAGAATACCTTGTACCTGCAAATGAATA GCCTTAAGCCCGAAGACACAGCGGTGTATTATTGT MARS GACGCAGATATTGCTTACACTGGCGCCGATTATTG SP_NEC_S_11_

[0635] GGGCCAGGGTACCCAGGTGACGGTCTCGAGCACAA

[0636] P3112 CD8

[0637] CAACTCCAGCCCCAAGACCACCTACGCCTGCACCT

[0638] Hinge_CD28

[0639] ACTATCGCATCTCAACCACTGTCCCTGCGCCCTGAG 177 tm_41BB_CD3

[0640] Z (nucleotide) GCATGCCGACCAGCAGCCGGTGGCGCGGTGCATAC CCGCGGACTGGACTTTGCCTGCGATTTTTGGGTGCT GGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTT GCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAA ACGGGGCAGAAAGAAACTCCTGTATATATTCAAAC AACCATTTATGCGACCAGTACAAACTACTCAAGAG GAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGA AGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGC AGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCC AGAACCAGCTCTATAACGAGCTCAATCTAGGACGA AGAGAGGAGTACGATGTTTTGGACAAGCGACGTGG CCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGG

[0641]

[0642] AAGAACCCTCAGGAAGGCCTGTACAATGAACTGCA Docket No. CNTY-033-WO-01

[0643] GAAAGATAAGATGGCGGAGGCCTACAGTGAGATT GGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGC ACGATGGCCTTTACCAGGGACTCAGTACAGCCACC AAGGACACCTACGACGCCCTTCACATGCAGGCCCT GCCCCCTCGC

[0644] ATGGCCAGATCTCCTGCTCAACTGCTGGGACTGCT GCTGCTGTGGCTTAGCGGAGCCAGATGCGAGGTAC AACTTTTGGAGTCAGGCGGTGGACTGGTACAACCG GGTGGTTCATTGCGTTTGAGCTGCGCTGCCTCTGGT TTTACGTACTCTTCTTATGCAATGGGCTGGTATCGC CAAGCGCCGGGCAAAGAACGCGAGTTGGTCTCCTC CATATCCGGTTCCGGTGGTAGCACCCGCTACGCGG ACTCGGTAAAAGGCCGTTTTACGATCAGTCGTGAT AATTCCAAGAATACCTTGTACCTGCAAATGAATAG CCTTAAGCCCGAAGACACAGCGGTGTATTATTGTG CCGTGGCGATTGGCGTAGGAGACTATTGGGGCCAG GGTACCCAGGTGACGGTCTCGAGCACAACAACTCC AGCCCCAAGACCACCTACGCCTGCACCTACTATCG MARS CATCTCAACCACTGTCCCTGCGCCCTGAGGCATGC SP_NEC_S_31_ CGACCAGCAGCCGGTGGCGCGGTGCATACCCGCGG P3113 CD8 ACTGGACTTTGCCTGCGATTTTTGGGTGCTGGTGGT

[0645] 178 Hinge_CD28 GGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGT tm_41BB_CD3 AACAGTGGCCTTTATTATTTTCTGGGTGAAACGGG

[0646] Z (nucleotide) GCAGAAAGAAACTCCTGTATATATTCAAACAACCA TTTATGCGACCAGTACAAACTACTCAAGAGGAAGA TGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAG GAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAG CGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACC AGCTCTATAACGAGCTCAATCTAGGACGAAGAGAG GAGTACGATGTTTTGGACAAGCGACGTGGCCGGGA CCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAAC CCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGA TAAGATGGCGGAGGCCTACAGTGAGATTGGGATGA AAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGG CCTTTACCAGGGACTCAGTACAGCCACCAAGGACA CCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTC GC MARS ATGGCCAGATCTCCTGCTCAACTGCTGGGACTGCT SP_NEC_S_55_ GCTGCTGTGGCTTAGCGGAGCCAGATGCGAGGTAC 179

[0647]

[0648] P3114 CD8 AACTTTTGGAGTCAGGCGGTGGACTGGTACAACCG Docket No. CNTY-033-WO-01

[0649] Hinge_CD28 GGTGGTTCATTGCGTTTGAGCTGCGCTGCCTCTGGT tm_41BB_CD3 TTTACCCTGTCTTCGTATGCAATGGGCTGGTACCGC

[0650] Z (nucleotide) CAAGCGCCGGGCAAAGAACGCGAGCTCGTTTCCAG CATCAGTGGGTCGGGTGGCTCTACCCGGTACGCGG ACTCGGTAAAAGGCCGTTTTACGATCAGTCGTGAT AATTCCAAGAATACCTTGTACCTGCAAATGAATAG CCTTAAGCCCGAAGACACAGCGGTGTATTATTGTG CCGCTTACATCGGCGGAGATTACTTGGGCCAGGGT ACCCAGGTGACGGTCTCGAGCACAACAACTCCAGC CCCAAGACCACCTACGCCTGCACCTACTATCGCAT CTCAACCACTGTCCCTGCGCCCTGAGGCATGCCGA CCAGCAGCCGGTGGCGCGGTGCATACCCGCGGACT GGACTTTGCCTGCGATTTTTGGGTGCTGGTGGTGGT TGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAAC AGTGGCCTTTATTATTTTCTGGGTGAAACGGGGCA GAAAGAAACTCCTGTATATATTCAAACAACCATTT ATGCGACCAGTACAAACTACTCAAGAGGAAGATG GCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGA GGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCG CAGACGCCCCCGCGTACCAGCAGGGCCAGAACCA GCTCTATAACGAGCTCAATCTAGGACGAAGAGAGG AGTAC GATGTTTTGGAC AAGCGACGTGGCC GGGAC CCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACC CTCAGGAAGGCCTGTACAATGAACTGCAGAAAGAT AAGATGGCGGAGGCCTACAGTGAGATTGGGATGA AAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGG CCTTTACCAGGGACTCAGTACAGCCACCAAGGACA CCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTC GC ATGGCCAGATCTCCTGCTCAACTGCTGGGACTGCT GCTGCTGTGGCTTAGCGGAGCCAGATGCGAGGTAC AACTTTTGGAGTCAGGCGGTGGACTGGTACAACCG GGTGGTTCATTGCGTTTGAGCTGCGCTGCCTCTGGT MARS CTGAGCTTCTCATCTTATTCAATGGGGTGGTATCGC SP_NEC_M_5_ CAAGCGCCGGGCAAAGAACGCGAGTTTGTTTCTGC P3106_CD8 AATCTCAGGCTCATCAGGTTCAACGAATTACGCGG 180 Hinge_CD28 ACTCGGTAAAAGGCCGTTTTACGATCAGTCGTGAT tm_41BB_CD3 AATTCCAAGAATACCTTGTACCTGCAAATGAATAG

[0651] Z (nucleotide) CCTTAAGCCCGAAGACACAGCGGTGTATTATTGTG CCGCCGAACACCGGGTGACCACTTCCGGAGTTTTC TACGACTATTGGGGCCAGGGTACCCAGGTGACGGT CTCGAGCACAACAACTCCAGCCCCAAGACCACCTA

[0652]

[0653] CGCCTGCACCTACTATCGCATCTCAACCACTGTCCC Docket No. CNTY-033-WO-01

[0654] TGCGCCCTGAGGCATGCCGACCAGCAGCCGGTGGC GCGGTGCATACCCGCGGACTGGACTTTGCCTGCGA TTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGC TTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTAT TTTCTGGGTGAAACGGGGCAGAAAGAAACTCCTGT ATATATTCAAACAACCATTTATGCGACCAGTACAA ACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATT TCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGA GTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTA CCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCA ATCTAGGACGAAGAGAGGAGTACGATGTTTTGGAC AAGCGACGTGGCCGGGACCCTGAGATGGGGGGAA AGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTA CAATGAACTGCAGAAAGATAAGATGGCGGAGGCC TACAGTGAGATTGGGATGAAAGGCGAGCGCCGGA GGGGCAAGGGGCACGATGGCCTTTACCAGGGACTC AGTACAGCCACCAAGGACACCTACGACGCCCTTCA CATGCAGGCCCTGCCCCCTCGC ATGGCCAGATCTCCTGCTCAACTGCTGGGACTGCT GCTGCTGTGGCTTAGCGGAGCCAGATGCGAGGTAC AACTTTTGGAGTCAGGCGGTGGACTGGTACAACCG GGTGGTTCATTGCGTTTGAGCTGCGCTGCCTCTGGT TTCACTTTCAGCAGTTATGCGATGGGATGGTATCGC CAAGCGCCGGGCAAAGAACGCGAGTTTGTTAGCAG CATAAGCGGTTCAGGGGGGTTAACCCGGTACGCGG ACTCGGTAAAAGGCCGTTTTACGATCAGTCGTGAT AATTCCAAGAATACCTTGTACCTGCAAATGAATAG MARS CCTTAAGCCCGAAGACACAGCGGTGTATTATTGTG SP_NEC_M_8_ CCGTAACCACCGGCTACCAAGGCGGCGTATATGAC P3107_CD8 TACTGGGGCCAGGGTACCCAGGTGACGGTCTCGAG Hin CACAACAACTCCAGCCCCAAGACCACCTACGCCTG 181 ge_CD28

[0655] tm_41BB_CD3 CACCTACTATCGCATCTCAACCACTGTCCCTGCGCC

[0656] Z (nucleotide) CTGAGGCATGCCGACCAGCAGCCGGTGGCGCGGTG CATACCCGCGGACTGGACTTTGCCTGCGATTTTTGG GTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTAT AGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGG GTGAAACGGGGCAGAAAGAAACTCCTGTATATATT CAAACAACCATTTATGCGACCAGTACAAACTACTC AAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAA GAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGT TCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAG GGCCAGAACCAGCTCTATAACGAGCTCAATCTAGG

[0657]

[0658] ACGAAGAGAGGAGTACGATGTTTTGGACAAGCGA Docket No. CNTY-033-WO-01

[0659] CGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGA GAAGGAAGAACCCTCAGGAAGGCCTGTACAATGA ACTGCAGAAAGATAAGATGGCGGAGGCCTACAGT GAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCA AGGGGCACGATGGCCTTTACCAGGGACTCAGTACA GCCACCAAGGACACCTACGACGCCCTTCACATGCA GGCCCTGCCCCCTCGC ATGGCCAGATCTCCTGCTCAACTGCTGGGACTGCT GCTGCTGTGGCTTAGCGGAGCCAGATGCGAGGTAC AACTTTTGGAGTCAGGCGGTGGACTGGTACAACCG GGTGGTTCATTGCGTTTGAGCTGCGCTGCCTCTGGT TTCACTTACTCTGGGTATGCAATGGGGTGGTATCGC CAAGCGCCGGGCAAAGAACGCGAGCTAGTCTCAA GCATCTCAGGTTCGGGTACCCTTACTTCGTACGCGG ACTCGGTAAAAGGCCGTTTTACGATCAGTCGTGAT AATTCCAAGAATACCTTGTACCTGCAAATGAATAG CCTTAAGCCCGAAGACACAGCGGTGTATTATTGTG ACGTAGATATACCTGTGGGCGACGCTACAACCGTT GGTGATTACTGGGGCCAGGGTACCCAGGTGACGGT CTCGAGCACAACAACTCCAGCCCCAAGACCACCTA MARS CGCCTGCACCTACTATCGCATCTCAACCACTGTCCC SP_NEC_M_17 TGCGCCCTGAGGCATGCCGACCAGCAGCCGGTGGC P3108 CD8 GCGGTGCATACCCGCGGACTGGACTTTGCCTGCGA

[0660] 182 Hinge_CD28 TTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGC tm_41BB_CD3 TTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTAT

[0661] Z (nucleotide) TTTCTGGGTGAAACGGGGCAGAAAGAAACTCCTGT ATATATTCAAACAACCATTTATGCGACCAGTACAA ACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATT TCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGA GTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTA CCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCA ATCTAGGACGAAGAGAGGAGTACGATGTTTTGGAC AAGCGACGTGGCCGGGACCCTGAGATGGGGGGAA AGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTA CAATGAACTGCAGAAAGATAAGATGGCGGAGGCC TACAGTGAGATTGGGATGAAAGGCGAGCGCCGGA GGGGCAAGGGGCACGATGGCCTTTACCAGGGACTC AGTACAGCCACCAAGGACACCTACGACGCCCTTCA CATGCAGGCCCTGCCCCCTCGC MARS ATGGCCAGATCTCCTGCTCAACTGCTGGGACTGCT SP_NEC_M_44 GCTGCTGTGGCTTAGCGGAGCCAGATGCGAGGTAC 183

[0662]

[0663] P3109 CD8 AACTTTTGGAGTCAGGCGGTGGACTGGTACAACCG Docket No. CNTY-033-WO-01

[0664] Hinge_CD28 GGTGGTTCATTGCGTTTGAGCTGCGCTGCCTCTGGT tm_41BB_CD3 CGTACGCTAAGTAGTTACGCGATGGGGTGGTACCG

[0665] Z (nucleotide) CCAAGCGCCGGGCAAAGAACGCGAGCTCGTTAGTA GTATTTCCGGGAGTGGAGGCTCTACCCGTTACGCG GACTCGGTAAAAGGCCGTTTTACGATCAGTCGTGA TAATTCCAAGAATACCTTGTACCTGCAAATGAATA GCCTTAAGCCCGAAGACACAGCGGTGTATTATTGT GCCGTCTACATTCTGGAGCTGGCTCCTGGCGCGGA ATACTGGGGCCAGGGTACCCAGGTGACGGTCTCGA GCACAACAACTCCAGCCCCAAGACCACCTACGCCT GCACCTACTATCGCATCTCAACCACTGTCCCTGCGC CCTGAGGCATGCCGACCAGCAGCCGGTGGCGCGGT GCATACCCGCGGACTGGACTTTGCCTGCGATTTTTG GGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCT ATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCT GGGTGAAACGGGGCAGAAAGAAACTCCTGTATAT ATTCAAACAACCATTTATGCGACCAGTACAAACTA CTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCA GAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGA AGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAG CAGGGCCAGAACCAGCTCTATAACGAGCTCAATCT AGGACGAAGAGAGGAGTACGATGTTTTGGACAAG CGACGTGGCCGGGACCCTGAGATGGGGGGAAAGC CGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAA TGAACTGCAGAAAGATAAGATGGCGGAGGCCTAC AGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGG GCAAGGGGCACGATGGCCTTTACCAGGGACTCAGT ACAGCCACCAAGGACACCTACGACGCCCTTCACAT GCAGGCCCTGCCCCCTCGC ATGGCCAGATCTCCTGCTCAACTGCTGGGACTGCT GCTGCTGTGGCTTAGCGGAGCCAGATGCGAGGTAC AACTTTTGGAGTCAGGCGGTGGACTGGTACAACCG GGTGGTTCATTGCGTTTGAGCTGCGCTGCCTCTGGT MARS TACACATTTTCGGATTATGCAATGGGTTGGTATCGC SP_NEC_M_46 CAAGCGCCGGGCAAAGAACGCGAGCTCGTTAGTA P3110 CD8 GTATTTCCGGGAGTGGAGGCTCTACCCGTTACGCG 184 Hinge_CD28 GACTCGGTAAAAGGCCGTTTTACGATCAGTCGTGA tm_41BB_CD3 TAATTCCAAGAATACCTTGTACCTGCAAATGAATA

[0666] Z (nucleotide) GCCTTAAGCCCGAAGACACAGCGGTGTATTATTGT GCCGCAGTAATTCGGCAGCCTAGCACCGGTTTCTA TGAATACTGGGGCCAGGGTACCCAGGTGACGGTCT CGAGCACAACAACTCCAGCCCCAAGACCACCTACG

[0667]

[0668] CCTGCACCTACTATCGCATCTCAACCACTGTCCCTG Docket No. CNTY-033-WO-01

[0669] CGCCCTGAGGCATGCCGACCAGCAGCCGGTGGCGC GGTGCATACCCGCGGACTGGACTTTGCCTGCGATT TTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTT GCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTT TCTGGGTGAAACGGGGCAGAAAGAAACTCCTGTAT ATATTCAAACAACCATTTATGCGACCAGTACAAAC TACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTC CAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGT GAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACC AGCAGGGCCAGAACCAGCTCTATAACGAGCTCAAT CTAGGACGAAGAGAGGAGTACGATGTTTTGGACAA GCGACGTGGCCGGGACCCTGAGATGGGGGGAAAG CCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACA ATGAACTGCAGAAAGATAAGATGGCGGAGGCCTA CAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGG GGCAAGGGGCACGATGGCCTTTACCAGGGACTCAG TACAGCCACCAAGGACACCTACGACGCCCTTCACA TGCAGGCCCTGCCCCCTCGC GAAGTACAACTTTTGGAGTCAGGCGGTGGACTGGT ACAACCGGGTGGTTCATTGCGTTTGAGCTGCGCTG CCTCTGGTTTCACTTTCTCCTCCTATGCGATGGGAT GGTATCGCCAAGCGCCGGGCAAAGAACGCGAGTTT GTTAGCAGCATAAGCGGTTCAGGGGGGTTAACCCG CTACGCGGACTCGGTAAAAGGCCGTTTTACGATCA GTCGTGATAATTCCAAGAATACCTTGTACCTGCAA ATGAATAGCCTTAAGCCCGAAGACACAGCGGTGTA TTATTGTGCCGTAACCACCGGCTACCAAGGCGGCG TATATGACTACTGGGGCCAGGGTACCCAGGTGACG

[0670] M8-CD8h- GTCTCGAGCACGACGACTCCTGCTCCAAGGCCTCC CD28tmd- TACACCTGCACCAACCATTGCAAGTCAGCCGTTGA CD30 GCCTCCGGCCAGAAGCATGTCGCCCAGCCGCAGGC 394 -CD3z

[0671] (nucleic acid) GGGGCTGTACACACGAGAGGCTTGGATTTCGCATG TGACTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCT GGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTAT TATTTTCTGGGTGTGCCATAGGCGGGCTTGCAGAA AAAGAATAAGGCAAAAATTGCATTTGTGCTATCCA GTCCAAACGTCCCAGCCGAAGCTGGAGCTGGTAGA CAGCAGACCACGACGATCCTCCACGCAACTGAGAT CAGGGGCCTCAGTTACGGAGCCTGTCGCGGAGGAA AGGGGCCTCATGTCTCAGCCTCTGATGGAGACTTG CCATAGCGTAGGTGCTGCCTACTTGGAGTCACTGC CACTCCAGGATGCTAGTCCAGCAGGAGGCCCCTCC

[0672]

[0673] TCACCTAGGGATTTGCCAGAGCCTCGCGTGAGTAC Docket No. CNTY-033-WO-01

[0674] CGAGCACACCAATAACAAAATTGAAAAAATATATA TTATGAAGGCGGACACCGTCATCGTGGGAACGGTG AAGGCCGAGCTCCCGGAGGGCCGGGGGTTGGCCG GACCTGCGGAACCAGAACTCGAGGAAGAACTTGA AGCAGATCATACGCCTCACTACCCAGAACAAGAGA CGGAACCGCCTTTGGGTTCTTGCAGTGACGTGATG TTGTCTGTGGAAGAGGAAGGAAAGGAGGATCCTCT TCCGACCGCTGCGAGCGGGAAAAGAGTGAAGTTCA GCAGATCCGCCGATGCTCCCGCCTATCAGCAGGGC CAAAACCAGCTGTACAACGAGCTGAACCTGGGGA GAAGAGAAGAGTACGACGTGCTGGACAAGCGGAG AGGCAGAGATCCTGAAATGGGCGGCAAGCCCAGA CGGAAGAATCCTCAAGAGGGCCTGTATAATGAGCT GCAGAAAGACAAGATGGCCGAGGCCTACAGCGAG ATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGG GACACGATGGACTGTACCAGGGACTGAGCACCGCC ACCAAGGATACCTATGACGCCCTGCACATGCAGGC CCTGCCTCCAAGA

[0675]

[0676] In some embodiments, the antigen-binding domain of the second polypeptide binds to an antigen. The antigen-binding domain of the second polypeptide may bind to more than one antigen or more than one epitope in an antigen. For example, the antigen-binding domain of the second polypeptide may bind to two, three, four, five, six, seven, eight or more antigens. As another example, the antigen-binding domain of the second polypeptide may bind to two, three, four, five, six. seven, eight or more epitopes in the same antigen.

[0677] The choice of antigen-binding domain may depend upon the type and number of antigens that define the surface of a target cell. For example, the antigen-binding domain may be chosen to recognize an antigen that acts as a cell surface marker on target cells associated with a particular disease state. In certain embodiments, the CARs of the present disclosure can be genetically modified to target a tumor antigen of interest by way of engineering a desired antigen-binding domain that specifically binds to an antigen (e.g., on a tumor cell). Non-limiting examples of cell surface markers that may act as targets for the antigen-binding domain in the CAR of the disclosure include those associated with tumor cells or autoimmune diseases.

[0678] In some embodiments, the antigen-binding domain binds to at least one tumor antigen or autoimmune antigen. Docket No. CNTY-033-WO-01

[0679] In some embodiments, the antigen-binding domain binds to at least one tumor antigen. In some embodiments, the antigen-binding domain binds to two or more tumor antigens. In some embodiments, the two or more tumor antigens are associated with the same tumor. In some embodiments, the two or more tumor antigens are associated with different tumors.

[0680] In some embodiments, the antigen-binding domain binds to at least one autoimmune antigen. In some embodiments, the antigen-binding domain binds to two or more autoimmune antigens. In some embodiments, the two or more autoimmune antigens are associated with the same autoimmune disease. In some embodiments, the two or more autoimmune antigens are associated with different autoimmune diseases.

