Compositions and methods for enhancing engineered t cell therapy

Abstract

Aspects of the present disclosure include a modified T cell comprising: a) an antigen binding moiety expressed on the surface of the cell, wherein the antigen binding moiety comprises an affinity binding domain specific for a disease-associated antigen; b) at least one modification for modulating the disease microenvironment and / or at least one modification for modulating cytokine activity; and c) at least one allogeneic escape modification and / or at least one modification for modulating T cell metabolism.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefits of U.S. Provisional Application No. 63 / 627746, filed January 31, 2024, and U.S. Provisional Application No. 63 / 738411, filed December 23, 2024. The entire contents of the above applications are hereby incorporated herein by reference. Technical Field

[0003] This disclosure relates in general to cell therapies using modified T cells. Background Technology

[0004] Adoptive cell therapy has undergone nearly continuous iteration over more than three decades (30 years), from early focus on basic lymphokine activation and / or tumor infiltration to more recent strategies involving engineered immune cells expressing genetically engineered antigen receptors such as chimeric antigen receptors (CARs). While there have been some hints and indications of the curative potential of these approaches along the way, much work remains to be done. Specifically, the success of CAR-T lymphocytes in eradicating tumors depends on the persistence and effector function of CAR-T cells, but an overabundance of either can trigger graft-versus-host (GvH) effects in patients. Furthermore, while adoptive transfer of CAR-expressing T cells has shown some success in treating hematologic malignancies, it has demonstrated limited efficacy in other cancer types, particularly solid tumors. Solid tumors present unique challenges compared to hematologic disorders, including but not limited to highly immunosuppressive and metabolically challenging tumor microenvironments.

[0005] Gamma-delta (γδ) T cells are thymus-derived lymphocytes that differ from αβ T cells in their activation mechanisms, function, and anatomical distribution. Specifically, while αβ T cells function specifically in adaptive immunity, γδ T cells are innate-like immune cells that recognize malignant cells in an MHC-independent manner through a series of activating receptors, similar to NK cells (Welsh et al.). Immunol Rev. 1997; 159: 79-93). Therefore, and compared to αβ T cells, γδ T cells can potentially be used in allogeneic settings without inducing the risk of graft-versus-host disease (GvHD). Furthermore, recent studies have shown that engineered γδ-T cells produce fewer pro-inflammatory cytokines than αβ-T cells, thereby reducing the risk of CRS in patients (Harrer et al., 1997; 159: 79-93). BMC Cancer 2017; 17(1): 551).

[0006] Despite the rapid and robust development of CAR-T therapy, the optimal parameters for CAR-T cell-target interactions leading to in vivo and human efficacy remain unclear. Given the differences in the mechanisms of action and function between αβ T cells and γδ T cells, CAR function and efficacy exhibited in an αβ T cell context cannot predict CAR function and efficacy in a γδ T cell context. Therefore, the actual conversion of γδ T cells by CAR T therapy is, at best, uncertain.

[0007] In the context of solid cancers and hematologic cancers, autoimmune diseases and / or pathogenic diseases, cell therapy using γδ T cells still faces many pressing challenges, including improving disease cell targeting, enhancing efficacy, preventing on-target / off-tumor immune reactivity and reducing side effects (e.g., toxicity from secreted cytokines), and improving the activity, survival and / or expansion of host γδ T cells after administration. Summary of the Invention

[0008] This disclosure provides modified T cells (e.g., γδ T cells) comprising an antigen recognition portion (e.g., CAR) and one or more modifications that improve the activity, survival, and / or expansion of the modified T cells, and / or enhance efficacy and / or reduce side effects when the modified T cells are used as an immunocellular therapy. In embodiments, combinations of T cell modifications have been shown herein to significantly enhance the cytotoxicity of T cells and the potency of the corresponding T cell therapy.

[0009] In one aspect, this disclosure provides a modified T cell comprising: a) an antigen recognition portion expressed on the cell surface, wherein the antigen recognition portion includes an affinity-binding domain specifically for disease-associated antigens; b) at least one modification for modulating the disease microenvironment and / or at least one modification for modulating cytokine activity; and c) at least one alloevasion modification and / or at least one modification for modulating T cell metabolism. In another aspect, this disclosure provides a modified T cell comprising: a) an antigen recognition portion expressed on the cell surface, wherein the antigen recognition portion includes means for specifically binding to disease-associated antigens; b) at least one modification for modulating the disease microenvironment and / or at least one modification for modulating cytokine activity; and c) at least one alloevasion modification and / or at least one modification for modulating T cell metabolism.

[0010] In this embodiment, the modified T cells are γδ T cells. Specifically, the γδ T cells are δ1, δ2, δ3, or δ4 γδ T cells, preferably δ2. -γδ T cells, more preferably δ1 γδ T cells.

[0011] In the embodiments, at least one modification for regulating the disease microenvironment includes disruption of the CBL-B gene, disruption of the REGNASE-1 gene, disruption of the roquin gene, disruption of the TGF-β receptor type 2 (TGFβR2) gene, expression of dominant-negative TGF-β receptor type 2 (dnTGFβR2), or any combination thereof. In the embodiments, at least one modification for regulating the disease microenvironment includes disruption of the TGFβR2 gene or expression of dnTGFβR2.

[0012] In the embodiments, at least one modification for regulating cytokine activity includes the expression of membrane-bound IL-12, disruption of the CISH gene, expression of a cytokine switching receptor, or any combination thereof. In the embodiments, at least one modification for regulating cytokine activity includes the expression of membrane-bound IL-12.