[0681] In some embodiments, the tumor antigen is associated with glioblastoma, ovarian cancer, cervical cancer, head and neck cancer, liver cancer, prostate cancer, pancreatic cancer, renal cell carcinoma, bladder cancer, or hematologic malignancy. Non-limiting examples of tumor antigen associated with glioblastoma include HER2, EGFRvIII, EGFR, CD133. PDGFRA, FGFR1, FGFR3, MET, CD70, ROBOland IL13Ra2. Non-limiting examples of tumor antigens associated with ovarian cancer include FOLR1, FSHR, MUC16, MUC1, Mesothelin, CA125, EpCAM, EGFR, PDGFRa, Nectin4, and B7H4. Non-limiting examples of the tumor antigens associated with cervical cancer or head and neck cancer include GD2, MUC1, Mesothelin, HER2, and EGFR. Non-limiting examples of tumor antigen associated with liver cancer include Claudin 18.2, GPC-3, EpCAM, cMET, and AFP. Nonlimiting examples of tumor antigens associated with hematological malignancies include CD22, CD79, BCMA, GPRC5D, SLAM F7, CD33, CLL1, CD123, and CD70. Non-limiting examples of tumor antigens associated with bladder cancer include Nectin4 and SLITRK6. Non-limiting examples of tumor antigens associated with glioblastoma include Cdl33, EGFr, CD70, and IL13Ra2. Non-limiting examples of tumor antigens associated with renal cell carcinoma include Nectin4, SLITRK6, CD70, and FOLR1. Non-limiting examples of tumor antigens associated with ovarian cancer include Nectin4, mesothelin, FSHR, and FOLR1. A non-limiting example of a tumor antigen associated with hepatocellular carcinoma includes GPC3.

[0682] Additional examples of antigens that may be targeted by the antigen-binding domain include, but are not limited to, alpha-fetoprotein, A3, antigen specific for A33 antibody. Ba 733, BrE3-antigen. carbonic anhydrase EX, CD1, CDla, CD3, CD5, CD15, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD33, CD38, CD45, Docket No. CNTY-033-WO-01

[0683] CD74, CD79a, CD80, CD123, CD138, colon-specific antigen-p (CSAp), CEA (CEACAM5), CEACAM6, CSAp, EGFR. EGP-E EGP-2, Ep-CAM, EphAl, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphAl 0, EphBl, EphB2, EphB3, EphB4, EphB6, FIt-I, Flt-3, folate receptor, HLA-DR, human chorionic gonadotropin (HCG) and its subunits, hypoxia inducible factor (HIF-I), la, IL-2, IL-6, IL-8, insulin growth factor- 1 (IGF-I), KC4-antigen, KS-1 -antigen, KS1-4, Le-Y, macrophage inhibition factor (MIF), MAGE, MUC2. MUC3, MUC4, NCA66. NCA95, NCA90, Nectin4, antigen specific for PAM-4 antibody, placental growth factor, p53, prostatic acid phosphatase, PSA, PSMA, RS5, S100, TAC, TAG-72, tenascin, TRAIL receptors, Tn antigen, Thomson-Friedenreich antigens, tumor necrosis antigens, VEGF, ED-B fibronectin, 17-1 A- antigen, an angiogenesis marker, an oncogene marker or an oncogene product.

[0684] In one embodiment, the antigen targeted by the antigen-binding domain is Nectin4. In one embodiment, the antigen-binding domain comprises an anti-Nectin4 VHH. In other embodiments, the antigen-binding domain comprises an anti-Nectin4 scFv. In one embodiment, the anti-Nectin4 antigen binding domain comprises the amino acid sequence set forth in one of SEQ ID NOs: 105-130, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity with one of SEQ ID NOs: 105-130. In one embodiment, the anti-Nectin4 antigen binding domain comprises the amino acid sequence encoded by the polynucleotide sequence set forth in one of SEQ ID NOs: 131-156, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity with one of SEQ ID NOs: 131-156.

[0685] In some embodiments, the antigen is associated with an autoimmune disease or disorder. Such antigens may be derived from cell receptors and cells which produce “self '-directed antibodies. In some embodiments, the antigen is associated with an autoimmune disease or disorder such as Rheumatoid arthritis (RA), multiple sclerosis (MS), Sjogren's syndrome, Systemic lupus ery thematosus, sarcoidosis, Type 1 diabetes mellitus, insulin dependent diabetes mellitus (IDDM), autoimmune thyroiditis, reactive arthritis, ankylosing spondylitis, scleroderma, polymyositis, dermatomyositis, psoriasis, vasculitis, Wegener's granulomatosis, Myasthenia gravis, Docket No. CNTY-033-WO-01

[0686] Hashimoto's thyroiditis, Graves' disease, chronic inflammatory demyelinating polyneuropathy, Guillain-Barre syndrome, Crohn's disease or ulcerative colitis.

[0687] In some embodiments, autoimmune antigens that may be targeted by the CAR disclosed herein include but are not limited to platelet antigens, myelin protein antigen, Sm antigens in snRNPs, islet cell antigen, Rheumatoid factor, and anticitrullinated protein, citrullinated proteins and peptides such as CCP-1, CCP-2 (cyclical citrullinated peptides), fibrinogen, fibrin, vimentin, fillaggrin, collagen I and II peptides, alphaenolase, translation initiation factor 4G1, perinuclear factor, keratin, Sa (cytoskeletal protein vimentin), components of articular cartilage such as collagen II, IX, and XI, circulating serum proteins such as RFs (IgG, IgM), fibrinogen, plasminogen, ferritin, nuclear components such as RA33 / hnRNP A2, Sm, eukaryotic translation elogation factor 1 alpha 1, stress proteins such as HSP-65, -70, -90, BiP, inflammatory / immune factors such as B7-H1, IL-1 alpha, and IL-8, enzymes such as calpastatin, alphaenolase, aldolase-A, dipeptidyl peptidase, osteopontin, glucose-6-phosphate isomerase, receptors such as lipocortin 1, neutrophil nuclear proteins such as lactoferrin and 25-35 kD nuclear protein, granular proteins such as bactericidal permeability increasing protein (BPI), elastase, cathepsin G, myeloperoxidase, proteinase 3, platelet antigens, myelin protein antigen, islet cell antigen, rheumatoid factor, histones, ribosomal P proteins, cardiolipin, vimentin, nucleic acids such as dsDNA, ssDNA, and RNA, ribonuclear particles and proteins such as Sm antigens (including but not limited to SmD's and SmB' / B), U1RNP, A2 / B1 hnRNP, Ro (SSA), and La (SSB) antigens.

[0688] In various embodiments, a CAR of the present disclosure can comprise an scFv domain or fragment thereof, and the scFv domain or fragment thereof used in the CAR may include a linker between the VH and VL domains. The linker can be a peptide linker and may include any naturally occurring amino acid. Exemplary amino acids that may be included into the linker are Gly, Ser Pro, Thr, Glu, Lys, Arg, He, Leu, His and The. The linker should have a length that is adequate to link the VH and the VL in such a way that they form the correct conformation relative to one another so that they retain the desired activity. such as binding to an antigen. The linker may be about 5-50 amino acids long. In some embodiments, the linker is about 10-40 amino acids long. In some embodiments, the linker is about 10-35 amino acids long. In some embodiments, the linker is about 10-30 amino acids long. In some embodiments, the linker is about 10-25 amino acids long. In some embodiments, the linker is about 10-20 amino acids long. In some embodiments, the linker is about 15-20 amino acids long. Docket No. CNTY-033-WO-01

[0689] Exemplary linkers that may be used are Gly rich linkers, Gly and Ser containing linkers, Gly and Ala containing linkers, Ala and Ser containing linkers, and other flexible linkers.

[0690] In one embodiment, a CAR can comprise a linker, and the linker is a Whitlow linker. In one embodiment, the Whitlow linker comprises the amino acid sequence set forth in SEQ ID NO: 3, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identify with SEQ ID NO: 3. In another embodiment, the linker is a (G4S)3 linker. In one embodiment, the (G4S)3 linker comprises the amino acid sequence set forth in SEQ ID NO: 25, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70. at least 75. at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identify' with SEQ ID NO: 25.

[0691] Other linker sequences may include portions of immunoglobulin hinge area, CL or CHI derived from any immunoglobulin heavy’ or light chain isotype. Exemplary linkers that may be used include any of SEQ ID NOs: 3, 25-56, 99, and 102-104 in Table 1. Additional linkers are described for example in Int. Pat. Publ. No. W02019 / 060695, incorporated by reference herein in its entirety.

[0692] II. Artificial Cell Death Polypeptide

[0693] According to embodiments of the application, an iPSC or a derivative cell thereof may comprise a second exogenous polynucleotide encoding an artificial cell death polypeptide.

[0694] As used herein, the term "artificial cell death polypeptide” refers to an engineered protein designed to prevent potential toxicity or otherwise adverse effects of a cell therapy. The artificial cell death polypeptide could mediate induction of apoptosis, inhibition of protein synthesis, DNA replication, growth arrest, transcriptional and post-transcriptional genetic regulation and / or antibody-mediated depletion. In some instance, the artificial cell death polypeptide is activated by an exogenous molecule, e.g. an antibody, that when activated, triggers apoptosis and / or cell death of a therapeutic cell.

[0695] In certain embodiments, an artificial cell death polypeptide comprises an inactivated cell surface receptor that comprises an epitope specifically recognized by an antibody, particularly a monoclonal antibody, which is also referred to herein as a Docket No. CNTY-033-WO-01

[0696] monoclonal antibody-specific epitope. When expressed by iPSCs or derivative cells thereof the inactivated cell surface receptor is signaling inactive or significantly impaired, but can still be specifically recognized by an antibody. The specific binding of the antibody to the inactivated cell surface receptor enables the elimination of the iPSCs or derivative cells thereof by ADCC and / or ADCP mechanisms, as well as, direct killing with antibody drug conjugates with toxins or radionuclides.

[0697] In certain embodiments, the inactivated cell surface receptor comprises an epitope that is selected from epitopes specifically recognized by an antibody, including but not limited to, ibritumomab, tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, alemtuzumab, bevacizumab. certolizumab pegol, daclizumab. eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, polatuzumab vedotin, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, avelumab, ofatumumab. panitumumab, or ustekinumab. In certain embodiments, the inactivated cell surface receptor comprises an epitope that is specifically recognized by¬ cetuximab. In certain embodiments, the inactivated cell surface receptor comprises an epitope that is specifically recognized by trastuzumab. In certain embodiments, the inactivated cell surface receptor comprises an epitope that is specifically recognized by bevacizumab. In certain embodiments, the inactivated cell surface receptor comprises an epitope that is specifically recognized by avelumab. In certain embodiments, the inactivated cell surface receptor comprises an epitope that is specifically recognized by ipilimumab.

[0698] Epidermal growth factor receptor, also known as EGFR, ErbBl and HERL is a cell -surface receptor for members of the epidermal growth factor family of extracellular ligands. As used herein, ‘’truncated EGFR,” “tEGFR,” “short EGFR” or “sEGFR” refers to an inactive EGFR variant that lacks the EGF-binding domains and the intracellular signaling domains of the EGFR. An exemplary tEGFR variant contains residues 322-333 of domain 2, all of domains 3 and 4 and the transmembrane domain of the native EGFR sequence containing the cetuximab binding epitope. Expression of the tEGFR variant on the cell surface enables cell elimination by an antibody that specifically binds to the tEGFR, such as cetuximab (Erbitux®), as needed. Due to the absence of the EGF-binding domains and intracellular signaling domains, tEGFR is inactive when expressed by iPSCs or derivative cell thereof. Docket No. CNTY-033-WO-01

[0699] An exemplary' inactivated cell surface receptor of the application comprises a tEGFR variant. In certain embodiments, expression of the inactivated cell surface receptor in an engineered immune cell expressing a chimeric antigen receptor (CAR) induces cell suicide of the engineered immune cell when the cell is contacted with an anti-EGFR antibody. Methods of using inactivated cell surface receptors are described in W02019 / 070856, WO2019 / 023396, WO2018 / 058002. the disclosure of which is incorporated herein by reference. For example, a subject who has previously received an engineered immune cell of the present disclosure that comprises a heterologous polynucleotide encoding an inactivated cell surface receptor comprising a tEGFR variant can be administered an anti-EGFR antibody in an amount effective to ablate in the subject the previously administered engineered immune cell.

[0700] In certain embodiments, the anti-EGFR antibody is cetuximab, matuzumab, necitumumab or panitumumab, preferably the anti-EGFR antibody is cetuximab.

[0701] In certain embodiments, the tEGFR variant comprises or consists of an amino acid sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 71, preferably the amino acid sequence of SEQ ID NO: 71.

[0702] In some embodiments, the inactivated cell surface receptor comprises one or more epitopes of CD79b, such as an epitope specifically recognized by polatuzumab vedotin. In certain embodiments, the CD79b epitope comprises or consists of an amino acid sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 78, preferably the amino acid sequence of SEQ ID NO: 78.

[0703] In some embodiments, the inactivated cell surface receptor comprises one or more epitopes of CD20, such as an epitope specifically recognized by rituximab. In certain embodiments, the CD20 epitope comprises or consists of an amino acid sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 80, preferably the amino acid sequence of SEQ ID NO: 80.

[0704] In some embodiments, the inactivated cell surface receptor comprises one or more epitopes of Her 2 receptor or ErbB, such as an epitope specifically recognized by trastuzumab. In certain embodiments, the monoclonal antibody-specific epitope comprises or consists of an amino acid sequence at least 90%, such as at least 90%, Docket No. CNTY-033-WO-01

[0705] 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 82, preferably the amino acid sequence of SEQ ID NO: 82.

[0706] III. Cytokine Expression

[0707] In some embodiments the iPSC cell or a derivative cell thereof optionally comprises an exogenous polynucleotide encoding a cytokine, such as interleukin- 15 or interleukin-2.

[0708] As used herein '‘Interleukin- 15” or “IL- 15” refers to a cytokine that regulates T and NK cell activation and proliferation, or a functional portion thereof. A “functional portion” (“biologically active portion”) of a cytokine refers to a portion of the cytokine that retains one or more functions of full length or mature cytokine. Such functions for IL- 15 include the promotion of NK cell survival, regulation of NK cell and T cell activation and proliferation as well as the support of NK cell development from hematopoietic stem cells. As will be appreciated by those of skill in the art, the sequence of a variety of IL-15 molecules are known in the art. In certain embodiments, the IL- 15 is a wild-type IL- 15. In certain embodiments, the IL- 15 is a human IL-15. In certain embodiments, the IL-15 comprises an amino acid sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 72, preferably the amino acid sequence of SEQ ID NO: 72.

[0709] In some embodiments, the IL-15 is a membrane bound form, where all or a functional portion of the IL-15 protein is fused to all or a portion of a transmembrane protein that anchors the expressed IL- 15 as a cell membrane-bound polypeptide (mbIL15)”. for example the construct described in US Patent US9629877B2, hereby incorporated by reference into the present application.

[0710] As used herein “Interleukin-2” refers to a cytokine that regulates T and NK cell activation and proliferation, or a functional portion thereof. In certain embodiments, the IL-2 is a wild-type IL-2. In certain embodiments, the IL-2 is a human IL-2. In certain embodiments, the IL-2 comprises an amino acid sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 76, preferably the amino acid sequence of SEQ ID NO: 76.

[0711] In certain embodiments, an inactivated cell surface receptor comprises a monoclonal antibody-specific epitope operably linked to a cytokine, preferably by an Docket No. CNTY-033-WO-01

[0712] autoprotease peptide sequence. Examples of the autoprotease peptide include, but are not limited to, a peptide sequence selected from the group consisting of porcine teschovirus-1 2 A (P2A), a foot-and-mouth disease virus (FMDV) 2 A (F2A), an Equine Rhinitis A Virus (ERAV) 2A (E2A), a Thosea asigna virus 2A (T2A), a cytoplasmic polyhedrosis virus 2 A (BmCPV2A), a Flacherie Virus 2 A (BmIFV2A), and a combination thereof. In one embodiment, the autoprotease peptide is an autoprotease peptide of porcine teschovirus-1 2A (P2A). In certain embodiments, the autoprotease peptide comprises an amino acid sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 73, preferably the amino acid sequence of SEQ ID NO: 73.

[0713] In certain embodiments, an inactivated cell surface receptor comprises a truncated epithelial growth factor (tEGFR) variant operably linked to an interleukin-15 (IL- 15) or IL-2 by an autoprotease peptide sequence. In a particular embodiment, the inactivated cell surface receptor comprises an amino acid sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%. 99% or 100%, identical to SEQ ID NO: 74, preferably the amino acid sequence of SEQ ID NO: 74.

[0714] In some embodiments, an inactivated cell surface receptor further comprises a signal sequence. In certain embodiments, the signal sequence comprises an amino acid sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%. 99% or 100%, identical to SEQ ID NO: 77, preferably the amino acid sequence of SEQ ID NO: 77.

[0715] In some embodiments, an inactivated cell surface receptor further comprises a hinge domain. In some embodiments, the hinge domain is derived from CD8. In one embodiment, the CD8 hinge domain comprises the amino acid sequence set forth in SEQ ID NO: 21, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity7with SEQ ID NO: 21.

[0716] In certain embodiments, an inactivated cell surface receptor further comprises a transmembrane domain. In some embodiments, the transmembrane domain is derived from CD8. In one embodiment, the CD8 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 23, or a variant thereof having at least 50, at least 55, at least 60, at least 65. at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity7with SEQ ID NO: 23. Docket No. CNTY-033-WO-01

[0717] In certain embodiment, an inactivated cell surface receptor comprises one or more epitopes specifically recognized by an antibody in its extracellular domain, a transmembrane region and a cytoplasmic domain. In some embodiments, the inactivated cell surface receptor further comprises a hinge region between the epitope(s) and the transmembrane region. In some embodiments, the inactivated cell surface receptor comprises more than one epitopes specifically recognized by an antibody, the epitopes can have the same or different ammo acid sequences, and the epitopes can be linked together via a peptide linker, such as a flexible peptide linker have the sequence of (GGGGS)n, wherein n is an integer of 1-8 (SEQ ID NO: 25). In some embodiments, the inactivated cell surface receptor further comprises a cytokine, such as an IL- 15 or IL-2. In certain embodiments, the cytokine is in the cytoplasmic domain of the inactivated cell surface receptor. Preferably, the cytokine is operably linked to the epitope(s) specifically recognized by an antibody, directly or indirectly, via an autoprotease peptide sequence, such as those described herein. In some embodiments, the cytokine is indirectly linked to the epitope(s) by connecting to the transmembrane region via the autoprotease peptide sequence.

[0718] Non-limiting exemplary inactivated cell surface receptor regions and sequences are provided in Table 2.

[0719] Table 2.

[0720] Regions Sequence SEQ ID NO

[0721] tEGFR-IL15:

[0722] tEGFR MRPSGTAGAALLALLAALCPASRAGVRKCKKCEGPCRK 71

[0723] VCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAF RGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTD LHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEIS DGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGE NSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRE CVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTG RGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKY ADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATG MVGALLLLLVVALGIGLFM

[0724] P2A ATNFSLLKQAGDVEENPGP 73 IL- 15 MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSA 72

[0725]

[0726] GLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVH Docket No. CNTY-033-WO-01

[0727] PSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANN SLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFIN TS CD79b-IL15:

[0728] Signal MEFGLSWVFLVALFRGVQC 77 Sequence

[0729] CD79b ARSEDRYRNPKGSACSRIWQS 78 epitope

[0730] CD8 (AA TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGL 21 136-182) DFACD

[0731] CD8 (AA IYIWAPLAGTCGVLLLSLVIT 23 183-203)

[0732] P2A ATNFSLLKQAGDVEENPGP 73 IL- 15 MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSA 72

[0733] GLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVH PSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANN SLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFIN TS CD20 mimitope-IL15:

[0734] Signal MEFGLSWVFLVALFRGVQC 77 Sequence

[0735] CD20 ACPYANPSLC 80 mimitope

[0736] Linker GGGSGGGS 27 CD8 (AA TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGL 21 136-182) DFACD

[0737] CD8 (AA IYIWAPLAGTCGVLLLSLVIT 23 183-203)

[0738] P2A ATNFSLLKQAGDVEENPGP 73 IL- 15 MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSA 72

[0739] GLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVH

[0740]

[0741] PSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANN Docket No. CNTY-033-WO-01

[0742] SLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFIN TS

[0743] ErbB epitope-IL15:

[0744] Signal MEFGLSWVFLVALFRGVQC 77 Sequence

[0745] ErbB EGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEE 82 epitope CRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEA DQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDE EGACQPCPINCTHSCVDLDDKGCPAEQRASPLTSIISAVV GILLV VVLGVVFGILIGGGGS GG

[0746] P2A ATNFSLLKQAGDVEENPGP 73 IL-15 MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSA 72

[0747] GLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVH PSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANN SLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFIN TS

[0748]

[0749] In a particular embodiment, the inactivated cell surface receptor comprises an amino acid sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 79, preferably the amino acid sequence of SEQ ID NO: 79.

[0750] In a particular embodiment, the inactivated cell surface receptor comprises an amino acid sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 81, preferably the amino acid sequence of SEQ ID NO: 81.

[0751] In a particular embodiment, the inactivated cell surface receptor comprises an amino acid sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 83, preferably the amino acid sequence of SEQ ID NO: 83.

[0752] IV. HLA Expression

[0753] In one aspect, MHC I and / or MHC II knock-out and / or knock down can be incorporated in the cells for use in “allogeneic” cell therapies, in which cells are harvested from a subject, modified to knock-out or knock-down, e.g., disrupt, B2M, Docket No. CNTY-033-WO-01

[0754] TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP gene expression, and then returned to a different subject. Knocking out or knocking down the B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes as described herein can: (1) prevent Graft versus Host response; (2) prevent Host versus Graft response; and / or (3) improve cell safety and efficacy. Accordingly, in certain embodiments, a presently disclosed invention comprises independently knocking out and / or knocking down one or more genes selected from the group consisting of B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes in an iPSC cell. In certain embodiments, a presently disclosed method comprises independently knocking out and / or knocking down two genes selected from the group consisting B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA. RFX5 and RFXAP genes in an iPSC cell, in particular, B2M and CIITA to achieve class I and II HLA disruption.. In certain embodiments, an iPSC or derivative cell thereof of the application can be further modified by introducing an exogenous polynucleotide encoding one or more proteins related to immune evasion, such as non-classical HLA class I proteins (e.g., HLA-E and HLA-G). In particular, disruption of the B2M gene eliminates surface expression of all MHC class I molecules, leaving cells vulnerable to lysis by NK cells through the “missing self’ response. Exogenous HLA-E expression can lead to resistance to NK-mediated lysis (Gomalusse et al., Nal Biotechnol. 2017; 35(8): 765-772).

[0755] Incorporating MHC I and / or MHC II knock-out and / or knock down in the cells for use in “allogeneic” cell therapies will allow the cell product candidates to escape recognition and destruction by the host immune system. The reduction in allogeneic reactivity7enabled by use of this technology will allow repeat dosing of the CAR-modified cell therapies to improve their therapeutic potential. In combination with the extended killing capability of optimized immune cells derived from single genetically engineered cell cloning, the cells will have the capacity7for repeat dosing to maximize durability of response and efficacy. Additionally, this technology may permit dosing in patients with limited or no immune preconditioning regimens.

[0756] Accordingly, in certain embodiments, an iPSC or derivative cell thereof of the application can be further modified by introducing a third exogenous polynucleotide encoding one or more proteins related to immune evasion, such as non-classical HLA class I proteins (e.g., HLA-E and HLA-G).

[0757] In certain embodiments, the iPSC or derivative cell thereof comprises a third exogenous polypeptide encoding at least one of a human leukocyte antigen E (HLA- Docket No. CNTY-033-WO-01

[0758] E) and human leukocyte antigen G (HLA-G). In a particular embodiment, the HLA-E comprises an amino acid sequence at least 90%. such as at least 90%. 91%. 92%. 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 65, preferably the amino acid sequence of SEQ ID NO: 65. In a particular embodiment, the HLA-G comprises an amino acid sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 68, preferably SEQ ID NO: 68.

[0759] In certain embodiments, the third exogenous polynucleotide encodes a polypeptide comprising a signal peptide operably linked to a mature B2M protein that is fused to an HLA-E via a linker. In a particular embodiment, the third exogenous polypeptide comprises an amino acid sequence at least sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 66.

[0760] In other embodiments, the third exogenous polynucleotide encodes a polypeptide comprising a signal peptide operably linked to a mature B2M protein that is fused to an HLA-G via a linker. In a particular embodiment, the third exogenous polypeptide comprises an amino acid sequence at least sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 69.

[0761] V. Other Optional Genome Edits

[0762] In one embodiment of the above described cell, the genomic editing at one or more selected sites may comprise insertions of one or more exogenous polynucleotides encoding other additional artificial cell death polypeptides, targeting modalities, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, or proteins promoting engrafiment, trafficking, homing, viability, self-renewal, persistence, and / or survival of the genome-engineered iPSCs or derivative cells thereof.

[0763] In some embodiments, the exogenous polynucleotides for insertion are operatively linked to (1) one or more exogenous promoters comprising CMV, EFla, PGK, CAG, UBC, or other constitutive, inducible, temporal-, tissue-, or cell typespecific promoters: or (2) one or more endogenous promoters comprised in the selected sites comprising AAVS1, CLYBL, CCR5, ROSA26. collagen. HTRP. Hll, beta-2 microglobulin, GAPDH, TCR or RUNX1, or other locus meeting the criteria of Docket No. CNTY-033-WO-01

[0764] a genome safe harbor. In some embodiments, the genome-engineered iPSCs generated using the above method comprise one or more different exogenous polynucleotides encoding proteins comprising caspase, thymidine kinase, cytosine deaminase, B-cell CD20, ErbB2 or CD79b wherein when the genome-engineered iPSCs comprise two or more suicide genes, the suicide genes are integrated in different safe harbor locus comprising AAVS1, CCR5, ROSA26, collagen, HTRP, Hll, Hll, beta-2 microglobulin, GAPDH, TCR or RUNX1. Other exogenous polynucleotides encoding proteins may include those encoding PET reporters, homeostatic cytokines, and inhibitory checkpoint inhibitory' proteins such as PD1, PD-L1, and CTLA4 as well as proteins that target the CD47 / signal regulatory protein alpha (SIRPa) axis.

[0765] In one aspect, the cell may comprise an exogenous polynucleotide encoding a CD 16 protein and / or an NKG2D protein, wherein the CD 16 protein and the NKG2D protein may be operably linked by an autoprotease peptide as disclosed in co-pending patent application PCT / US23 / 68079. Accordingly, in some aspects, cells of the present invention may comprise genetically engineered iPSCs and cells derived therefrom that exogenously express recombinant CD 16 and recombinant NKG2D. The surface receptor CD16 (FcyRIIIA) affects human natural killer (NK) cells during maturation. NK cells bind the Fc portion of IgG via CD16, and execute antibodydependent cellular cytotoxicity, which is critical for the effectiveness of several antitumor monoclonal antibody therapies. NKG2D is an stimulatory / activating receptor that is mostly expressed on cells of the cytotoxic arm of the immune system including NK cells and subsets of T cells. NKG2D is crucial in diverse aspects of innate and adaptive immune functions. In some embodiments, CD 16 and NKG2D are expressed from in a single polynucleotide construct as it is advantageous to reduce the number of gene edits of a cell.

[0766] In some embodiments, the polynucleotide construct encoding the CD 16 protein and the NKG2D protein also includes a polynucleotide sequence encoding an autoprotease peptide or self-cleaving peptide. In some embodiments, an exogenous polynucleotide construct encoding the CD 16 protein, the NKG2D protein and the selfcleaving peptide is introduced into the iPSC or derivative cell thereof. The exogenous or isolated polynucleotide construct can be introduced into a gene locus of the iPSC or derivative cell thereof. Docket No. CNTY-033-WO-01

[0767] In some embodiments, the exogenous polynucleotide construct comprises the nucleic acid sequence of SEQ ID NO: 185. In some embodiments, the exogenous polynucleotide construct encodes for the amino acid sequence of SEQ ID NO: 186.

[0768] In some embodiments, the CD16 protein (which is also referred to as “low affinity immunoglobulin gamma Fc region receptor III-A'’ or “Fc gamma receptor Illa”) is a wildtype CD16 protein. In some embodiments, the human wildtype CD16 protein has the amino acid sequence set forth in NCBI Ref. Seq. No. NP_000560.7 or UniProtNo. P08637. In some instance, the coding sequence of human wildtype CD16 is set forth in NCBI Ref. No. NM_000569.8.

[0769] In some embodiments, the CD 16 protein is a CD 16 variant protein. In some instances, the CD 16 variant protein has an amino acid sequence having at least 90%. e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to wildty pe CD16 such as that of SEQ ID NO: 187. In some instances, the CD16 variant is a high affinity CD16 variant. In other instances, the CD16 variant is a non-cleavable CD16 variant. In some instances, the CD 16 variant is a high affinity and non-cleavable CD 16 variant.

[0770] In some embodiments, the CD 16 variant comprises one or more amino acid substitutions selected from the group consisting of F158V, F176V, S197P, D205A. S219A. T220A, and any combination thereof. In some embodiments, the CD 16 variant has an Fl 58V substitution and one or more substitutions selected from Fl 76V, S197P, D205A, S219A, T220A, and any combination thereof. In one embodiment, the CD 16 variant has an Fl 76V substitution and one or more substitutions selected from Fl 58V, S197P, D205A, S219A, T220A, and any combination thereof. In many embodiments, the CD 16 variant has an S197P, substitution and one or more substitutions selected from F158V, F176V, D205A, S219A, T220A, and any combination thereof. In various embodiments, the CD16 variant has a D205A substitution and one or more substitutions selected from F158V, F176V, S197P, S219A, T220A, and any combination thereof. In some embodiments, the CD 16 variant has a substitution and one or more substitutions selected from F158V, F176V, S197P, D205A, S219A, T220A, and any combination thereof. In some embodiments, the CD16 variant has an S219A substitution and one or more substitutions selected from Fl 58V. Fl 76V, S197P, D205A, T220A. and any combination thereof. In some embodiments, the CD 16 variant has a T220A substitution and one or more Docket No. CNTY-033-WO-01

[0771] substitutions selected from F158V, F176V, S197P, D205A, S219A. T220A, and any combination thereof. In some embodiments, the variant CD 16 protein has the sequence of SEQ ID NO: 188. In some embodiments, the nucleic acid sequence encoding the variant CD16 protein has the sequence of SEQ ID NO: 189. In some embodiments, the wildty pe CD16 protein has the sequence of SEQ ID NO: 187.

[0772] In some embodiments, the NKG2D protein (which is also referred to as NK. G2-D type II integral membrane protein, CD314, killer cell lectin-like receptor subfamily KI member 1 or KLRK1) is a wildtype NKG2D protein. In some embodiments, the human wildtype NKG2D protein has the amino acid sequence set forth inNCBI Ref. Seq. Nos. NP_001186734.1 or NP_031386.2 or UmProtNo. P26718. In some instance, the coding sequence of human wildtype NKG2D is set forth in NCBI Ref. Nos. NM_001199805.1 or NM_007360.3. In some embodiments, the NKG2D protein is aNKG2D variant protein. In some instances, the NKG2D variant protein has an amino acid sequence having at least 90%, e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to wildtype NKG2D such as that of SEQ ID NO: 190. In some embodiments, the NKG2D protein has the amino acid sequence of SEQ ID NO: 190. In some embodiments, the nucleic acid sequence encoding the NKG2D protein has sequence of SEQ ID NO: 191.

[0773] As discussed above, provided herein are constructs containing autoprotease peptide sequences including 2A peptides that can induce ribosomal skipping during translation of an polypeptide. 2A peptides function to “cleave” an mRNA transcript by making the ribosome skip the synthesis of a peptide bond at the C-terminus, between the glycine (G) and proline (P) residues, thereby leading to separation between the end of the 2A sequence and the next peptide downstream. 2A peptides include, but are not limited to, a porcine teschovirus-1 2A (P2A) peptide, a foot-and-mouth disease virus (FMDV) 2 A (F2A) peptide, an Equine Rhinitis A Virus (ERAV) 2A (E2A) peptide, a Thosea asigna virus 2A (T2A) peptide, a cytoplasmic polyhedrosis virus 2 A (BmCPV2A) peptide, and a Flacherie Virus 2A (BmIFV2A) peptide.

[0774] An exemplary' P2A peptide can include an amino acid sequence having at least 90%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 192. In some embodiment, the P2A peptide has the amino acid sequence of SEQ ID NO: 192. Docket No. CNTY-033-WO-01

[0775] Another optional genome edit is the insertion of a polynucleotide encoding a membrane-bound interleukin 12 (IL- 12) comprising a first polypeptide comprising an IL-12 alpha subunit p35, a second polypeptide comprising an IL-12 beta subunit p40 and a transmembrane fused to the terminus of the first and / or second IL- 12 subunit polypeptide as disclosed in co-pending patent application PCT / US23 / 68105. In certain embodiments, the polynucleotide encoding the membrane bound IL- 12 is fused to a polynucleotide encoding an ADAM 17 protease cleavage site peptide for the activation induced release of the IL-12 through the protease ADAM17. ADAM17 is expressed by activated lymphocytes and is directly involved in the liberation of other immune mediators like TNFa that are similarly presented as a membrane anchored form. When this membrane tethered IL- 12 is expressed on engineered iNK or T cells, it remains cell associated. Upon cell activation and the increased expression of ADAM17, the protease cleaves the membrane stalk and releases IL-12 into the extracellular space. This ty pe of regulation ensures that the activities of the IL-12 are confined to spaces surrounding the tumor where the engineered immune cells engage their targets on the tumor cells that cause their activation. Accordingly, the cell of the invention may further comprise (i) an exogenous polynucleotide encoding a membrane-bound interleukin 12 (IL- 12) comprising a first polypeptide comprising an IL- 12 alpha subunit p35 or a polypeptide at least 90% similar thereto, a second polypeptide comprising an IL- 12 beta subunit p40 or a polypeptide at least 90% similar thereto, and a transmembrane domain fused to the terminus of the first and / or second IL- 12 subunit polypeptide.

[0776] In some other embodiments, the genome-engineered iPSCs generated using the method provided herein comprise in / del at one or more endogenous genes associated with targeting modality, receptors, signaling molecules, transcription factors, drug target candidates, immune response regulation and modulation, or proteins suppressing engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival of the iPSCs or derivative cells thereof.

[0777] VI. Targeted Genome Editing at Selected Locus in iPSCs

[0778] According to embodiments of the application, one or more of the exogenous polynucleotides are integrated at one or more loci on the chromosome of an iPSC.

[0779] Genome editing, or genomic editing, or genetic editing, as used interchangeably herein, is a type of genetic engineering in which DNA is inserted, Docket No. CNTY-033-WO-01

[0780] deleted, and / or replaced in the genome of a targeted cell. Targeted genome editing (interchangeable with "‘targeted genomic editing” or “targeted genetic editing”) enables insertion, deletion, and / or substitution at pre-selected sites in the genome. When an endogenous sequence is deleted or disrupted at the insertion site during targeted editing, an endogenous gene comprising the affected sequence can be knocked-out or knocked-down due to the sequence deletion or disruption. Therefore, targeted editing can also be used to disrupt endogenous gene expression with precision. Similarly used herein is the term “targeted integration,” referring to a process involving insertion of one or more exogenous sequences at pre-selected sites in the genome, with or without deletion of an endogenous sequence at the insertion site.

[0781] Targeted editing can be achieved either through a nuclease-independent approach, or through a nuclease-dependent approach. In the nuclease-independent targeted editing approach, homologous recombination is guided by homologous sequences flanking an exogenous polynucleotide to be inserted, through the enzymatic machinery of the host cell.

[0782] Alternatively, targeted editing could be achieved with higher frequency through specific introduction of double strand breaks (DSBs) by specific rare-cutting endonucleases. Such nuclease-dependent targeted editing utilizes DNA repair mechanisms including non-homologous end joining (NHEJ), which occurs in response to DSBs. Without a donor vector containing exogenous genetic material, the NHEJ often leads to random insertions or deletions (in / dels) of a small number of endogenous nucleotides. In comparison, when a donor vector containing exogenous genetic material flanked by a pair of homology arms is present, the exogenous genetic material can be introduced into the genome during homology directed repair (HDR) by homologous recombination, resulting in a “targeted integration.”

[0783] Available endonucleases capable of introducing specific and targeted DSBs include, but not limited to, zinc-fmger nucleases (ZFN). transcription activator-like effector nucleases (TALEN), RNA-guided CRISPR (Clustered Regular Interspaced Short Palindromic Repeats) systems. Additionally, DICE (dual integrase cassette exchange) system utilizing phiC31 and Bxbl integrases is also a promising tool for targeted integration.

[0784] ZFNs are targeted nucleases comprising a nuclease fused to a zinc finger DNA binding domain. By a “zinc finger DNA binding domain” or “ZFBD” it is meant a Docket No. CNTY-033-WO-01

[0785] polypeptide domain that binds DNA in a sequence-specific manner through one or more zinc fingers. A zinc finger is a domain of about 30 amino acids within the zinc finger binding domain whose structure is stabilized through coordination of a zinc ion. Examples of zinc fingers include, but not limited to, C2H2 zinc fingers, C3H zinc fingers, and C4 zinc fingers. A “designed” zinc finger domain is a domain not occurring in nature whose design / composition results principally from rational criteria, e.g., application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP designs and binding data. See, for example, U. S. Pat. Nos. 6,140,081; 6,453,242; and 6,534,261; see also WO 98 / 53058; WO 98 / 53059; WO 98 / 53060; WO 02 / 016536 and WO 03 / 016496. A “selected” zinc finger domain is a domain not found in nature whose production results primarily from an empirical process such as phage display, interaction trap or hybrid selection. ZFNs are described in greater detail in U. S. Pat. No. 7,888,121 and U. S. Pat. No. 7,972,854, the complete disclosures of which are incorporated herein by reference. The most recognized example of a ZFN in the art is a fusion of the Fokl nuclease with a zinc finger DNA binding domain.

[0786] A TALEN is a targeted nuclease comprising a nuclease fused to a TAL effector DNA binding domain. By “transcription activator-like effector DNA binding domain”, “TAL effector DNA binding domain”, or “TALE DNA binding domain” it is meant the polypeptide domain of TAL effector proteins that is responsible for binding of the TAL effector protein to DNA. TAL effector proteins are secreted by plant pathogens of the genus Xanthomonas during infection. These proteins enter the nucleus of the plant cell, bind effector-specific DNA sequences via their DNA binding domain, and activate gene transcription at these sequences via their transactivation domains. TAL effector DNA binding domain specificity depends on an effector-variable number of imperfect 34 amino acid repeats, which comprise polymorphisms at select repeat positions called repeat variable-diresidues (RVD). TALENs are described in greater detail in U. S. Patent Application No. 2011 / 0145940, which is herein incorporated by reference. The most recognized example of a TALEN in the art is a fusion polypeptide of the Fokl nuclease to a TAL effector DNA binding domain.

[0787] Another example of a targeted nuclease that finds use in the subject methods is a targeted Spoil nuclease, a polypeptide comprising a Spol 1 polypeptide having nuclease activity fused to a DNA binding domain, e.g. a zinc finger DNA binding Docket No. CNTY-033-WO-01

[0788] domain, a TAL effector DNA binding domain, etc. that has speci ficity for a DNA sequence of interest. See, for example. U. S. Application No. 61 / 555,857, the disclosure of which is incorporated herein by reference.

[0789] Additional examples of targeted nucleases suitable for the present application include, but not limited to Bxbl, phiC3 1, R4, PhiBTl, and Wp / SPBc / TP901-l, whether used individually or in combination.

[0790] Other non-limiting examples of targeted nucleases include naturally occurring and recombinant nucleases; CRISPR related nucleases from families including cas, cpf, cse, csy, csn, csd, cst, csh, csa, csm, and cmr; restriction endonucleases; meganucleases; homing endonucleases, and the like. As an example, CRISPR / Cas9 requires two major components: (1) a Cas9 endonuclease and (2) the crRNA-tracrRNA complex. When co-expressed, the two components form a complex that is recruited to a target DNA sequence comprising PAM and a seeding region near PAM. The crRNA and tracrRNA can be combined to form a chimeric guide RNA (gRNA) to guide Cas9 to target selected sequences. These two components can then be delivered to mammalian cells via transfection or transduction. As another example. CRISPR / Cpfl comprises two major components: (1) a CPfl endonuclease and (2) a crRNA. When co-expressed, the two components form a ribobnucleoprotein (RNP) complex that is recruited to a target DNA sequence comprising PAM and a seeding region near PAM. The crRNA can be combined to form a chimeric guide RNA (gRNA) to guide Cpfl to target selected sequences. These two components can then be delivered to mammalian cells via transfection or transduction.

[0791] MAD7 is an engineered Cas 12a variant originating from the

[0792] bacterium Eubacterium rectale that has a preference for 5'-TTTN-3' and 5'-CTTN-3' PAM sites and does not require a tracrRNA. See, for example, PCT Publication No.

[0793] 2018 / 236548, the disclosure of which is incorporated herein by reference.

[0794] DICE mediated insertion uses a pair of recombinases, for example, phiC31 and Bxbl, to provide unidirectional integration of an exogenous DNA that is tightly restricted to each enzymes’ own small attB and attP recognition sites. Because these target att sites are not naturally present in mammalian genomes, they must be first introduced into the genome, at the desired integration site. See, for example, U. S. Application Publication No. 2015 / 0140665, the disclosure of which is incorporated herein by reference. Docket No. CNTY-033-WO-01

[0795] One aspect of the present application provides a construct comprising one or more exogenous polynucleotides for targeted genome integration. In one embodiment, the construct further comprises a pair of homologous arm specific to a desired integration site, and the method of targeted integration comprises introducing the construct to cells to enable site specific homologous recombination by the cell host enzymatic machinery’. In another embodiment, the method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides to the cell, and introducing a ZFN expression cassette comprising a DNA-binding domain specific to a desired integration site to the cell to enable a ZFN-mediated insertion. In yet another embodiment, the method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides to the cell, and introducing a TALEN expression cassette comprising a DNA-binding domain specific to a desired integration site to the cell to enable a TALEN-mediated insertion. In another embodiment, the method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides to the cell, introducing a Cpfl expression cassette, and a gRNA comprising a guide sequence specific to a desired integration site to the cell to enable a Cpfl -mediated insertion. In another embodiment, the method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides to the cell, introducing a Cas9 expression cassette, and a gRNA comprising a guide sequence specific to a desired integration site to the cell to enable a Cas9-mediated insertion. In still another embodiment, the method of targeted integration in a cell comprises introducing a construct comprising one or more att sites of a pair of DICE recombinases to a desired integration site in the cell, introducing a construct comprising one or more exogenous polynucleotides to the cell, and introducing an expression cassette for DICE recombinases, to enable DICE-mediated targeted integration.

[0796] Sites for targeted integration include, but are not limited to, genomic safe harbors, which are intragenic or extragemc regions of the human genome that, theoretically, are able to accommodate predictable expression of newly integrated DNA without adverse effects on the host cell or organism. In certain embodiments, the genome safe harbor for the targeted integration is one or more loci of genes selected from the group consisting of AAVS1, CLYBL. CCR5. ROSA26, collagen, HTRP, Hll, GAPDH, TCR and RUNX1 genes. Docket No. CNTY-033-WO-01

[0797] In other embodiments, the site for targeted integration is selected for deletion or reduced expression of an endogenous gene at the insertion site. As used herein, the term ‘'deletion’’ with respect to expression of a gene refers to any genetic modification that abolishes the expression of the gene. Examples of “deletion” of expression of a gene include, e.g., a removal or deletion of a DNA sequence of the gene, an insertion of an exogenous polynucleotide sequence at a locus of the gene, and one or more substitutions within the gene, which abolishes the expression of the gene.

[0798] Genes for target deletion include, but are not limited to, genes of major histocompatibility complex (MHC) class I and MHC class II proteins. Multiple MHC class I and class II proteins must be matched for histocompatibility in allogeneic recipients to avoid allogeneic rejection problems. “MHC deficient”, including MHC-class I deficient, or MHC-class II deficient, or both, refers to cells that either lack, or no longer maintain, or have reduced level of surface expression of a complete MHC complex comprising a MHC class I protein heterodimer and / or a MHC class II heterodimer, such that the diminished or reduced level is less than the level naturally detectable by other cells or by synthetic methods. MHC class I deficiency can be achieved by functional deletion of any region of the MHC class I locus (chromosome 6p21), or deletion or reducing the expression level of one or more MHC class-I associated genes including, not being limited to, beta-2 microglobulin (B2M) gene, TAP 1 gene, TAP 2 gene and Tapasin genes. For example, the B2M gene encodes a common subunit essential for cell surface expression of all MHC class I heterodimers. B2M null cells are MHC-I deficient. MHC class II deficiency can be achieved by functional deletion or reduction of MHC-II associated genes including, not being limited to, RFXANK, CIITA, RFX5 and RFXAP. CIITA is a transcriptional coactivator, functioning through activation of the transcription factor RFX5 required for class II protein expression. CIITA null cells are MHC-II deficient. In certain embodiments, one or more of the exogenous polynucleotides are integrated at one or more loci of genes selected from the group consisting of B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes to thereby delete or reduce the expression of the gene(s) with the integration. Other genes that may be targeted for deletion include NKG2A, CD38, CD70 and CD33.

[0799] In certain embodiments, the exogenous polynucleotides are integrated at one or more loci on the chromosome of the cell, preferably the one or more loci are of genes selected from the group consisting of AAVS1, CLYBL, CCR5, ROSA26, Docket No. CNTY-033-WO-01

[0800] collagen, HTRP, Hl 1, GAPDH, RUNX1, B2M, TAPI, TAP2, Tapasin, NLRC5, CIITA, RFXANK, CIITA. RFX5, RFXAP, TCR a or b constant region, NKG2A, NKG2D, CD33, CD38, CIS, CBL-B, S0CS2, PD1, CTLA4, LAG3, TIM3, or TIGIT genes, provided at least one of the one or more loci is of a MHC gene, such as a gene selected from the group consisting of B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes. Preferably, the one or more exogenous polynucleotides are integrated at a locus of an MHC class-I associated gene, such as a beta-2 microglobulin (B2M) gene, TAP 1 gene, TAP 2 gene or Tapasin gene; and at a locus of an MHC-II associated gene, such as a RFXANK, CIITA, RFX5, RFXAP, or CIITA gene; and optionally further at a locus of a safe harbor gene selected from the group consisting of AAVS1. CLYBL, CCR5, ROSA26. collagen. HTRP. Hll, GAPDH, TCR and RUNX1 genes. More preferably, the one or more of the exogenous polynucleotides are integrated at the loci of CIITA, AAVS1 and B2M genes.

[0801] In certain embodiments, (i) the first exogenous polynucleotide is integrated at a locus of AAVS1 gene or CLYBL gene; (ii) the second exogenous polypeptide is integrated at a locus of CIITA gene; and (iii) the third exogenous polypeptide is integrated at a locus of B2M gene; wherein integrations of the exogenous polynucleotides delete or reduce expression of CIITA and B2M genes.

[0802] In certain embodiments, (i) the first exogenous polynucleotide comprises the polynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more sequences selected from the group consisting of SEQ ID NOs: 131-156, and 171-184; (ii) the second exogenous polynucleotide comprises the polynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 75; and (iii) the third exogenous polynucleotide comprises the polynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 67.

[0803] In certain embodiments, (i) the first exogenous polynucleotide comprises the polynucleotide sequence of one or more sequences selected from the group consisting of SEQ ID NOs: 131-156, and 171-184; (ii) the second exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 75; and (iii) the third exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 67. Docket No. CNTY-033-WO-01

[0804] VII. Derivative Cells

[0805] In another aspect, the invention relates to a cell derived from differentiation of an iPSC, a derivative cell. As described above, the genomic edits introduced into the iPSC are retained in the derivative cell. In certain embodiments of the derivative cell obtained from iPSC differentiation, the derivative cell is a hematopoietic cell, including, but not limited to, HSCs (hematopoietic stem and progenitor cells), hematopoietic multipotent progenitor cells, T cell progenitors, NK cell progenitors, T cells, NKT cells, NK cells, B cells, antigen presenting cells (APC), monocytes and macrophages. In certain embodiments, the derivative cell is an immune effector cell, such as a NK cell or a T cell.

[0806] In certain embodiments, the application provides a natural killer (NK) cell or a T cell comprising: (i) a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (ii) a second exogenous polynucleotide encoding a truncated epithelial growth factor (tEGFR) variant and an interleukin 15 (IL- 15), wherein the tEGFR variant and IL- 15 are operably linked by an autoprotease peptide sequence, such as autoprotease peptide sequence of porcine teseho virus- 1 2A (P2A); and (iii) a deletion or reduced expression of an MHC class I associated gene and an MHC class II associated gene, such as an MHC class-I associated gene selected from the group consisting of a B2M gene, TAP 1 gene, TAP 2 gene and Tapasin gene, and an MHC-II associated gene selected from the group consisting of a RFXANK gene, CIITA gene, RFX5 gene, RFXAP gene, and CIITA gene, preferably the B2M gene and CIITA gene.

[0807] In certain embodiments, the NK cell or T cell further comprises a third exogenous polynucleotide encoding at least one of a human leukocyte antigen E (HLA-E) and a human leukocyte antigen G (HLA-G).

[0808] Also provided is aNK cell or a T cell comprising: (i) a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR) having the amino acid sequence of one or more selected from the group consisting of SEQ ID NOs: 157-170; (ii) a second exogenous polynucleotide encoding a truncated epithelial growth factor (tEGFR) variant having the amino acid sequence of SEQ ID NO: 71, an autoprotease peptide having the amino acid sequence of SEQ ID NO: 73, and interleukin 15 (IL-15) having the amino acid sequence of SEQ ID NO: 72; and (iii) a third exogenous polynucleotide encoding a human leukocyte antigen E (HLA-E) having the amino acid sequence of SEQ ID NO: 66; Docket No. CNTY-033-WO-01

[0809] wherein the first, second and third exogenous polynucleotides are integrated at loci of AAVS1, CIITA and B2M genes, respectively, to thereby delete or reduce expression ofCIITA and B2M.

[0810] In certain embodiments, the first exogenous polynucleotide comprises the polynucleotide sequence of one or more selected from the group consisting of SEQ ID NOs: 131-156, and 171-184; the second exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 75; and the third exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 67.

[0811] Also provided is a CD34+ hematopoietic progenitor cell (HPC) derived from an induced pluripotent stem cell (iPSC) comprising: (i) a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (ii) a second exogenous polynucleotide encoding an inactivated cell surface receptor that comprises a monoclonal antibody-specific epitope and an interleukin 15 (IL-15), wherein the inactivated cell surface receptor and IL- 15 are operably linked by an autoprotease peptide sequence; and (iii) a deletion or reduced expression of one or more of B2M, TAP 1. TAP 2, Tapasin, RFXANK, CIITA. RFX5 and RFXAP genes.

[0812] In certain embodiments, the CD34+ HPC further comprises a third exogenous polynucleotide encoding a human leukocyte antigen E (HLA-E) and / or human leukocyte antigen G (HLA-G).

[0813] In certain embodiments, the CAR comprises (i) a signal peptide; (ii) an extracellular domain comprising a binding domain that specifically binds to Nectin4; (iii) a hinge region; (iv) a transmembrane domain; (v) an intracellular signaling domain; and (vi) a co-stimulatory domain, such as a co-stimulatory domain comprising a CD28 signaling domain.

[0814] Also provided is a method of manufacturing the derivative cell. The method comprises differentiating the iPSC under conditions for cell differentiation to thereby obtain the derivative cell.

[0815] An iPSC of the application can be differentiated by any method known in the art. Exemplary methods are described in US8846395, US8945922, US8318491, WO2010 / 099539, W02012 / 109208, W02017 / 070333, WO2017 / 179720, W02016 / 010148, WO2018 / 048828, WO2019 / 157597, WO2022 / 120334, WO2022 / 133169, WO2022 / 216624, WO2022 / 216514, and WO2022 / 216524, each of which are herein incorporated by reference in its entirety. The differentiation protocol may use feeder cells or may be feeder-free. As used herein, “feeder cells’’ or Docket No. CNTY-033-WO-01

[0816] “feeders’" are terms describing cells of one type that are co-cultured with cells of a second type to provide an environment in which the cells of the second type can grow, expand, or differentiate, as the feeder cells provide stimulation, growth factors and nutrients for the support of the second cell type.

[0817] In another embodiment of the invention, the iPSC derivative cells of the invention are NK cells which are prepared by a method of differentiating an iPSC into an NK cell by subjecting the cells to a differentiation protocol including the addition of recombinant human IL-12p70 for the final 24 hours of culture. By including the IL-12 in the differentiation protocol, cells that are primed with IL- 12 demonstrate more rapid cell killing compared to those that are differentiated in the absence of IL-12. In addition, the cells differentiated using the IL- 12 conditions demonstrate improved cancer cell growth inhibition.

[0818] VIII. Polynucleotides, vectors, and host cells

[0819] (1) Nucleic acids encoding a CAR

[0820] In another general aspect, the invention relates to an isolated nucleic acid encoding a chimeric antigen receptor (CAR) useful for an invention according to embodiments of the application. It will be appreciated by those skilled in the art that the coding sequence of a CAR can be changed (e.g., replaced, deleted, inserted, etc.) without changing the amino acid sequence of the protein. Accordingly, it will be understood by those skilled in the art that nucleic acid sequences encoding CARs of the application can be altered without changing the amino acid sequences of the proteins.

[0821] In certain embodiments, the isolated nucleic acid encodes a CAR targeting Nectin4. In a particular embodiment, the isolated nucleic acid encoding the CAR comprises a polynucleotide sequence at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to one or more sequences selected from SEQ ID NOs: 131-156, and 171-184.

[0822] In another general aspect, the application provides a vector comprising a polynucleotide sequence encoding a CAR useful for an invention according to embodiments of the application. Any vector known to those skilled in the art in view of the present disclosure can be used, such as a plasmid, a cosmid, a phage vector or a viral vector. In some embodiments, the vector is a recombinant expression vector such Docket No. CNTY-033-WO-01

[0823] as a plasmid. The vector can include any element to establish a conventional function of an expression vector, for example, a promoter, ribosome binding element, terminator, enhancer, selection marker, and origin of replication. The promoter can be a constitutive, inducible, or repressible promoter. A number of expression vectors capable of delivering nucleic acids to a cell are known in the art and can be used herein for production of a CAR in the cell. Conventional cloning techniques or artificial gene synthesis can be used to generate a recombinant expression vector according to embodiments of the application.

[0824] In a particular aspect, the application provides vectors for targeted integration of a CAR useful for an invention according to embodiments of the application. In certain embodiments, the vector comprises an exogenous polynucleotide having, in the 5’ to 3’ order, (a) a promoter; (b) a polynucleotide sequence encoding a CAR according to an embodiment of the application; and (c) a terminator / polyadenylation signal.

[0825] In certain embodiments, the promoter is a CAG promoter. In certain embodiments, the CAG promoter comprises the polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 63. Other promoters can also be used, examples of which include, but are not limited to, EFla, UBC, CMV, SV40, PGK1, and human beta actin.

[0826] In certain embodiments, the terminator / polyadenylation signal is a SV40 signal. In certain embodiments, the SV40 signal comprises the polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 64. Other terminator sequences can also be used, examples of which include, but are not limited to, BGH, hGH, and PGK.

[0827] In certain embodiments, the polynucleotide sequence encoding a CAR comprises the polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to one or more selected from the group consisting of SEQ ID NOs: 131-156, and 171-184.

[0828] In some embodiments, the vector further comprises a left homology arm and a right homology7arm flanking the exogenous polynucleotide. As used herein, “left homology' arm"’ and “right homology arm"’ refers to a pair of nucleic acid sequences that flank an exogenous polynucleotide and facilitate the integration of the exogenous polynucleotide into a specified chromosomal locus. Sequences of the left and right Docket No. CNTY-033-WO-01

[0829] arm homology arms can be designed based on the integration site of interest. In some embodiments, the left or right arm homology arm is homologous to the left or right side sequence of the integration site.

[0830] In certain embodiments, the left homology arm comprises the polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 84, 87, 90, 193. 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, or 215. In certain embodiments, the right homology arm comprises the polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 85, 88, 91, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, or 216.

[0831] In a particular embodiment, the vector comprises a polynucleotide sequence at least 85%, such as at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 92, preferably the polynucleotide sequence of SEQ ID NO: 92. Table 3 provides a list of exemplary homology arm sequences and corresponding guide sequences for facilitating integration of an exogenous polynucleotide at various loci.

[0832] Table 3.

[0833] guide Locus gRNA LHA RHA sequenc

[0834]

[0835] e Docket No. CNTY-033-WO-01

[0836] ACTCTGCCCCAG CCACCCCACAGTG GCCTCCTTACCAT GGGCCACTAGGGA TCCCCTTCGACCT CAGGATTGGTGAC ACTCTCTTCCGCA AGAAAAGCCCCAT TTGGAGTCGCTTT CCTTAGGCCTCCTC AACTGGCCCTGG CTTCCTAGTCTCCT CTTTGGCAGCCTG GATATTGGGTCTAA TGCTGACCCATGC CCCCCACCTCCTGT AGTCCTCCTTACC TAGGCAGATTCCTT ATCCCTCCCTCGA ATCTGGTGACACA CTTCCCCTCTTCC CCCCCATTTCCTGG GATGTTGAGCCC AGCCATCTCTCTCC CTCCAGCCGGTCC TTGCCAGAACCTCT TGGACTTTGTCTC AAGGTTTGCTTACG CTTCCCTGCCCTG ATGGAGCCAGAGA CCCTCTCCTGAAC GGATCCTGGGAGG CTGAGCCAGCTC GAGAGCTTGGCAG CCATAGCTCAGTC GGGGTGGGAGGGA TGGTCTATCTGCC AGGGGGGGATGCG TGGCCCTGGCCAT TGACCTGCCCGGTT TGTCACTTTGCGC CTCAGTGGCCACCC TGCCCTCCTCTCG TGCGCTACCCTCTC TCTGT CCCCCGAGTGCC CCAGAACCTGAGC CCCCT CTTGCTGTGCCGC TGCTCTGACGCGGC CCACC AAVS1 AAS1

[0837] CGGAACTCTGCC CGTCTGGTGCGTTT CCACA

[0838] (PPP1R gRNA2

[0839] CTCTAACGCTGCC CACTGATCCTGGTG

[0840] 12C) intron 1

[0841] GTCTCTCTCCTGA CTGCAGCTTCCTTA (SEQ ID GTCCGGACCACTT CACTTCCCAAGAG NO: TGAGCTCTACTGG GAGAAGCAGTTTG 217) CTTCTGCGCCGCC GAAAAACAAAATC TCTGGCCCACTGT AGAATAAGTTGGT TTCCCCTTCCCAG CCTGAGTTCTAACT GCAGGTCCTGCTT TTGGCTCTTCACCT TCTCTGACCTGCA TTCTAGTCCCCAAT TTCTCTCCCCTGG TTATATTGTTCCTC GCCTGTGCCGCTT CGTGCGTCAGTTTT TCTGTCTGCAGCT ACCTGTGAGATAA TGTGGCCTGGGTC GGCCAGTAGCCAG ACCTCTACGGCTG CCCCGTCCTGGCAG GCCCAGATCCTTC GGCTGTGGTGAGG CCTGCCGCCTCCT AGGGGGGTGTCCG TCAGGTTCCGTCT TGTGGAAAACTCC TCCTCCACTCCCT CTTTGTGAGAATGG CTTCCCCTTGCTC TGCGTCCTAGGTGT TCTGCTGTGTTGC TCACCAGGTCGTG TGCCCAAGGATG GCCGCCTCTACTCC CTCTTTCCGGAGC CTTTCTCTTTCTCC ACTTCCTTCTCGG ATCCTTCTTTCCTT CGCTGCACCACG AAAGAGTCCCCAG

[0842]

[0843] TGATGTCCTCTGA TGCTATCTGGGACA Docket No. CNTY-033-WO-01 GCGGATCCTCCCC TATTCCTCCGCCCA GTGTCTGGGTCCT GAGCAGGGTCCCG CTCCGGGCATCTC CTTCCCTAAGGCCC TCCTCCCTCACCC TGCTCTGGGCTTCT AACCCCATGCCG GGGTTTGAGTCCTT TCTTCACTCGCTG GGCAAGCCCAGGA GGTTCCCTTTTCC GAGGCGCTCAGGC TTCTCCTTCTGGG TTCCCTGTCCCCCT GCCTGTGCCATCT TCCTCGTCCACCAT CTCGTTTCTTAGG CTCATGCCCCTGGC ATGGCCTTCTCCG TCTCCTGCCCCTTC ACGGATGTCTCCC CCTACAGGGGTTCC TTGCGTCCCGCCT TGGCTCTGCTCTTC CCCCTTCTTGTAG AGACTGAGCCCCG GCCTGCATCATCA TTCCCCTGCATCCC CCGTTTTTCTGGA CGTACCCCTGCATC CAACCCCAAAGT CCCCTTCCCCTGCA ACCCCGTCTCCCT TCCCCCAGAGGCC GGCTTTAGCCACC CCAGGCCACCTACT TCTCCATCCTCTT TGGCCTGGACCCC GCTTTCTTTGCCT ACGAGAGGCCACC GGACACCCCGTT CCAGCCCTGTCTAC CTCCTGTGGATTC CAGGCTGCCTTTTG GGGTCACCTCTCA GGTGGATTCTCCTC CTCCTTTCATTTG CAACTGTGGGGTG GGCAGCTCCCCT ACTGCTTGGCAAA ACCCCCCTTACCT CTCACTCTTCGGGG CTCTAGTCTGTGC TATCCCAGGAGGC TAGCTCTTCCAGC CTGGAGCATTGGG CCCCTGTCATGGC GTGGGCTGGGGTT ATCTTCCAGGGGT CAGAGAGGAGGGA CCGAGAGCTCAG TTCCCTTCTCAGGT CTAGTCTTCTTCC TACGTGGCCAAGA TCCAACCCGGGC AGCAGGGGAGCTG CCCTATGTCCACT GGTTTGGGTCAGGT TCAGGACAGCAT CTGGGTGTGGGGT GTTTGCTGCCTCC GACCAGCTTATGCT AGGGATCCTGTGT GTTTGCCCAGGAC CCCCGAGCTGGG AGCCTAG ACCACCTTATATT CCCAGGGCCGGT (SEQ ID NO: 194) TAATGTGGCTCTG GTTCTGGGTACTT TTATCTGTCCCCT

[0844] (SEQ ID NO: 193)

[0845]

[0846] Docket No. CNTY-033-WO-01

[0847] GCATATAAAACC ATCCAGCAGAGAA TCAGCAGAAATA TGGAAAGTCAAAT AAGAGGTTTTGTT TTCCTGAATTGCTA GTTTGGTAAGAA TGTGTCTGGGTTTC CATACCTTGGGTT ATCCATCCGACATT GGTTGGGCACGG GAAGTTGACTTACT TGGCTCGTGCCTG GAAGAATGGAGAG TAATCCCAACACT AGAATTGAAAAAG TTGGGAGGCCAA TGGAGCATTCAGA GGCAGGCTGATC CTTGTCTTTCAGCA ACTTGAAGTTGG AGGACTGGTCTTTC GAGTTCAAGACC TATCTCTTGTACTA AGCCTGGCCAAC CACTGAATTCACCC ATGGTGAAATCC CCACTGAAAAAGA CGTCTCTACTGAA TGAGTATGCCTGCC AATACAAAAATT GTGTGAACCATGT CATTC AACCAGGCATGG GACTTTGTCACAGC TCTGC TGGTGTGTGCCTG CCAAGATAGTTAA TGGAT TAGTCCCAGGAA GTGGGGTAAGTCTT GACGT TCACTTGAACCCA ACATTCTTTTGTAA GGAGGCGGAGGT GCTGCTGAAAGTT

[0848] (SEQ ID TGCAGTGAGCTG GTGTATGAGTAGTC NO: AGATCTCACCACT ATATCATAAAGCT

[0849] 218) GCACACTGCACT GCTTTGATATAAAA

[0850] B2M

[0851] CCAGCCTGGGCA AAGGTCTATGGCC

[0852] B2M gRNA2 or

[0853] ATGGAATGAGAT ATACTACCCTGAAT

[0854] 4 exon 2

[0855] TCCATCCCAAAA GAGTCCCATCCCAT CTCAC AATAAAAAAATA CTGATATAAACAA GTCAT AAAAAATAAAGA TCTGCATATTGGGA CCAGC ACATACCTTGGGT TTGTCAGGGAATGT AGAGA TGATCCACTTAGG TCTTAAAGATCAG AACCTCAGATAA ATTAGTGGCACCTG

[0856] (SEQ ID TAACATCTGCCAC CTGAGATACTGAT NO: GTATAGAGCAAT GCACAGCATGGTTT

[0857] 240) TGCTATGTCCCAG CTGAACCAGTAGTT GCACTCTACTAG TCCCTGCAGTTGAG ACACTTCATACA CAGGGAGCAGCAG GTTTAGAAAATC CAGCACTTGCACA AGATGGGTGTAG AATACATATACACT ATCAAGGCAGGA CTTAACACTTCTTA GCAGGAACCAAA CCTACTGGCTTCCT AAGAAAGGCATA CTAGCTTTTGTGGC AACATAAGAAAA AGCTTCAGGTATAT AAAATGGAAGGG TTAGCACTGAACG GTGGAAACAGAG AACATCTCAAGAA TACAATAACATG GGTATAGGCCTTTG AGTAATTTGATGG TTTGTAAGTCCTGC GGGCTATTATGA TGTCCTAGCATCCT ACTGAGAAATGA ATAATCCTGGACTT

[0858]

[0859] ACTTTGAAAAGT CTCCAGTACTTTCT Docket No. CNTY-033-WO-01 ATCTTGGGGCCA GGCTGGATTGGTAT AATCATGTAGACT CTGAGGCTAGTAG CTTGAGTGATGTG GAAGGGCTTGTTCC TTAAGGAATGCT TGCTGGGTAGCTCT ATGAGTGCTGAG AAACAATGTATTC AGGGCATCAGAA ATGGGTAGGAACA GTCCTTGAGAGC GCAGCCTATTCTGC CTCCAGAGAAAG CAGCCTTATTTCTA GCTCTTAAAAATG ACCATTTTAGACAT CAGCGCAATCTC TTGTTAGTACATGG CAGTGACAGAAG TATTTTAAAAGTAA ATACTGCTAGAA ACATCTTTTTTATATTTGCTTCCTCTAC ATCTGCTAGAAA AAAAACAAAAAA CTGTCTTTTTCATA GGCATGTATAGA GATCGAGACATGT GGAATTATGAGG AAGCAGCATCATG GAAAGATACCAA GAGGTAAGTTTTTG GTCACGGTTTATT AGTCTCTTTTTGGATGTATACAAACTTCTTCAAAATGGA GGTGGCTTGTTGG GTCCTGAGGACTAT GAAGGTGGAAGC TTATAGACAGCTCT TCATTTGGCCAGA AACATGATAACCC GTGGAAATGGAA TCACTATGTGGAG TTGGGAGAAATC AACATTGACAGAG GATGACCAAATG TAACATTTTAGCAG TAAACACTTGGTG CCTGATATAGCTT (SEQ IDNO: 196) GACACCAAGTTA GCCCCAAGTGAA ATACCCTGGCAA TATTAATGTGTCT TTTCCCGATATTC CTCAGGTACTCCA AAGATTCAGGTTT ACTCACGTC

[0860]

[0861] (SEQ IDNQ: 195) Docket No. CNTY-033-WO-01

[0862] ACAGGAGGTAAC AGCCCCAAGGTAA CATTTAACAAGA AAAGGCCGGGAAA AAGCAGAGTGAT GCATCTTAATTTAG GTTAGATTATAGC CGTGCAGTCTCAGC AAGATACTGTTG TGGTCCTGCCATTC ACTGTAGAAGGC CAGATAAACAGAG TCTGAGGCTAGA AAACCATTCTGAAT GAGCTGCTTTCTA TGGGGATGGGGGT TAAAACAGAGTG GAGGATGGGAACA ATCATATATTAGA GGAGTCTGTGTCCT AGAGGTGTTAAA GCTGGGGCAGGCC GACATGTTCACA ATTGGAAGATGTG CCAAGCTGAGAC AAAGAGTTGTCTAT TTCCTCCTTGATA TTCCTTCCACCGGA CCACCAGGAGGA GGGAGACTTCAGG TGGGCAGAGACT TCAGCCAGGTGTCT GGAAAAGACACT GGAGTATGAACCA AACTTTCTCCCTA TGTATCAGCACCG TGGGAGTCAGTA AAAGGTTCTAGAA TTATTTAGCATCA GTCAGACTTTCGGG CTTTGGCGGGTCA CAGTGTGTCACTAA CCCCAAACCATCT CTCTCAGCATGCTG CCTTG GACTACAAGGGT GCCTGGCTCGGCCC GGGCT ACCATATTTGGGT ACAGCAAGGTCTT CTGAC CIITA CIITA TAACACTCTTTTG CTCGCCTCCCTTTG AGGTA

[0863] gRNAl

[0864] exl GTATAATTTATGT GGTAAATACTGAG

[0865] exon 1

[0866] TTTAGTCCAATGT GGGTGCCTCTGCA (SEQ ID CTTGGGATGAAA GGACGGGACCTCT NO: ATGACAGGTGGG GCCAGACTCCACTC 219) CCACTTATGATCT CATACCCAGAGAA CCAGAGAAATTC GCAGGGAAACCAA AGGGCAATTTGG AATTGGAGTCAGC TGTGGGAGTAGG CTTGAGGTGTAGCT CATGGTAGAGGA GTTGAGCCCTCAGC GAGCAGCATCTA AGCTGGGGAGAGC AGAAGTCCCCAG TGGCGGATGCTGC CAGAGGCTCTCA CCTCCCCCCAGTTT GCTTGTCTTGAGG CCTAATGGTGTTGT CATCTGGGCGGA TTAAAAAGGGTCA GGGCTATGATACT GGGGACGGGGGAA GGCCCCATCCTGC CAGATGGTGGGAA AGAAGGTGGCAG GAGCACAGTGCAG ATATTGGCAGCTG ACACCTGGCACCG GCACCAGTGCGG GCTCTGAAGGCAG TTCCATTGTGATC CATGGCAGCTACA ATCATTTCTGAAC CCGTTGGCTGGGA GTCAGACTGTTGA AGGGTGTGCCCCT AGGTTCCCCCAA GAAGAAGTCGTTT CAGACTTTCTGTG ACATTCTCGAGTCA

[0867]

[0868] CAACTTTCTGTCT ATTTTCCTGGAGTG Docket No. CNTY-033-WO-01 TCACCAAATTCA TACAATGGACCTGT GTCCACAGTAAG GGGAAAGCCTGTA GAAGTGAAATTA TGAAAGGGTAATG ATTTCAGAGGTGT ATGAGGGACCTAG GGGGAGGGCTTA CACAGTGTCCAAT AGGGAGTGTGGT ATTTTATAGGAACT AAAATTAGAGGG GGAATTGAGCTCA TGTTCAGAAACA TAGGAGCTCAATTT GAAATCTGACCG TATTGGCATTGCTG CTTGGGGCCACCT TTGTTGGATGGTTA TGCAGGGAGAGT AAGGGGTGGTATCTTTTTTGATGATCCCTTTTCTCAGACT CCTCACTTGTTTC CCCCTGAAATGTAT TTTGCATGTTGGC GGTTTGCTTTGAAC TTAGCTTGGCGGG CCAGAGACTGATG CTCCCAACTGGTG ACAGGTCTGCCGG ACTGGTTAGTGAT TGTGGTTGGGTGCA GAGGCTAGTGAT GCCTTAAGTTGCTA GAGGCTGTGTGCT CGGGAAAGTGTTG TCTGAGCTGGGC GAGGGGGAGAAGT ATCCGAAGGCAT CAGAGGTAACCTT CCTTGGGGAAGC GCCCCCTCCCTCAA TGAGGGCACGAG TTCCAGATGAGGA GAGGGGCTGCCA AATTCAGGCCTGA GACTCCGGGAGC AAAGGGA TGCTGCCTGGCTG GGATTCCTACAC (SEQ IDNO: 198) AATGCGTTGCCTG GCTCCACGCCCTG CTGGGTCCTACCT GTCAG

[0869]

[0870] (SEQ TDNO: 197) Docket No. CNTY-033-WO-01

[0871] AAACTTCTACCAC AAGTTGGGCAGAA CGTCACCTATCTC AAGTCAGAAAAGA TCAGGGTTTCCTA CGTGAGTGAGCCC AACATACTCTGA CTCCCTGATCCAAC ACAAGTTTCCTCA CTAGCCTTGCTTGA CTCTGCCACTGTG GACCTGGCCTTTCC ACCCAGAAAGCT TTGACTCCAAAGCC ATCTGTTCTCCTT TGCTGTGGGTCCAA CTCCCAGACTCTG CTTGCTTCCCTCGC CCTCATCTTTCAA TAAGTCCTGTCTGG GGCCTGGTTCCA TTGGGAGGCCCTTT GGATCCCTTCCTC AAAAGCCAACAGG CAGGAAGCCTTC AGCCTTAAAATGT CCTGATTGCCCTA ACATCTGATTATTT TTCCACCATACAC CATGGCCCTGATA CTTTTTTCTCGGA ACCCTCCAATGGCT CTTCATGTCATGG ACAAAATACATGC TGCCTATATTCCA CACAAGGCCTGTA AGGGCTCTGAGC TGGCCCTTCCTCCC CATGTACCCCTTT TCTCCAAACCCACT ATATAGTTACCAC ATGAGACACCACA TATTTACTGAGTG CTTCAGCCACCACC TGCCC CCTACTGTATACC AGCTTCTCCACTCC AACTT AGCTACTGTGTTG TATAACCCATGTGC CTGCT CIITA CIITA GATGCTCTAGATG CCTTTCAAGCCTCG GGCAT

[0872] gRNA4

[0873] ex5 TAGAACCTCTAAT GGGCCTTTGCAGTT

[0874] exon 5

[0875] TATCACCATCGAT GCTATAGTCTCTAT (SEQ ID TCCTGGGTAGTAG CTGGAATGCCCTTC NO: GCATTATTTATTC CCCCAGTTCTTCCC 220) ATCTTACACAGAT ATGGCTGACTCCTT GAGAAAATGGAG TGAATCTTTCTGGT GCCCACAGTGGT GTTGGCTAAACTGT TAAATAAGTAGC CACCTCTTCCTGGA CCAAGATTGCAC ACCCTTCTCTGACC AGCTAGTAGGGC ATCCTTCCATGTAG TAGTGGAAAGTA ATTAGCTCAGTTAT GAGGTGGAATTT TCTCACCTTGTGTG GAACTCAAATCC TCTTTTTCCTTGCA TGCAGTAACTCTA GTTTAGCACTCATT CCATTCTGCCTTG ACCATCTGGACAT CTCTTCTTTGTAG ATTTTACGCCTTGC CAGTAGATAAGT TCTCCCACTGTGAG TTTCATGGATACA GACAGGGACCTTG TACCTCATCCTTT TCTTTCTTGCTCGT TGATTAGATTAAG GACTGTTTCCCCAG GGCCCCTGGAGT CATCTAGTGCAGTG GTCAGTGTTCATT CCTGGTATGCAGTA CATTTGTTTGATC GCACCTCAGTAGA ATTCATTCATTCA TATCTGTTGAATGA

[0876]

[0877] ACAAACATTTCTT AAACATCTGTAAA Docket No. CNTY-033-WO-01 GAGTCCCCACTGT ATGGGTGTAACAG GTGCCAGGCCCA TTAACTGAGTACTT GAGGTTCCCCAG ATTATGGGTCTGAC CCCAAGGCCTGG CATGTGTAAGTCCT CACACAGTGGGC GTATCTATTTATTC CTTCAGTTAGACC AGTTCTTAAACAG TTGTTGATTGACT GTGAATCGCACAC GCGCTTTTCCTTG AGGGTATGAGATTT TCTGGGCAGCGG TAAAAGTGCAAAG AACTGGACCAGT AATATTCAGTGAA ATGTCTTCCAGGA GGCTGGGCGCAGC CTCCCAGCTGGA GGCTCACACCTGTA GGGCCTGAGCAA ATCCCAGCAGTTTG GGACATTTTCAGT GGAGGCCAAGGGG AAGTTTGTGGTGG GACGGATCACTTG GTGGGGAGGTCT AGGTCAGGAGTTT TGGCTCAGCCTGC GATACCTGCCTGGC ATTTCCTGCCTTG CAACATGGTGAAA TTCCCTGGGGGGT CCGCGTCTCTACCA GCCCTAATACCTG AAAAATACAAAAA ACGACCATTCATT TTAGCCGGGTGTG GATGGGCAGTCA GTGGTGCACGCCT GACCCCTCTCCCC GTAATCCCAGCTAC AAGGTGGGTACA TCGGGAGGCTGAG ATAGAGACTCAC GCAGGAGAATCGC CTTGGGCTTTCAT TTGAACCCAGGAG TGATTGTGTGAGT GTGGAGGTTGCAG TGGTCTCTGGTTT TGAG TTCTCAAAGTAGA GCACATAGGACC (SEQ ID NO: 200) AGATGAAGTGAT CGGTGAGAGTAT GGAGATGCCAGC AG

[0878]

[0879] (SEQ IDNO: 199) Docket No. CNTY-033-WO-01

[0880] AAAAATAAAAAA CGCGGGCTTGGTG TAAAAATATTAA ATCTGCCTCGTGGT CTTAATTTAACTT GTGCATCCAGCGCT TAAACAAAAAAG TCGCACAGGCTCA CAGGTGGTCTCC GCAGCAGCTGCCG AAGAATGCAGGA CTCGAGTCACTTGG GATAACTGACCG GGTGAGTTGAGAT GGTGCAGTGTCTC GGAAAAGTTGGGA ATGCCTTTAATCC AGAAAACATAGAG CAGCACTTTGGA AGGCGCGTGACCG AGGCCAAGGCGG AAAAGACAGAATG GTGGATCACCCA AGATGGGTACAAA AGGTCAGGAGTT GAGGCCAGAGAGG CAAGTCCAGCCT AAGATCTGGTAGG GGCCAACATGGT GCAGAGACAGAGA GAAACCCCATCT CCAGAACAGGGAG CTACTAAAAATA GCGAGGCGGGGAC CAAAAAATTAGC CAGGCTGCCCGGT CAGGCATGGTGG GTAGGGGCTACGA CGCGCGCATGTT GACAGGCAGCCCT ACTCCCAGCTACT GCCAGGAGGTACA CGCGAGGCTCAG GGGAGATCCCGGG GTCCC ACAGGAGAATCG ATGGGAAAGGTAG ATTGG CTTGAACCCAGG GCACACATGGAAA TCGCG CD70

[0881] AGATCGAGGTTG TGGAAGATGACTC GGCTT CD70 gRNAl

[0882] CGGCGAGCTGAG GGCTCTGGTGTTCC

[0883] exon 1

[0884] ATGGCGCCACTG CCCGGCAGGCTGA (SEQ ID CACTCCAGCCTG CTCAGAGGCTGCT NO: GGTGACAGAGGG GGGGGCTTCACAA 221) AGACCTCCGTCTC GGCTGGGCGTGGG AAAAACAAAACA GGCTTCCTGGGGCC AATCAAAAAAAT TCCTAGGACGGGA GCAGGAGAGGGG TGGCCCCAGCCACT TACACGAATATTT CGCTCCGGGTGGG GGGGAGCACCCC GGAGGGGTCCCTTT CAATTCTTGGATG GGGGACCGCGCCG TCTGCTGTATCCC GGCGCCTTTGCAGC CAGTGCACAGCA GTAGAGAGTCCGC CAATCTAATCCCT TGCGCGCGGTGCTC AATAAATGTGCA TCGCGCCCAGTGA GTGGAGGTTTGTT CATCCAGGAAAAC GAATAAATGAAT GATTCGGGAAACG GGGCCCCAGAAG AAGAAGTTCTTTTG AATGAGGTGGAG AAGGTCTCGACTTC AGGGGAATAGGA ACGTTCCCCGCTGG AGATTGAATGTCT TTCAGACCTGCTTC CCTGCCTGAAGG CTCTTTAAGAAGTC TCGGGCGGGGAG TTAAGAGTAAAAA GGGTTGGGGGCA AAAATAAAATGAA

[0885]

[0886] GGCAACTCTGAG ATAAAATCACCAG Docket No. CNTY-033-WO-01 GCTCACCCGGGG TGCGCGCCGTGGG CCACTGCCTGCAT ATGAGAGGTGGAA CCTGGCAACTGC AGGAGGATGGACA CTCCACCCACTTT GAGAAAAGAGAGC AGGATCTTCAGA TCCTGGCACAGGG CTGGCAGCGGTT GACACATAGAACC GGAGGGAATTTC TCTCTGCTTACGTC CCCTCGCCAATTG CGTGCCCTGTTTTC CTCAAGTCCCTCC TGGTCTTTTCTTCC CCTCGACCGGCC AGTGGGACGTAGC GGACATCCCCAG TGAGCTGCAGCTG AGAGGGGCAGGC AATCACACAGGTA TGGTCCCCTGACA ACACGGGGGACGT GGTTGAAGCAAG GGAGGGACGGGGA TAGACGCCCAGG GAAGAAGAGGCAC AGCCCCGGGAGG AGAGAGAGAAGGA GGGCTGCAGTTTC AGGAGAGGTAGAA CTTCCTTCCTTCT AGACAAGTGGGGA CGGCAGCGCTCC GAGACAGAGAGAA GCGCCCCCATCG AGAGACACAGACA CCCCTCCTGCGCT GAGACGGAGGGAG AGCGGAGGTGAT AGAGGGAGGGAGA CGCCGCGGCGAT GATAGGGAGGGAA GCCGGAGGAGGG ACGGAGAGGGGGA TTCGGGCTGCTCG GACAGAGAGAAGA GTGCGGCGCAGG CAGAGAGG CCCTATGGGTGC GTCCTGCGGGCT (SEQ ID NO: 202) GCTTTGGTCCCAT TGGT

[0887]

[0888] (SEQ ID NO: 201) Docket No. CNTY-033-WO-01

[0889] GTCCAGACTCAG ATCATGTCTGCTCG GAGGACTTAGTTC ACAGCTCTGACTA TCTTCATAAAAAG AACACTGTGCCCC TTGATCACCTTTG AAAGTGTTGAGGA CTTTCTCCTTTGC ATTGGGAAAACCT ATGGGCAATAAA AGCTGAGTTAGTG GAAGTGAAAAAT GTCTCTTTTCTGTT AAATTCCCAGTG ACAATAAAGCTCA AGCTTGCATTCTG TAATGAAAATTAG CTTGGGAACAAC CCTTCTTTGTTCTT ATAGCACATCCA CCCCAAGTCTTTCT TTTTCTAGAGAGC TTCTAGACGAAACT TGTCCAGTCCCCA ACTTTCAACTGTTT TTTGAGGGCTGCT TAACTTCCTTACTG AGCCACATGTCA TTAACTTC CAT ATT AGCAGGAACCCA TCTAGATAGTATGG GAGTATAACCAG TCAGACTGTTATTT GAAAATGTCTGT CTTGACTTTTAAAT GGTAGACCCCAG GTAAGATATTATCT TGATTCATGCCTC ACTGACTTCCTTCT CCATCTCCACACC ATGTAAGATGAGG CTCCTGTAGTCCC ATTGAGCTCTCTTA GTCAG CTCCCACGCTGAT CCCTTCTCCCATTT AGCTG TCTGGACTAGGC CCTCATCCTTCCAA TGATC CLYBL CACATGGCTTGG CATAAATATATTTT ACTCT CLYBL gRNA2

[0890] CCAGTGGAACAC GGGATTATATCAA

[0891] intron 2

[0892] ATGCAGGCTTGC CATTCAATGTTACT (SEQ ID ACGTCTGGAACT TAAAGTGACCTTGT NO: CTGCCACCTAATT AAATATTTTCACAA 222) TAGGATCCCAGA CTGAGCCATGTTTG CTCTCCTACTGGA ATTTGTATACTTAT GACACAGGTCCT GTTTACTTTACTGT TAGTGACAGTCTG TTTTCCTGAAGTTA CACCACCATTCA ATAATTGCCTTGAA GACAAGTCAGTA TTTATTTATTTCTTT GGGCCATCTTAG AAAAATGTTTCATT ATCATCCAGCCCT ACTCAGGACTGTA AGTCAAGCCACC GTTTACATTACGAT AGATAACTGTAC TCTTTGTGTTATAC CCACATAAGTGA AGTTGATGGGTTTC CCCCTGGCGAGA TTTTCTTTCTTAATT CCAGCAGGAGAA TCTTTAAAAAATAG TCATGCCAATGG AGATGGGGTCTTA GCCAATATACATT CTATATTACCCAGG CTGACCCACAGTT CTGGTCTTGAAGTC TCATAATAAAAT CTGGGCTCAAGTG AAAATGGTTGTG ATCTTCCTGTCTCA GTTGTAAGCCACT GCCTACCAAGTAG ATGTTTCAGAGTG CTGAGACTATAGG

[0893]

[0894] GTTTGTTACACAG TGCAAAAAAGCCA Docket No. CNTY-033-WO-01 CAATAAATAACT CTATACCTGGCTAG AATATAGTAGGC TTTACAGGTTTTAA ATACCATCAAGT CAAATGCATTATGC CCAAAGTAGGTA CACGTATCCATTAT GAGAAGAATGTA TACAGGATCACAC AATAGCAGAGCA AAGATATTTTCATT AAACAGCATGAC ACCCTGAAAGCAT TGGTGGCTGGGA CCCTGTGTTCCACC GGCTTAAAACTG AATTCATCCTGCCT GGACAGGATCAG CCATGAGCCGCTG AGTCATGAAAGA GCAACCACTGATCT AGTCAAAGAAAT CTATAGTTTTGCCT GGTTCAGAAGTA TTTCTAAAATGTCA AGGCTGAGACTG TATAATTGGAATCA ACTTACAAAAGC TACAGTCTGTAGCA TGAAAGTCCCTTT TTTTCAGACTAGCT AAGTTGGTGTTTG TTTAAAATTTGGCA GTGCATTGGCAG AT ATGC ATTT AAGG GGGCAGGTATGG TTCCTCCTTAAATG TGACTTAAAAGA TGAAGGGCATAGC GCCATGCTCAAC CAATGTGGCTTGAT AAGATCAAGCAC AGCTCATTTCTTTT AACACAATCACG TATTGGTGAATATT GGTCACCCCAGC TCATTGTCTGGATG AGACCTTAGCGA TTCCACAGTTTGTT GTCTAGCCATTTC TATCCTCGAGAGCT TTTGGTGGTGGTC TGGCGT ACAGTCATGCTTC AGCCCAGTTTCCA (SEQ ID NO: 204) CTTGGACAAATG GTACATATTTTCA ATGAGATGAAAA TTAAGATACAATC CATGTGCTCAGA GAGTGATCA

[0895]

[0896] (SEQ ID NO: 203) Docket No. CNTY-033-WO-01

[0897] TTATGTAGTGTTC AATCTGCCCCCAA CTAATACTATTTA ACCCAAAGAGGCA GGTTATGCATAA GCAACGAAAACCT GTTAACTGTGTCA AAAGGCAATAAAA CATAGCATTTTCT ACTCCATTTTAGCA GTC AT AT AC A A AT ACTGAACAGGAAA ATTTAATTTTTTA TAACCTATGCGGA TAATTTTTAATAA ATTAAACCTTCAAA ATAATTTTTAATA AAGCTTCTCAGGAT ATTTCTTAGAACA TTTCAAGGGAATG TTTCAGCCATTAA ACAAAACCTATCA GAAAAGACCATT CTGCAAAGGTAAA TAATTACCTCTAT GCATTTAAAAGAT TGATTTTCATTAA CCTCAATATAACA TGTGTATAAATTA GTCTAGGATGTGC CTAATTAAAAGT AGCTTGGGGTACA AATGTAGTGCAA GGAATGTGGGGAA GAAATTTTTATAT AGAGAAGGGAGTG ATATATATGTCAA CTCATATATCTTCT CTTTACTATTACT ATTTGCAAAGATC CATGTGCAGACC AGAATTCCAAGTT ACTCA ACATAGTCTTAAC GAGATATGCTATTT GACCT CATTACTGGATTT CAATGTAAAGTAT GAATC CAAAATTAACAT GAAGACTGATTGA NKG2A TGCCC AAAAATGAGTGC ACTCATTGTTGAAG NKG2A gRNAl C AGAAGTTTTTCTA TTTGTAGTCTTTGT

[0898] exon 3

[0899] ATACAACATTTTA CAAATAATTCATG

[0900] (SEQ ID ATTTTTAACACAG GAGCATTATTTTTC NO: GTAAAACATCAG CTGAAAATTCAAT

[0901] 223) ACTTTAAGAAAT GGTATATTATTCTG ATATTTTTATTCC AGAAAAAGATTAC TGAACTTCTTTAT AATGGGAGATGAG TCCTTAGTAATTT GGTTTGGGGTCCA ATTGCTTCTCATT AGTTTCTCTGTATG GCCCCAGCAATA ATTCCTGTGCATTC ATATTTTGTCAAA AGGTTCTCTTGTCT TGCAGAAAATTT GTGAATCTTCTAAA ATCTTTTTTTTTTG CGACTGTATCCACC AGTCGGAGTCTC TCTCCTTTCGCACT ACTCTGTCGCCCA GTTCCCATTTCTCT GGCTGGAGTGCA CCCTGCAGATTTAC GTGGCACAATCT CATCAGCTCCAGA CCGCTCACTGCA GAAGCTCATTGTTG ACCTCTGCCGCCC GGATCCTGGGAAT ATGTTCAAGAGA TATCTGTCTTATCT TTCTCCTGCCTCA TAATGGCCTCTGTG GCCTCCCGAGTA GTAACGATAGTTGT GCTAGGACTACA TATTCCCTGTAAGT GGCGCCTGACAT CTATTTTCGAAGAT

[0902]

[0903] CATGCCCGACTA TACAAGGGGAATT Docket No. CNTY-033-WO-01CTTTTTGTATTTTTTTCACGTTAATGAT AGTAGAGACGGA TGAATGTGCCTCTA GTTTCACCGTGTT AACATTTCATATTT AGCCAGGATGGT TCAGGGAATAGAG CTCGATCTCCTGA TTCTCATTGTAATG CCTCGTGATCGGC TATATATTTGGACT ATGCCTCGGCCTC AAATGTGGAATGA CCAAAGTGCTGG TTATTCTGAATTTG GATTACAGGCGT TCAAAGAATAAAT GAGCCACCGCGC GAAAGAATAATTG CCGGCCTAAAAA TTGAAAGTATTCGC TCTTTTTTTAAAA TTCTGATGCAATCG CAAATATTCATAA TATGTATATATTTG GAAACGTGTTTA GATTTCATAACTCA GGCTTGAAGAAA AAAATATGTTCTAG ATCAGAGAAAGA GAGTCTGAAAAAC ACTTTAGATTATT CTTACTGAGAAAT TAATGCAAAATG AGAAATTAATTTTT AGCTCCAATACTC GAAAGTAGTTAAA GTTCTCCACCTCA TCAAGAATTATAA CCCTTTTAATTGC GAACTATATGAGA ACTAGGGAATCC TGGTGAAATTTGGT TGTATATAAACCA TCTTTAGATCTATG TTTATTAACTTCT AAATACTTTTCCAA TAACTACTGTTAT AAAACCACCATTA TATAGAGTACAG CTTTATC TCCCTGACATCAC ACACTGCAGAGA (SEQ ID NO: 206) TGGATAACCAAG GAGTAATCTACTC AGACCTG

[0904]

[0905] (SEQ ID NO: 205) Docket No. CNTY-033-WO-01

[0906] CTCAAAAGGCAG TGAGAGACTCTAA GAGGTCGGAAAG ATCCAGTGACAAG AATAAACAATGA TCTGTCTGCCTATT GAGTCACATTAA CACCGATTTTGATT AAACACAAAATC CTCAAACAAATGT CTACGGAAATAC GTCACAAAGTAAG TGAAGAATGAGT GATTCTGATGTGTA CTCAGCACTAAG TATCACAGACAAA GAAAAGCCTCCA ACTGTGCTAGACAT GCAGCTCCTGCTT GAGGTCTATGGAC TCTGAGGGTGAA TTCAAGAGCAACA GGATAGACGCTG GTGCTGTGGCCTGG TGGCTCTGCATGA AGCAACAAATCTG CTCACTAGCACTC ACTTTGCATGTGCA TATCACGGCCAT AACGCCTTCAACA ATTCTGGCAGGGT ACAGCATTATTCCA CAGTGGCTCCAA GAAGACACCTTCTT CTAACATTTGTTT CCCCAGCCCAGGT GGTACTTTACAGT AAGGGCAGCTTTG TTATTAAATAGAT GTGCCTTCGCAGGC GTTTATATGGAGA TGTTTCCTTGCTTC AGCTCTCATTTCT AGGAATGGCCAGG GAGTC TTCTCAGAAGAG TTCTGCCCAGAGCT TCTCA CCTGGCTAGGAA CTGGTCAATGATGT GCTGG TRAC GGTGGATGAGGC CTAAAACTCCTCTG TACAC TRAC gRNAl

[0907] ACCATATTCATTT ATTGGTGGTCTCGG

[0908] exon 6

[0909] TGCAGGTGAAAT CCTTATCCATTGCC (SEQ ID TCCTGAGATGTAA ACCAAAACCCTCTT NO: GGAGCTGCTGTG TTTACTAAGAAAC 224) ACTTGCTCAAGG AGTGAGCCTTGTTC CCTTATATCGAGT TGGCAGTCCAGAG AAACGGTAGTGC AATGACACGGGAA TGGGGCTTAGAC AAAAGCAGATGAA GCAGGTGTTCTGA GAGAAGGTGGCAG TTTATAGTTCAAA GAGAGGGCACGTG ACCTCTATCAATG GCCCAGCCTCAGTC AGAGAGCAATCT TCTCCAACTGAGTT CCTGGTAATGTGA CCTGCCTGCCTGCC TAGATTTCCCAAC TTTGCTCAGACTGT TTAATGCCAACAT TTGCCCCTTACTGC ACCATAAACCTC TCTTCTAGGCCTCA CCATTCTGCTAAT TTCTAAGCCCCTTC GCCCAGCCTAAG TCCAAGTTGCCTCT TTGGGGAGACCA CCTTATTTCTCCCT CTCCAGATTCCAA GTCTGCCAAAAAA GATGTACAGTTTG TCTTTCCCAGCTCA CTTTGCTGGGCCT CTAAGTCAGTCTCA TTTTCCCATGCCT CGCAGTCACTCATT GCCTTTACTCTGC AACCCACCAATCA

[0910]

[0911] CAGAGTTATATTG CTGATTGTGCCGGC Docket No. CNTY-033-WO-01 CTGGGGTTTTGAA ACATGAATGCACC GAAGATCCTATTA AGGTGTTGAAGTG AATAAAAGAATA GAGGAATTAAAAA AGCAGTATTATTA GTCAGATGAGGGG AGTAGCCCTGCA TGTGCCCAGAGGA TTTCAGGTTTCCT AGCACCATTCTAGT TGAGTGGCAGGC TGGGGGAGCCCAT CAGGCCTGGCCG CTGTCAGCTGGGA TGAACGTTCACTG AAAGTCCAAATAA AAATCATGGCCT CTTCAGATTGGAAT CTTGGCCAAGATT GTGTTTTAACTCAG GATAGCTTGTGCC GGTTGAGAAAACA TGTCCCTGAGTCC GCTACCTTCAGGAC CAGTCCATCACG AAAAGTCAGGGAA AGCAGCTGGTTTC GGGCTCTCTGAAG TAAGATGCTATTT AAATGCTACTTGA CCCGTATAAAGC AGATACCAGCCCT ATGAGACCGTGA ACCAAGGGCAGGG CTTGCCAGCCCCA AGAGGACCCTATA CAGAGCCCCGCC GAGGCCTGGGACA CTTGTCCATCACT GGAGCTCAATGAG GGCATCTGGACT AAAGGAGAAGAGC CCAGCCTGGGTT AGCAGGCATGAGT GGGGCAAAGAGG TGAATGAAGGAGG GAAATGAGATCA CAGG TGTCCTAACCCTG ATCCTCTTGTCCC (SEQ ID NO: 208) ACAGATATCCAG AACCCTGACCCT GCCGTGTACCAG

[0912] C

[0913]

[0914] (SEQ ID NO: 207) Docket No. CNTY-033-WO-01

[0915] CTTGCTGCTCTGG TGTCCGGGGACAA AACAGGCCAAGA ACCCTGCTGCCGGC GCTTTTCTGCCTC TCTCTAGGAGAGC AGAGTCTTTGCAC CCAACTCTGTCTTG CTGCCATTTCCTC GCGTCAGTATCCTG TGCTTGGGAAAT GTCCTGATCCTCGT GTTTGCCCCAAG CGTGGTGCTCGCG GGAGTTGGGTGA GTGGTCGTCCCGA CTTGATCGCTCAC GGTGGCGCCAGCA ATTACTTAGGTCT GTGGAGCGGTCCG CTGCTTGAATGTC GGCACCACCAAGC ACAGATGTTCTCT GCTTTCCCGAGACC TAATAAAGAAGA GTCCTGGCGCGAT GGCAAGAAAAGC GCGTCAAGTACAC CACTTTATTATTT TGAAATTCATCCTG ATTAAACTCCCGC AGATGAGGTGGGT ATAGAGTGCAGT TGGCGACTAAGGC ATTATTACTGTGT GCACCGGTGGGCA GCCAGACCCTGC CTGCGGGGACAGC TTCAAACACATTC AGGGCCCCGCGCG CATGGACTATAA CAGGGAAGCCGCC AATTGCATCTCTG CGGATCGCCCGGA CCGAG AGCAGCTCCTAG ACCGGGCATCTTCC ACCGT AGCTGGTAGTAA GTGGCGGGTCAGC CCTGG CD38

[0916] CAACTTACATTTA CGAGAGCCCGCCG CGCG CD38 gRNA2

[0917] CTGGGTGATTACC GGTGGTGCTGAGT

[0918] exon 1

[0919] ATGTGCCAGGTAT AGGGAGTCCCGGG (SEQ ID TGTGCTAAACAC CTCGGGGCTCCGC NO: GTTGTAGATATTA GGGCCGCTTTCAG 225) ACTCACTTAATCC GAGCAGCTGGCCT TCGTAACAATCCC TGGCACCGAGCGT ATGAAGTAGGTA GCCCGCGGGAGGC CTGCTACTATCCC GGGGGGGGGCGCT GGCTTTACATCTG GCTCGGTGGCTCTG AAGTACAGAGAG CTGCGTAGCCGGT GTTAAGTAACTTG GAACACTTGGCAC CCCCATGTCATCC CGATGCCCGCCTTC AGCAAGAACTAA TGGGCAAGGTGCC ATTTGAACCCAG CTGAGCCCAGCCC AGCTTAGCCACT CTCGCCGGGCTGC GATGCCTCTTGAG AGCCCACCCTCGG AGAAGGAGTCAG CGCGCTCAGCCCG ACTTAAGTTGAGT CTTCACCGCTTCAG CTTTAAAGGTGGT GGACGGAATAGAA TGACCAGGCATTT CTCGCAGATGCAG GTCAGAGTTAAG GGTGTCGCTGACAT AAAGAGAGGTAG TTTCAACTTTTTCT GACATCCTTTTCC GCGGTTTCCGCCCG AGGCAGAGGGCA CTGTCTCTGACCCG

[0920]

[0921] TTGTGTGCACACA AAAGTGCCCCCGG Docket No. CNTY-033-WO-01 CGTATAGAAGCA ACGGTTACAGAGG GGCAGCCCACCC ACACTTAAGTGGTT TCATGCTTTCCAG TGCAAAGCCTGTG GAAGCAAATGTG GTAGGGGAGGAGG GCTCAGGTGTAA GTGTAGAAGGGCC AGTGCCCGGTTG AAACCACGGAACT ATGAAGGGAGTT TAGTTTTATTCATT AGCGGAGGGAGT TATATAAAGCAGC ATAAGGATGTAC ACTCCGATTCTTTT TGTCTGCCCCCTT TGCGCGGCCTGAA AGGACACCTGCA ATGCATGTGACCA GAGGATTAAGGT GAGAAGTAATTAA GGCTGTTTCTCCC CAAAACAATGTCA TGGAGGTGGAGT ACTTCTAAAACCG GGGTGGGTCACT AGACATTACTTAG GCACAGGAGCCT ATGATAAGGCGCA ATAGTTGTTGGTC GCAACTCGGTGAA TTTTAAACTCTTA TCTGTACAAACCTT TTGGTGTAACCAG GGAAAAAAAACAC CCACGGAACTCT ATTAGTCTATGGGA GAGGCAAGGGGT CCTTCCAGTTTTCT TGGGGGTGGGAA CATGCTCCTTTCCA GGGAAACAGAGA GCTACTAACCTCTC AAAGGCAAGTGA CTAAAGGGAACAA AACAGAAGGGGA CCACTTTTTGGATT GGTGCAGTTTCAG TGATTCCCAGGCCT AACCCAGCCAGC CGCTTTCACCGGGA CTCTCTCTTGCTG AATTATCGTTGCTT CCTAGCCTCCTGC GTAAAACAGA CGGCCTCATCTTC GCCCAGCCAACC (SEQ ID NO: 210) CCGCCTGGAGCC CTATGGCCAACT GCGAGTTCAGCC CGG

[0922]

[0923] (SEQ ID NO: 209) Docket No. CNTY-033-WO-01

[0924] GAAGGACTTTAG CCGGCCACTCCAA ACATGGGGTTCG AAACCTGACCTGCT CATGTCTCAGATG CTGTGTCCTGGGCC GCCCTGAAGGTA TGTGAGCAGGGAA CTGATCCAGGCTC CACCCCCGATCTTC TGGTGCTCCTGGA TCCTGGTTGTCAGC AGGCAAGACTCA TGCCCCCACCTCCC GATTCTGCTCCAT TGGGCCCCAGGAC CTCCTCCCATCTC TACTCACTCCTCGG TGGGCGGGTCTCT TGCTCATAATCACC GGCATCTCTGGCC CCACGGCCCCAGG CATGAGGGTCAA ACCACGGCACCAA TCTGTGTGGAGG CCTGACCTGTCAGG GGACAAGCTCTG TGAAGTTCGCTGG AGCATGTGTGGG AGCTGGTGTGACT TCTGC TCTGAGGTTCCTC ACGGAGAGAACCA AGGGA TTCCATGCAGGG TCCAGCTCAACGTC AACAA CTGAGGTCTCCTG ACCTGTAAGTGCTG GAGAC CTCCTCCCCAGCT GGCCAGGATGCTG C TCCTGTCCGGCCC GGGTCCCTGAGGG TGTAGTCCTTCCC TGTAGGGGAGACA

[0925] (SEQ ID CTCCACTCCCTTC GGATGGGCTGGTG NO: CTCTTTTCTGCTC CTGGGGACATTTA

[0926] 226) ACACAGGAAGCC GTGTCCTGGAGGC CD33

[0927] CTGGAAGCTGCTT CTGGCTGAGTTCGG CD33 gRNA

[0928] CCTCAGACATGC GAGCCAGAAGGAC

[0929] exon 3

[0930] CGCTGCTGCTACT ATGAGCCCTGTCCC GAGTG GCTGCCCCTGCTG TTCTGCATTTCTGT GCCGG TGGGCAGGTGAG GGTTTCTGGCAGG GTTCT TGGCTGTGGGGA AGTAAGGGGAAAT AGAGT GAGGGGTTGTCG GCCTACCCTTATCT G GGCTGGGCCGAG CATCTCTACCCCCA CTGACCCTCGTTT ACTGAAGGAAATC

[0931] (SEQ ID CCCCACAGGGGC CTCTCTTCCTCTCC NO: CCTGGCTATGGAT TAGATGTTCCACAG

[0932] 241) CCAAATTTCTGGC AACCCAACAACTG TGCAAGTGCAGG GTATCTTTCCAGGA AGTCAGTGACGG GATGGCTCAGGTA TACAGGAGGGTT GGAAGGAGCCTCC TGTGCGTCCTCGT CCGCCTGGGGCTGT GCCCTGCACTTTC TACTGACATTGAGT TTCCATCCCATAC CTGTGTCAGGTTTG CCTACTACGACA GTCAGATCTGGACT AGAACTCCCCAG TTCAGAGTCAAAT TTCATGGTTACTG GTTCAGAGGCAAG GTTCCGGGAAGG GCCTGCAGTTAGA AGCCATTATATCC CACGGGTAGACAT AGGGACTCTCCA CAGGCACCTTGGA GTGGCCACAAAC AAAGGATATTTGG

[0933]

[0934] AAGCTAGATCAA GGATGACTAGCAA Docket No. CNTY-033-WO-01 GAAGTACAGGAG CTTCCCCCTTGCCC GAGACTCAGGGC ATCCAAATAATGCT AGATTCCGCCTCC CTTTGTCTCCCTCC TTGGGGATCCCA TGTCTCTGAATGTC GTAGGAACAACT TTGGGGTATTTTAT GCTCCCTGAGCAT TTTTAATTGATATG CGTAGACGCCAG TAATAATAGTACAT GAGGAGGGATAA ATTTATGGATGGCA TGGTTCATACTTC TAGTGATGTTTC C A TTTCGGATGGAG TACTAATAATGTAT AGAGGAAGTACC AGTAATCAGATCA AAATACAGTTAC GGGTAATAGCATA AAATCTCCCCAG TCCATCATCTTGAA CTCTCTGTGCATG CATTTATTATTTCA TGACAGGTGAGG TTGTTGTTGGGAAC CACAGGCTTCAG ATTCAATATCCCCT AAGTGGCCGCAA TTCTAGCTATTTGA GGGAAGTTCATG AGCTATCTATTATT GGTACTGCAGGG GTTAAGCATAGTC CAGGGCTGGGAT ATCCTACAGTGGTA GGGACCCTGGTA TAGAACACCAGAA CTGGGAGGGGTT CTTATTCTTCCTTT TAGGGGTAAAGC CCAGGTGTAATCTA CTGTCGTGCTTAG GTATCCTTTAACAA CGGGGGAGCTTG ATCTCTCTCCTTAT ACCAGAGGTTGA CATTGTTCCCCTAA TCTTCTCTCAGGC CCTTCCCAGCCCTT CCTCACCTGGACC ATTATT CTCCCTCCTGATT CTGCATCCCCTCT (SEQ ID NO: 212) TTCTCCTCACTAG ACTTGACCCACA GGCCCAAAATCC TCATCCCTGGCAC TCTAGAAC

[0935]

[0936] (SEQ ID NO: 211) Docket No. CNTY-033-WO-01

[0937] TGCTTCATAAGTG TCCAAAATTGAAC AAGGAGAAATAA ACATAGTTGGAAG AATATAGACAAG TAAAGCACTCCTCA TGAACGCTGAAA GCAAATGTAAAAG GATTTTGTCACCA AACAGAAAGTACA CCAGGCCTGCCCT ACAAACTGTCTCTC ACAAGAGCTCCT AGACCACAGTGCA GAAGGAAGCGCT ATCAAACTAAAAC AAACATGGAAAG TCAGGATTAAGAA GAACAACCGGTA ACTCACTCAAAAC CCAGCCACTGCA CGCTCAACTACATG AAAACATGCCAA GAAACTGAACAAC ATTGTAAACACC CTGCTCCTGAATGA ATTGAGGCCAGG CTACTGGGTACATA AAGAAACTGCAT ACGAAATGAAGGC CAACTAACGAGC AGAAGTAAAGATG AAAATAACCAGC TTCTTTGAAACCAA TAACATCATCATG CGAGAACAAAGAC ACAGGATCAAAT ACAACATACCAGA TCACACATAACA ATCTCTGGGACAC ATATTAACCTTAA ATTCAAAGCAGTG ATGTAAATAGGC TGTAGAGGAAAAT CGGCA TAAATGCTCCAAT TTATAGCACTAAAT CCACA TAAAAGACACAG GCCCACAAGAGAA CCACA

[0938] 5' end 5' end IL2 ACTGGCAAACTG AGCAGGAAAGATC CCGAT ofIL2 gRNAl GATAAAGAGTCA TAAAATTGACAGC AGACCCATCAGT CTAACATCACAATT (SEQ ID GTGCTGTATTCAG AAAAGAACTAGAG NO: GAAACCCATCTC AAACAAGAGCAAA 227) ACGTGCAGAGAC CACATTCAAAAGC ACACATAGGCTC TAGCAGAAGGCAA AAAATAAAGGGA GAAATAACTACAA TGGAGGAAGATC TCAGAGCAGAACT TACCAAGCAAAT GAAGGAGATAGAG GGAAAACAAAAA ACATACAAAAAAC AAGGCAGGGGTT CCTTCAAAAAATC GCAATCCTAGTCT AATGAATCCAGGA CTGATAAAACAG GCTGGTTTTTTGAA ACTTTAAACCAA AAGATCAACAAAA CAAAGATCAAAA TTGATAGACCACTA GAGACACAGAAG GCAAAACTAATAC GCCATTACATAAT AGAAGAGAGAAGA GGTAAAGGGATC ATCAAATAGACAC AATTCAACAAGA AATAAAAAATGAT AGAGTTAACTATC AAACGGGATATCA CTAAATATATATG CCACTGATCCCACA CACCCAATACAG GAAATACAAACTA GAGCACCTAGAT CCATCAAAGAATA TCATAAAGCAAG CTATAAACACCTCT

[0939]

[0940] TCCTTAGAGACCT ATGCAAATAAACT Docket No. CNTY-033-WO-01

[0941] ACAAAGAGACTT AGAAAATCTAGAA AGACTCCCACAC GAAATGGATAAAT AATAATAATGGG TCCTCGACACATAC AGACTTTAACAC ACCCTCCCAAGACT CCCACTGTCAAC AAACCAGGAAGAA ATTAGACAGATC GCTGAATCTCTGAA AATGAGACAGAA TAGACCAATAACA AATTAACAAGGA GGCTCTGAAATTG TATCCAGGAATTG AGGCAACAATTAA AACTCAACTCTGC CACCTTACCAACCA ACCAAGCGGACC ATAA TAATAGACATCTA CAGAACTCTCCA (SEQ ID NO: 214) CCCCAAATCAAC AGAATATACATTC TTTT

[0942] (SEQ ID NO: 213)

[0943] GGTGCCTGCCAC GAAACATAAAACTACATTCTGTCTCTAGGTGATTTTATCCGAGGTACTTCCG GCTCCCTCCAGTGT TTAGTAGAGACA CTGAGGGTAATCT GGGTTTCACCGTG GCAGGACTGAGGG TTAGCCAGGATG TAATTGCAGATGCT GTCTCGATCTCCT AGCTGTTAGCAGC GACCTCATGATCC AAACTATGCAAAA ACCCATCTTGGCC GCTGAGGACTGGC TCCCAAAGTGCT TTATAAAGCCAGA GGGATTACAGGC TCTGGGTGAGTCAT ATGAACCACTGC GTTTCCTGTCAACA GCCCGGCCGCAT TCCTCTGCTGGGCC ACCTC CGCTAGTTTTTAA CATAACACATGCA GTTCC AAACTTTTTGTAG ACCCCAAACTTCCA ACAAC

[0944] 3' end of

[0945] 3' end AGACAGATTCTTA TTACAAGTTCAAA CCTGG B2M

[0946] ofB2M CTATGTTGCCAAG GTTTCTAAGGGGAT gRNAl

[0947] GCTGGTCTCAAA AGCATTACAGTGT (SEQ ID CTCCTGGCCTCAA GTATGATATTGGAC NO: GAGATCCTCCAG TCAGACCTGAGTTT 228) TCTTCGGCCTCCC GAATCCTAATTCCA AAAAAGATGGGA CAAAAGAAATTGG TTACAGGCATGA AAAAGAGTCATAT GCCACCTCACCTG TGCTGACTTGACCC GCCTCTTTTTTTT TTTGTCACCATATC GTATATTACCTGA CATAAAATGGGAT TCTCAGGTATTCT AATTATTCCTATAT GCTATAGCAACA CATAAATTTACTTA GAAAGACGAAGA TTTATTCACTTAGT CAGAATCCTTAG CATTTGTTAAATAA CTGTCTGCAAGTG ATATGGAGTGTCTA

[0948]

[0949] TGCATGCCATTTT CTTTGTGCCGGGCA Docket No. CNTY-033-WO-01

[0950] CATCATCTGAAG CTCTTTTTAGGGTG AGTCAGCGAGTG GTTCTGAGAAGGG TCTTAGGTGGAGT GATGGCAATGAGA CTTGCAAAAGCA AGGGCTCTCTAAG GGCCCTGAGCCA ATGCAAGACTCCA AAGATTTGGATG GGCAACTGCTTTTA CAAATGACTTGTT CTTCCAGTGGTTCT AAGAAAGGGCTC TTATTTTCACAGCT TTCGAGACCGTG CATTAGAGCAAAT CCATTGCACTCCA TACCACAGCAGGG GCCCGGGCAAGA AGATACAGGTTGA AGAGTGAAACTC GTATCCCTTATCCG TGTTTCAAAAAA AAAAGCCTGGCAC AAAAAAGTGGGG CAGAAGTGTTTTAA GGCTCCTAGGAA ATTTTGGATTTTTT AAGAACAGTAAA TTTGATTTTTGGAA GGAGTGGGGGAT TATTTGTTAATTAT GAAGGACAGGGA CAGTTGAGCATCTC ATGGGAAGAAGC TAATGTGAAAATCT CAAGCGAGAGCA AAAATCCAAAATG TGATTTCCGAAGT CCCCAGTGACCCTT CCTACACTCAGCC TCCTTTGAGCATCA TGATCACACGGG TGTTGGTGCTCAAA AAGCTTTAGAAC AAGTTTGAGATGTT AAAGAACACACC GGAGCATTTAGGA TCAGAGTTTTTCC TTTCA TGCCTCAACACA AAGGAGCTGGGC (SEQ ID NO: 216) TTTGGTGCTCTTC ATCAGCCTGTCTT TGGCTAT

[0951]

[0952] (SEQ ID NO: 215)

[0953] (2) Nucleic acids encoding an inactivated cell surface receptor

[0954] In another general aspect, the invention relates to an isolated nucleic acid encoding an inactivated cell surface receptor useful for an invention according to embodiments of the application. It will be appreciated by those skilled in the art that the coding sequence of an inactivated cell surface receptor can be changed (e.g., replaced, deleted, inserted, etc.) without changing the amino acid sequence of the protein. Accordingly, it will be understood by those skilled in the art that nucleic acid sequences encoding an inactivated cell surface receptor of the application can be altered without changing the amino acid sequences of the proteins. Docket No. CNTY-033-WO-01

[0955] In certain embodiments, an isolated nucleic acid encodes any inactivated cell surface receptor described herein, such as that comprises a monoclonal antibodyspecific epitope, and / or a cytokine, such as an IL- 15 or IL-2, wherein the monoclonal antibody-specific epitope and the cytokine are optionally operably linked by an autoprotease peptide sequence.

[0956] In some embodiments, the isolated nucleic acid encodes an inactivated cell surface receptor comprising an epitope specifically recognized by an antibody, such as ibritumomab, tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, alemtuzumab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab. palivizumab. ranibizumab. tocilizumab, trastuzumab, vedolizumab. adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, avelumab. ofatumumab, panitumumab, or ustekinumab. In some embodiments, the isolated nucleic acid encodes an inactivated cell surface receptor comprising an epitope specifically recognized by cetuximab. In some embodiments, the isolated nucleic acid encodes an inactivated cell surface receptor comprising an epitope specifically recognized by trastuzumab. In some embodiments, the isolated nucleic acid encodes an inactivated cell surface receptor comprising an epitope specifically recognized by bevacizumab. In some embodiments, the isolated nucleic acid encodes an inactivated cell surface receptor comprising an epitope specifically recognized by avelumab. In some embodiments, the isolated nucleic acid encodes an inactivated cell surface receptor comprising an epitope specifically recognized by ipilimumab.

[0957] In certain embodiments, the isolated nucleic acid encodes an inactivated cell surface receptor having a truncated epithelial growth factor (tEGFR) variant.

[0958] Preferably, the inactivated cell surface receptor comprises an epitope specifically recognized by cetuximab, matuzumab, necitumumab or panitumumab, preferably cetuximab.

[0959] In certain embodiments, the isolated nucleic acid encodes an inactivated cell surface receptor having one or more epitopes of CD79b, such as an epitope specifically recognized by polatuzumab vedotin.

[0960] In certain embodiments, the isolated nucleic acid encodes an inactivated cell surface receptor having one or more epitopes of CD20, such as an epitope specifically recognized by rituximab. Docket No. CNTY-033-WO-01

[0961] In certain embodiments, the isolated nucleic acid encodes an inactivated cell surface receptor having one or more epitopes of Her 2 receptor, such as an epitope specifically recognized by trastuzumab

[0962] In certain embodiments, the autoprotease peptide sequence is porcine tesehovirus-1 2 A (P2A).

[0963] In certain embodiments, the truncated epithelial growth factor (tEGFR) variant consists of an amino acid sequence having at least 90%, 91%, 92%, 93%. 94%, 95%.

[0964] 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 71.

[0965] In certain embodiments, the monoclonal antibody -specific epitope specifically- recognized by polatuzumab vedotin consists of an amino acid sequence at least 90%. such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 78.

[0966] In certain embodiments, the monoclonal antibody-specific epitope specifically recognized by rituximab consists of an amino acid sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 80.

[0967] In certain embodiments, the monoclonal antibody-specific epitope specifically recognized by trastuzumab consists of an amino acid sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 82.

[0968] In certain embodiments, the IL-15 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 72.

[0969] In certain embodiments, the autoprotease peptide has an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 73.

[0970] In certain embodiments, the polynucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 74.

[0971] In a particular embodiment, the isolated nucleic acid encoding the inactivated cell surface receptor comprises a polynucleotide sequence at least 90%. such as at Docket No. CNTY-033-WO-01

[0972] least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 75. preferably the polynucleotide sequence of SEQ ID NO: 75.

[0973] In certain embodiments, the polynucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity7to the amino acid sequence of SEQ ID NO: 79.

[0974] In another general aspect, the application provides a vector comprising a polynucleotide sequence encoding an inactivated cell surface receptor useful for an invention according to embodiments of the application. Any vector known to those skilled in the art in view of the present disclosure can be used, such as a plasmid, a cosmid, a phage vector or a viral vector. In some embodiments, the vector is a recombinant expression vector such as a plasmid. The vector can include any element to establish a conventional function of an expression vector, for example, a promoter, ribosome binding element, terminator, enhancer, selection marker, and origin of replication. The promoter can be a constitutive, inducible, or repressible promoter. A number of expression vectors capable of delivering nucleic acids to a cell are known in the art and can be used herein for production of a inactivated cell surface receptor in the cell. Conventional cloning techniques or artificial gene synthesis can be used to generate a recombinant expression vector according to embodiments of the application.

[0975] In a particular aspect, the application provides a vector for targeted integration of an inactivated cell surface receptor useful for an invention according to embodiments of the application. In certain embodiments, the vector comprises an exogenous polynucleotide having, in the 5' to 3’ order, (a) a promoter; (b) a polynucleotide sequence encoding an inactivated cell surface receptor, such as an inactivated cell surface receptor comprising a truncated epithelial growth factor (tEGFR) variant and an interleukin 15 (IL- 15), wherein the tEGFR variant and IL- 15 are operably linked by an autoprotease peptide sequence, such as porcine teschovirus-1 2A (P2A), and (c) a terminator / polyadenylation signal.

[0976] In certain embodiments, the promoter is a CAG promoter. In certain embodiments, the CAG promoter comprises the polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 63. Other promoters can also be used, examples of which Docket No. CNTY-033-WO-01

[0977] include, but are not limited to, EFla, UBC, CMV, SV40, PGK1, and human beta actin.

[0978] In certain embodiments, the terminator / polyadenylation signal is a SV40 signal. In certain embodiments, the SV40 signal comprises the polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 64. Other terminator sequences can also be used, examples of which include, but are not limited to BGH, hGH, and PGK.

[0979] In certain embodiments, the polynucleotide sequence encoding an inactivated cell surface receptor comprises the polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 75.

[0980] In some embodiments, the vector further comprises a left homology arm and a right homology7arm flanking the exogenous polynucleotide.

[0981] In certain embodiments, the left homology arm comprises the polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%.

[0982] 98% or 100%, identical to SEQ ID NO: 84. In certain embodiments, the right homology arm comprises the polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 85

[0983] In a particular embodiment, the vector comprises a polynucleotide sequence at least 85%, such as at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 86, preferably the polynucleotide sequence of SEQ ID NO: 86.

[0984] (3) Nucleic acids encoding an HLA construct

[0985] In another general aspect, the invention relates to an isolated nucleic acid encoding an HLA construct useful for an invention according to embodiments of the application. It will be appreciated by those skilled in the art that the coding sequence of an HLA construct can be changed (e.g.. replaced, deleted, inserted, etc.) without changing the amino acid sequence of the protein. Accordingly, it will be understood by those skilled in the art that nucleic acid sequences encoding an HLA construct of the application can be altered without changing the amino acid sequences of the proteins.

[0986] In certain embodiments, the isolated nucleic acid encodes an HLA construct comprising a signal peptide, such as an HLA-G signal peptide, operably linked to an Docket No. CNTY-033-WO-01

[0987] HLA coding sequence, such as a coding sequence of a mature B2M, and / or a mature HLA-E. In some embodiments, the HLA coding sequence encodes the HLA-G and B2M, which are operably linked by a 4X GGGGS linker, and / or the B2M and HLA-E, which are operably linked by a 3X GGGGS linker. In a particular embodiment, the isolated nucleic acid encoding the HLA construct comprises a polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 67, preferably the polynucleotide sequence of SEQ ID NO: 67. In another embodiment, the isolated nucleic acid encoding the HLA construct comprises a polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 70, preferably the polynucleotide sequence of SEQ ID NO: 70.

[0988] In another general aspect, the application provides a vector comprising a polynucleotide sequence encoding a HLA construct useful for an invention according to embodiments of the application. Any vector known to those skilled in the art in view of the present disclosure can be used, such as a plasmid, a cosmid, a phage vector or a viral vector. In some embodiments, the vector is a recombinant expression vector such as a plasmid. The vector can include any element to establish a conventional function of an expression vector, for example, a promoter, ribosome binding element, terminator, enhancer, selection marker, and origin of replication. The promoter can be a constitutive, inducible, or repressible promoter. A number of expression vectors capable of delivering nucleic acids to a cell are known in the art and can be used herein for production of a HLA construct in the cell. Conventional cloning techniques or artificial gene synthesis can be used to generate a recombinant expression vector according to embodiments of the application.

[0989] In a particular aspect, the application provides vectors for targeted integration of a HLA construct useful for an invention according to embodiments of the application. In certain embodiments, the vector comprises an exogenous polynucleotide having, in the 5’ to 3’ order, (a) a promoter; (b) a polynucleotide sequence encoding an HLA construct; and (c) a terminator / polyadenylation signal.

[0990] In certain embodiments, the promoter is a CAG promoter. In certain embodiments, the CAG promoter comprises the polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 63. Other promoters can also be used, examples of which Docket No. CNTY-033-WO-01

[0991] include, but are not limited to, EFla, UBC, CMV, SV40, PGK1, and human beta actin.

[0992] In certain embodiments, the terminator / polyadenylation signal is a SV40 signal. In certain embodiments, the SV40 signal comprises the polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 64. Other terminator sequences can also be used, examples of which include, but are not limited to BGH, hGH, and PGK.

[0993] In certain embodiments, a polynucleotide sequence encoding a HLA construct comprises a signal peptide, such as a HLA-G signal peptide, a mature B2M, and a mature HLA-E, wherein the HLA-G and B2M are operably linked by a 4X GGGGS linker (SEQ ID NO: 31) and the B2M transgene and HLA-E are operably linked by a 3X GGGGS linker (SEQ ID NO: 25). In particular embodiments, the HLA construct comprises the polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 67, preferably the polynucleotide sequence of SEQ ID NO: 67. In another embodiment, the HLA construct comprises the polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 70, preferably the polynucleotide sequence of SEQ ID NO: 70.

[0994] In some embodiments, the vector further comprises a left homology arm and a right homology arm flanking the exogenous polynucleoti...

Claims

Docket No. CNTY-033-WO-01CLAIMSclaimed:

1. An induced pluripotent stem cell (iPSC) or a derivative cell thereof comprising:one or more exogenous polynucleotides encoding a chimeric antigen receptor (CAR) comprising (i) a costimulatory domain comprising CD30 or a fragment thereof, and (ii) one or more antigen binding domains each targeting a tumor antigen independently selected from the group consisting of a Nectin- 4 antigen, a CD19 antigen, and a CD22 antigen; andat least one of:(i) a deletion or reduced expression of one or more of B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5, RFXAP genes; (ii) an exogenous polynucleotide encoding a natural killer (NK) cell receptor immunoglobulin gamma Fc region receptor III (FcyRIII), cluster of differentiation 16 (CD16) and / or an NKG2D protein;(iii) a deletion or reduced expression of one or more of NKG2A or CD70, CD38, and CD33 genes;(iv) an exogeneous polynucleotide encoding a cytokine; (v) an exogenous polynucleotide encoding a safety switch; (vi) an exogeneous polynucleotide encoding a PSMA cell tracer; and (vii) an exogeneous polynucleotide encoding a membrane bound IL- 12 polypeptide.

2. The iPSC or the derivative cell thereof according to claim 1, wherein:(i) the CD30 comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 395; or(ii) the one or more exogenous polynucleotides encoding the CAR comprising the costimulatory domain comprising CD30 comprises nucleotides having at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 396.

3. The iPSC or the derivative cell thereof according to claim 1 or 2, wherein:Docket No. CNTY-033-WO-01(i) the CAR is a dual-targeting CAR, wherein the one or more antigen binding domains comprises a first antigen binding domain targeting Nectin-4, and wherein the one or more antigen binding domains comprises an additional antigen-binding domain that specifically binds to an antigen selected from the group consisting of CD70, Folate Receptor alpha. FSHR, mesothelin, and SLITRK6; or(ii) the CAR is a dual -targeting CAR, wherein the one or more antigen binding domains comprises a first antigen binding domain targeting CD 19, and wherein the one or more antigen binding domains comprises an additional antigen-binding domain that specifically binds to CD22.

4. The iPSC or the derivative cell thereof according to claim 1 or 2, wherein the one or more antigen binding domains comprises an anti-Nectin4 VHH domain.

5. The iPSC or the derivative cell thereof according to any one of claims 1-4, wherein the cytokine comprises an IL- 15.

6. The iPSC or derivative cell according to claim 5, further comprising an inactivated cell surface receptor that comprises a monoclonal antibody-specific epitope, wherein the inactivated cell surface receptor and the IL- 15 are operably linked by an autoprotease peptide.

7. The iPSC or the derivative cell thereof according to claim 5, wherein the IL-15 comprises an IL-15 and an IL-15 receptor alpha (IL-15Ra) fusion polypeptide.

8. The iPSC or the derivative cell thereof according to any one of claims 5-7, wherein the IL- 15 comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 72.

9. The iPSC or the derivative cell thereof according to any one of claims 1-8.comprising the deletion or reduced expression of one or more of B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes.Docket No. CNTY-033-WO-0110. The iPSC or the derivative cell thereof according to any one of claims 1-9, wherein the CD 16 is a CD 16 variant protein.

11. The iPSC or the derivative cell thereof according to claim 10, wherein the CD16 variant protein is a high affinity CD16 variant.

12. The iPSC or the derivative cell thereof according to claim 10 or 11, wherein the CD 16 variant protein is a non-cleavable CD 16 variant.

13. The iPSC or the derivative cell thereof according to any one of claims 10-12, wherein the CD16 variant protein comprises wild-type CD16 having one or more amino acid substitutions selected from the group consisting of Fl 58V, F176V, S197P, D205A, S219A, T220A.

14. The iPSC or the derivative cell thereof according to any one of claims 10-13, wherein the CD 16 variant protein comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOS: 187 and 188.

15. The iPSC or the derivative cell thereof according to any one of claims 1-14.comprising an exogenous polynucleotide encoding the CD16 protein and the NKG2D protein, wherein the CD 16 protein and the NKG2D protein are operably linked by an autoprotease peptide.

16. The iPSC or the derivative cell thereof according to claim 15, wherein the NKG2D protein is a wildtype NKG2D protein.

17. The iPSC or the derivative cell thereof according to claim 15, wherein the NKG2D protein comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 190.

18. The iPSC or the derivative cell thereof according to any one of claims 15-17, wherein the autoprotease peptide is selected from the group consisting of aDocket No. CNTY-033-WO-01porcine teschovirus-1 2A (P2A) peptide, a foot-and-mouth disease virus 2A (F2A) peptide, an Equine Rhinitis A Virus (ERAV) 2A (E2A) peptide, a Thosea asigna virus 2A (T2A) peptide, a cytoplasmic polyhedrosis virus 2A (BmCPV2A) peptide, and a Flacherie Virus 2A (BmIFV2A) peptide.

19. The iPSC or the derivative cell thereof according to claim 18, wherein the autoprotease peptide is a P2A peptide comprising amino acids having at least 90% sequence identity to SEQ ID NO: 192.

20. The iPSC or the derivative cell thereof according to any one of claims 15-19, wherein the exogenous polynucleotide encoding the CD 16 protein and the NKG2D protein comprises polynucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%. 99% or 100% sequence identity to SEQ ID NO: 193.

21. The iPSC or the derivative cell thereof according to any one of claims 1-20, wherein one or more of the exogenous polynucleotides are integrated at one or more loci on the chromosome of the cell selected from the group consisting of AAVS1, CLYBL, CCR5, ROSA26, collagen, HTRP, Hl 1, GAPDH, RUNX1, B2M, TAPI, TAP2, Tapasin, NLRC5, RFXANK, CIITA, RFX5, RFXAP, TCR a or b constant region, NKG2A, NKG2D, CD33, CD38, CD70, TRAC, CIS, CBL-B, SOCS2, PD1, CTLA4. LAG3. TIM3, and TIGIT genes, provided at least one of the exogenous polynucleotides is integrated at a locus of a gene selected from the group consisting of B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes to thereby result in a deletion or reduced expression of the gene.

22. The iPSC or the derivative cell thereof according to any one of claims 1-21, wherein one or more of the exogenous polynucleotides are integrated at the loci of the AAVS1 and B2M genes.

23. The iPSC or the derivative cell thereof according to any one of claims 1-22 having a deletion or reduced expression of one or more of B2M or CIITA genes.Docket No. CNTY-033-WO-0124. The iPSC or the derivative cell thereof according to claim 23, comprising the deletion or reduced expression of B2M and CIITA genes.

25. The iPSC of any one of claims 1-24, wherein the iPSC is reprogrammed from whole peripheral blood mononuclear cells (PBMCs).

26. The iPSC of any one of claims 1-25, wherein the iPSC is derived from a reprogrammed T-cell.

27. The iPSC or the derivative cell thereof according to any one of claims 1-26, wherein the CAR comprises:(i) a signal peptide;(ii) an extracellular domain comprising a binding domain that specifically binds the Nectin4 antigen, the CD 19 antigen, and / or the CD22 antigen; (iii) a hinge region;(iv) a transmembrane domain;(v) an intracellular signaling domain; and / or(vi) the costimulatory domain comprising CD30 or a fragment thereof.

28. The iPSC or the derivative cell thereof according to claim 27, wherein the extracellular domain comprises a VHH single domain antibody that specifically binds the Nectin4 antigen.

29. The iPSC or the derivative cell thereof according to claim 27 or 28, wherein the extracellular domain comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 105-130, 379-381, and 384-388.

30. The iPSC or the derivative cell thereof according to claim 27 or 28 wherein (i) the extracellular domain is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 131-156, 382, 383. and 389-392; or (ii) the CAR is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%,Docket No. CNTY-033-WO-0196%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 171- 184, and 394.

31. The iPSC or the derivative cell thereof according to any one of claims 2-30, wherein the additional CAR comprises:(i) a signal peptide;(ii) an additional extracellular domain comprising a binding domain that specifically binds to an antigen selected from the group consisting of CD70, Folate Receptor alpha, FSHR, mesothelin, and SLITRK6; (iii) a hinge region;(iv) a transmembrane domain;(v) an intracellular signaling domain; and(vi) a co-stimulatory domain.

32. The iPSC or the derivative cell thereof according to claim 31. wherein the additional extracellular domain comprises a VHH or an scFv that specifically binds to an antigen selected from the group consisting of CD70, Folate Receptor alpha, FSHR, mesothelin, and SLITRK6.

33. The iPSC or the derivative cell thereof according to any one of claims 27-32.wherein the signal peptide comprises a GMCSFR signal peptide or a MARS signal peptide.

34. The iPSC or the derivative cell thereof according to any one of claims 27-33, wherein the hinge region for each of the CAR and the additional CAR are independently selected from the group consisting of a CD28 hinge region, an IgG4 hinge region, and a CD8 hinge region.

35. The iPSC or the derivative cell thereof according to any one of claims 27-34, wherein the transmembrane domain for each of the CAR and the additional CAR are independently selected from the group consisting of a CD28 transmembrane domain and a CD8 transmembrane domain.Docket No. CNTY-033-WO-0136. The iPSC or the derivative cell thereof according to any one of claims 27-35, wherein the intracellular signaling domain comprises a CD3^ intracellular domain.

37. The iPSC or the derivative cell thereof according to any one of claims 27-36, wherein the co-stimulatory domain for each of the CAR and the additional CAR are independently selected from the group consisting of a CD28 signaling domain, a 41BB signaling domain, and a DAP10 signaling domain.

38. The iPSC or the derivative cell thereof according to any one of claims 27-37, wherein in the CAR:(i) the signal peptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ IDNOs: 1, 97, or 98;(ii) the extracellular domain comprises amino acids having at least 90%, 91%, 92%, 93%. 94%. 95%. 96%. 97%. 98%. 99% or 100% sequence identity to SEQ ID NOs: 105-130, 379-381, and 384-388, or the extracellular domain is encoded by a polynucleotide having at least 90%, 91%, 92%. 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 131-156, 382, 383. and 389-392; (iii) the hinge region comprises amino acids having at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 21 or 96;(iv) the transmembrane domain comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 23 or 24;(v) the intracellular signaling domain comprises amino acids having at least 90%, 91%, 92%. 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:

6. or the intracellular signaling domain is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 101; andDocket No. CNTY-033-WO-01(vi) the costimulatory domain comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%. 98%. 99% or 100% sequence identity to SEQ ID NO: 395.

39. The iPSC or the derivative cell thereof according to claim 38, wherein in the CAR:(i) the signal peptide comprises amino acids having the sequence of SEQ ID NO: 1, 97, or 98;(ii) the extracellular domain comprises amino acids having the sequence of one of SEQ ID NOs: 105-130;(iii) the hinge region comprises amino acids having the sequence of SEQ ID NO: 21 or 96;(iv) the transmembrane domain comprises amino acids having the sequence of SEQ ID NO: 23 or 24;(v) the intracellular signaling domain comprises amino acids having the sequence of SEQ ID NO: 6, or the intracellular signaling domain is encoded by the polynucleotide having the sequence of SEQ ID NO: 101; and(vi) the co-stimulatory domain comprises amino acids having the sequence of SEQ ID NO: 395.

40. The iPSC or a derivative cell thereof according to any one of claims 1-39, comprising an exogenous polynucleotide encoding a safety switch.

41. The iPSC or a derivative cell thereof according to claim 40, wherein the safety switch comprises an exogenous polynucleotide encoding an inactivated cell surface receptor that comprises a monoclonal antibody-specific epitope.

42. The iPSC or the derivative cell thereof according to claim 41, wherein the inactivated cell surface receptor is selected from the group of monoclonal antibody specific epitopes selected from epitopes specifically recognized by ibritumomab, tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, alemtuzumab, bevacizumab, certolizumab pegol, daclizumab, eculizumab.Docket No. CNTY-033-WO-01efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, polatuzumab vedotin, ranibizumab, tocilizumab. trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, avelumab, ofatumumab, panitumumab, and ustekinumab.

43. The iPSC or the derivative cell thereof according to claim 42, wherein the inactivated cell surface receptor is a truncated epithelial growth factor (tEGFR) variant.

44. The iPSC or the derivative cell thereof according to claim 43, wherein the tEGFR variant consists of amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 71.

45. The iPSC or the derivative cell thereof according to any one of claims 40-44, wherein the safety switch comprises an intracellular domain having a herpes simplex virus thymidine kinase (HSV-TK).

46. The iPSC or the derivative cell thereof according to any one of claims 1-45 comprising the exogeneous polynucleotide encoding the PSMA cell tracer, wherein the PSMA cell tracer comprises an extracellular domain comprising a PSMA extracellular domain or fragment thereof.

47. The iPSC or the derivative cell thereof according to claim 46, comprising a combined artificial cell death / reporter system polypeptide comprising an intracellular domain having a herpes simplex virus thymidine kinase (HSV- TK) and a linker, a transmembrane region, and an extracellular domain comprising the PSMA extracellular domain or fragment thereof.

48. The iPSC or the derivative cell thereof according to any one of claims 45-47, wherein the HSV-TK comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 229 or 230.Docket No. CNTY-033-WO-0149. The iPSC or the derivative cell thereof according to claim 47, wherein the combined artificial cell death / reporter system polypeptide comprises the HSV- TK fused to a truncated variant PSMA polypeptide via the linker.

50. The iPSC or the derivative cell thereof according to claim 49, wherein the truncated variant PSMA polypeptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 231.

51. The iPSC or the derivative cell thereof according to any one of claims 47-50, wherein the linker comprises an autoprotease peptide sequence selected from the group consisting of P2A peptide sequence, T2A peptide sequence, E2A peptide sequence, and F2A peptide sequence.

52. The iPSC or the derivative cell thereof according to any one of claims 47-51, wherein the artificial cell death / reporter system polypeptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 232.

53. The iPSC or the derivative cell thereof according to claim 52. wherein the artificial cell death / reporter system polypeptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOS: 233-235.

54. The iPSC or the derivative cell thereof according to any one of claims 47-53, wherein the artificial cell death / reporter system polypeptide is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOS: 236-238.55.

56. The iPSC or the derivative cell thereof according to any one of claims 1- Error! Reference source not found, wherein:Docket No. CNTY-033-WO-01(i) the one or more exogenous polynucleotides encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain targeting aNectin4 antigen comprises nucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more selected from the group consisting of SEQ ID NOs: 171-184 and 396;(ii) the exogenous polynucleotide encoding an NK cell receptor immunoglobulin gamma Fc region receptor III (FcyRIII, cluster of differentiation 16 (CD16)) and / or an NKG2D protein comprises nucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more of SEQ ID NOs: 185, 189, and 191;(iii) the exogeneous polynucleotide encoding a cytokine comprises nucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 239; (iv)the exogenous polynucleotide encoding a safety switch comprises nucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more of SEQ ID NO: 236-238; and / or(v) the exogeneous polynucleotide encodes a PSMA cell tracer, and the PSMA cell tracer comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 231.

57. The iPSC or the derivative cell thereof according to any one of claims 1-56 wherein:(i) the one or more exogenous polynucleotides encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain targeting aNectin4 antigen comprises nucleotides having a sequence selected from the group consisting of SEQ ID NOs: 171-184 and 396; (ii) the exogenous polynucleotide encoding an NK cell receptor immunoglobulin gamma Fc region receptor III (FcyRIII, cluster of differentiation 16 (CD 16)) and / or anNKG2D protein comprises nucleotides having a sequence of SEQ ID NO: 185, 189, or 191;Docket No. CNTY-033-WO-01(iii) the exogeneous polynucleotide encoding the cytokine comprises nucleotides having a sequence of SEQ ID NO: 239; and / or (iv)the exogenous polynucleotide encoding the safety switch comprises nucleotides having a sequence of one of SEQ ID NOs: 236-238.

58. The iPSC or the derivative cell thereof according to claim 56 or 57, wherein the exogenous polynucleotides are integrated into a gene locus independently selected from the group consisting of an AAVS1 locus, a B2M locus, a CIITA locus, a CCR5 locus, a CD70 locus, a CLYBL locus, an NKG2A locus, an NKG2D locus, a CD33 locus, a CD38 locus, a TRAC locus, a TRBC1 locus, a ROSA26 locus, an HTRP locus, a GAPDH locus, a RUNX1 locus, a TAP1 locus, a TAP2 locus, a TAPBP locus, an NLRC5 locus, a RFXANK locus, a RFX5 locus, a RFXAP locus, a CISH locus, a CBLB locus, a SOCS2 locus, a PD1 locus, a CTLA4 locus, a LAG3 locus, a TIM3 locus, and aTIGIT locus.

59. The iPSC or the derivative cell thereof according to claim 58, wherein:(i) the one or more exogenous polynucleotides encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain targeting a Nectin4 antigen is integrated at a locus of the AAVS1 gene; (ii) the exogenous polynucleotide encoding an NK cell receptor immunoglobulin gamma Fc region receptor III (FcyRIII, cluster of differentiation 16 (CD16)) and / or anNKG2D is integrated at a locus of the CD70 gene;(iii) the exogeneous polynucleotide encoding the cytokine is integrated at the locus of the NKG2A gene;(iv)the exogenous polynucleotide encoding a safety switch is integrated at the locus of the CLYBL gene; and(v) there is a deletion or reduced expression of the CIITA gene.

60. The derivative cell of any one of claims 1-59, wherein the derivative cell is a natural killer (NK) cell or a T cell.

61. The derivative cell of claim 60, wherein the derivative cell is a T cell.Docket No. CNTY-033-WO-0162. The derivative cell of claim 61, wherein the T cell is a gamma delta T cell.

63. The derivative cell of claim 62, wherein the T cell is a gamma delta Vy9 / V51 T cell.

64. A composition comprising the derivative cell according to any one of claims 1- 63.

65. The composition according to claim 64, further comprising or being used in combination with, one or more therapeutic agents selected from the group consisting of a peptide, a cytokine, a checkpoint inhibitor, a mitogen, a growth factor, a small RNA, a dsRNA (double stranded RNA), siRNA, oligonucleotide, mononuclear blood cells, a vector comprising one or more polynucleic acids of interest, an antibody, a chemotherapeutic agent or a radioactive moiety, or an immunomodulatory drug (IMiD).

66. A CD34+ hematopoietic progenitor cell (HPC) derived from an induced pluripotent stem cell (iPSC) of any one of claims 1-59.

67. The CD34+ HPC according to claim 66. wherein:(i) the CD30 comprises amino acids having at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 395; or(ii) the one or more exogenous polynucleotides encoding the CAR comprising the costimulatory domain comprising CD30 comprises nucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 396.

68. The CD34+ HPC according to any one of claims 66-67, wherein one or more of the exogenous polynucleotides are integrated at one or more loci on the chromosome of the cell independently selected from the group consisting of AAVS1, CLYBL, CCR5, ROSA26, collagen, HTRP, Hl 1, GAPDH, RUNX1, B2M. TAPI, TAP2, Tapasin. NLRC5, RFXANK, CIITA, RFX5, RFXAP, TCR a or b constant region, NKG2A, NKG2D, CD33, CD38, CIS, CBL-B,Docket No. CNTY-033-WO-01SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT genes, provided at least one of the exogenous polynucleotides is integrated at a locus of a gene selected from the group consisting of B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes to thereby result in a deletion or reduced expression of the gene.

69. The CD34+ HPC according to claim 68, wherein one or more of the exogenous polynucleotides are integrated at the loci of the AAVS1 and B2M genes.

70. The CD34+ HPC according to any one of claims 66-69 having a deletion or reduced expression of one or more of B2M or CIITA genes.

71. The CD34+ HPC according to any one of claims 66-70, wherein the CAR comprises:(i) a signal peptide;(ii) an extracellular domain comprising a binding domain that specifically binds the Nectin4 antigen, the CD19 antigen, and / or the CD22 antigen; (iii) a hinge region;(iv) a transmembrane domain;(v) an intracellular signaling domain; and(vi) the co-stimulatory domain comprising CD30 or a fragment thereof.

72. The CD34+ HPC according to claim 71, wherein the extracellular domain comprises a VHH single domain antibody that specifically binds the Nectin4 antigen.

73. The CD34+ HPC according to claim 72, wherein the extracellular domain comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 105-130.

74. The CD34+ HPC according to any one of claims 66-73 having an additional CAR comprising:(i) a signal peptide;Docket No. CNTY-033-WO-01(ii) an additional extracellular domain comprising a binding domain that specifically binds an antigen selected from the group consisting of CD70, Folate Receptor alpha, FSHR, mesothelin, and SLITRK6; (iii) a hinge region;(iv) a transmembrane domain;(v) an intracellular signaling domain; and(vi) a co-stimulatory domain, such as a co-stimulatory domain comprising a CD28 signaling domain.

75. The CD34+ HPC according to claim 74, wherein the additional extracellular domain comprises a VHH that specifically binds the antigen selected from the group consisting of CD70, Folate Receptor alpha, FSHR, mesothelin, and SLITRK6.

76. The CD34+ HPC according to any one of claims 66-75, comprising an additional exogenous polynucleotide encoding a CD 16 protein and an NKG2D protein, wherein the CD 16 protein and the NKG2D protein are operably linked by an autoprotease peptide.

77. The CD34+ HPC according to claim 76. wherein the CD16 protein is a CD16 variant protein.

78. The CD34+ HPC according to claim 77, wherein the CD16 variant is a high affinity CD 16 variant.

79. The CD34+ HPC according to claim 77 or 78, wherein the CD16 variant is a non-cleavable CD 16 variant.

80. The CD34+ HPC according to any one of claims 77-79, wherein the CD16 variant comprises one or more amino acid substitutions selected from the group consisting of Fl 58V, Fl 76V, S197P, D205A, S219A, T220A, and any combination thereof.

81. A chimeric antigen receptor (CAR) polypeptide comprising (i) an extracellular domain comprising an antigen binding domain that specifically binds toDocket No. CNTY-033-WO-01Nectin4, CD19, and / or CD22, and (ii) a costimulatory domain comprising CD30.

82. The CAR according to claim 81, wherein the CAR is a dual -targeting CAR, and wherein the extracellular domain comprises an additional antigen-binding domain that specifically binds to an antigen selected from the group consisting of CD70, Folate Receptor alpha, FSHR, mesothelin, and SLITRK6.

83. The CAR according to any one of claims 81-82, wherein the CAR comprises:(i) a signal peptide;(ii) the extracellular domain comprising the antigen binding domain that specifically binds to the Nectin4 antigen, the CD19 antigen, and / or the CD22 antigen;(iii) a hinge region;(iv) one or more transmembrane domains;(v) an intracellular signaling domain; and / or(vi) the co-stimulatory domain comprising CD30 or a fragment thereof.

84. The CAR according to any one of claims 81-83. wherein the extracellular domain comprises a VHH single domain antibody that specifically binds to the Nectin4 antigen.

85. The CAR according to any one of claims 81-83. wherein the extracellular domain comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 105-130, 379-381, and 384-388.

86. The CAR according to any one of claims 81-83, wherein (i) the extracellular domain is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 131-156; or (ii) the CAR is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 171-184.Docket No. CNTY-033-WO-0187. The CAR according to any one of claims 83-86, wherein the signal peptide comprises a GMCSFR signal peptide or a MARS signal peptide.

88. The CAR according to any one of claims 83-87, wherein the hinge region for each of the CAR and the additional CAR are independently selected from the group consisting of a CD28 hinge region, an IgG4 hinge region, and a CD8 hinge region.

89. The CAR according to any one of claims 83-88, wherein the transmembrane domain for each of the CAR and the additional CAR are independently selected from the group consisting of a CD28 transmembrane domain and a CD8 transmembrane domain.

90. The CAR according to any one of claims 83-89, wherein the intracellular signaling domain comprises a CD3q intracellular domain.

91. The CAR according to any one of claims 83-90, wherein the co-stimulatory domain for each of the CAR and the additional CAR are independently selected from the group consisting of a CD28 signaling domain, a 4 IBB signaling domain, and a DAP 10 signaling domain.

92. The CAR according to any one of claims 83-91, wherein in the CAR:(i) the signal peptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%. 98%, 99% or 100% sequence identity to SEQ ID NOs: 1, 97, or 98;(ii) the extracellular domain comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 105-130, or the extracellular domain is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 131- 156;(iii) the hinge region comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%. 98%, 99% or 100% sequence identity to SEQ ID NO: 21 or 96;Docket No. CNTY-033-WO-01(iv) the transmembrane domain comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%. 98%. 99% or 100% sequence identity to SEQ ID NO: 23 or 24;(v) the intracellular signaling domain comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:

6. or the intracellular signaling domain is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 101; and(vi) the co-stimulatory domain comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%. 96%. 97%. 98%. 99% or 100% sequence identity to SEQ ID NO: 395.

93. The CAR according to claim 92, wherein in the CAR:(i) the signal peptide comprises amino acids having the sequence of SEQ ID NO: 1, 97, or 98;(ii) the extracellular domain comprises amino acids having the sequence of one of SEQ ID NOs: 105-130;(iii) the hinge region comprises amino acids having the sequence of SEQ ID NO: 21 or 96:(iv) the transmembrane domain comprises amino acids having the sequence of SEQ ID NO: 23 or 24;(v) the intracellular signaling domain comprises amino acids having the sequence of SEQ ID NO: 6, or the intracellular signaling domain is encoded by the polynucleotide of SEQ ID NO: 101; and(vi)the co-stimulatory domain comprises amino acids having the sequence of SEQ ID NO: 395.

94. A pharmaceutical composition comprising the derivative cell according to any one of claims 60-63.

95. A method of treating cancer in a subject in need thereof, comprising administering the derivative cell according to any one of claims 1-63, or the composition according claim 64, 65, or 94, to a subject in need thereof.Docket No. CNTY-033-WO-0196. The method of treatment according to claim 95. wherein the cancer is selected from the group consisting of leukemias, such as AML, CML, ALL and CLL, lymphomas, such as Hodgkin lymphoma, non-Hodgkin lymphoma and multiple myeloma, and solid cancers such as sarcomas, skin cancer, melanoma, bladder cancer, brain cancer, breast cancer, uterus cancer, ovarian cancer, prostate cancer, lung cancer, colorectal cancer, cervical cancer, liver cancer, head and neck cancer, esophageal cancer, pancreatic cancer, renal cancer, adrenal cancer, stomach cancer, testicular cancer, cancer of the gall bladder and biliary tracts, thyroid cancer, thymus cancer, cancer of bone, and cerebral cancer, as well as cancer of unknow n primary (CUP).

97. The method according to claim 96, wherein the cancer is selected from the group consisting of bladder, breast, lung, pancreatic, ovarian, head & neck, and esophageal cancers.

98. The method according to any one of claims 95-97, wherein the subject has minimal residual disease (MRD) after an initial cancer treatment.

99. The method according to any one of claims 95-98, wherein the subject has no minimal residual disease (MRD) after one or more cancer treatments or repeated dosing.

100. The method according to any one of claims 95-99, further comprising administering to the subject a therapeutic agent selected from the group consisting of ibritumomab, tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, alemtuzumab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, polatuzumab vedotin, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, avelumab, ofatumumab, panitumumab, and ustekinumab.Docket No. CNTY-033-WO-01101. The method according to any one of claims 95-99, further comprising administering to the subject a therapeutic agent, wherein the therapeutic agent is avelumab.

102. The method according to claim 100 or 101, wherein the cell and the therapeutic agent are administered concurrently.

103. The method according to claim 100 or 101, wherein the cell and the therapeutic agent are administered sequentially.

104. A method of manufacturing the derivative cell of any one of claims 60- 63, comprising differentiating the iPSC according to any one of claims 1-59 under conditions for cell differentiation to thereby obtain the derivative cell.

105. The method according to claim 104, wherein the iPSC is obtained by genetically engineering an unmodified iPSC, wherein the genetic engineering comprises targeted editing of the genome of the iPSC.

106. The method according to claim 105, wherein the targeted editing comprises deletion, insertion, or in / del carried out by CRISPR, ZFN, TALEN, homing nuclease, homology recombination, or any other functional variation of these methods.

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