[0013] In the embodiments, at least one allogeneic escape modification includes disruption of the ICAM-1 gene, disruption of the CD58 gene, disruption of the Fas gene, expression of a dominant-negative Fas receptor (dnFas), expression of a chimeric antigen receptor (CAR) that binds to CD70, or any combination thereof. In the embodiments, at least one allogeneic escape modification includes disruption of the Fas gene or expression of dnFas.

[0014] In one embodiment, at least one modification for regulating T cell metabolism includes disruption of the RASA2 gene, disruption of the MED12 gene, or any combination thereof. In another embodiment, the modified T cells contain dnTGFβR2. In yet another embodiment, at least one modification for regulating T cell metabolism includes disruption of the MED12 gene.

[0015] In the implementation scheme, the modified T cells contain disruption of the CISH gene, disruption of the CBL-B gene, disruption of the ICAM-1 gene, disruption of the CD58 gene, disruption of the Roquin-1 gene, disruption of the Regnase-1 gene, disruption of the TGFβR2 gene, disruption of the RASA2 gene, disruption of the MED12 gene, or a combination thereof.

[0016] In one embodiment, the modified T cells comprise at least one modification for regulating cytokine activity, including the expression of membrane-bound IL-12; and at least one modification for regulating T cell metabolism, including disruption of the MED12 gene. In another embodiment, the modified T cells further comprise disruption of the Fas gene or expression of dnFas. In yet another embodiment, the modified T cells further comprise disruption of the TGFβR2 gene or expression of dnTGFβR2.

[0017] In one embodiment, the modified T cells comprise at least one modification for regulating the disease microenvironment, including disruption of the TGFβR2 gene or expression of dnTGFβR2; and at least one modification for regulating T cell metabolism, including disruption of the MED12 gene. In another embodiment, the modified T cells further comprise disruption of the Fas gene or expression of dnFas. In yet another embodiment, the modified T cells comprise at least one modification for regulating cytokine activity, including expression of membrane-bound IL-12.

[0018] In one embodiment, the modified T cells contain at least one heterologous cytokine receptor expressed on the cell surface. In another embodiment, the modified T cells express exogenous stem cell factor (SCF).

[0019] In an embodiment, the antigen recognition moiety is selected from the group consisting of: TCR, αβ TCR, γδ TCR, chimeric antigen receptor (CAR), whole antibody or its antigen-binding fragment, single-chain variable fragment (scFv), heavy chain or light chain single-domain antibody (sdAb), Fab, F(ab)2, or any combination thereof, said antigen recognition moiety binding to: (i) cell surface tumor antigen, (ii) a peptide derived from a cell surface expressed as a tumor antigen in a complex with MHC (peptide-MHC complex), (iii) a cell surface antigen associated with an autoimmune disease or pathogen, or (iv) a peptide derived from a cell surface expressed as a complex with MHC (peptide-MHC complex) associated with an autoimmune disease or pathogen. In an embodiment, the antigen recognition moiety is a CAR. In an embodiment, the CAR further comprises a transmembrane (TM) domain, a hinge domain, a co-stimulatory domain, and an intracellular signal transduction domain. In an embodiment, the hinge domain is a CD8α hinge domain. In an embodiment, the TM domain includes the CD8α TM domain. In the implementation scheme, the intracellular signal transduction domain is the CD3ζ intracellular signal transduction domain.

[0020] In the implementation plan, the disease-related antigen is a tumor antigen, an autoimmune disease-related antigen, or a pathogen antigen.

[0021] In another aspect, this disclosure provides a pharmaceutical composition comprising a plurality of modified T cells as described herein. In an embodiment, the plurality of modified T cells are a plurality of γδ T cells comprising a composition of at least about 60%, at least about 80%, or at least about 80% to about 90% δ1 γδ T cells, and a pharmaceutically acceptable carrier. In an embodiment, the plurality of T cells comprises at least about 10 7 γδ T cells, optionally about 10 8 To about 1010 One γδ T cell.

[0022] In another aspect, this disclosure provides a method for treating or preventing a disease in a subject, comprising administering the pharmaceutical composition herein to the subject in need. In one embodiment, the disease is an autoimmune disease. In another embodiment, the disease is cancer or a precancerous condition.

[0023] In another embodiment, this disclosure provides a method for enhancing the efficacy of T-cell therapy, comprising disrupting the MED12 gene in T cells and expressing membrane-bound IL-12 in the T cells. In an embodiment, the method further comprises disrupting the Fas gene in the T cells or expressing dnFas in the T cells. In an embodiment, the modified T cells also include an antigen recognition portion expressed on the cell surface, wherein the antigen recognition portion includes an affinity-binding domain specific to disease-associated antigens. In an embodiment, the T cells are γδ T cells. In an embodiment, the γδ T cells are δ1, δ2, δ3, or δ4 γδ T cells, preferably δ2. - γδ T cells, more preferably δ1 γδ T cells. In the embodiment, the T cells are αβ T cells.

[0024] In another aspect, this disclosure provides a method for enhancing the cytotoxicity of T cells, comprising disrupting the MED12 gene in T cells and expressing membrane-bound IL-12 in the T cells. In embodiments, the method further comprises disrupting the Fas gene in the T cells or expressing dnFas in the T cells. In embodiments, the modified T cells also include an antigen recognition portion expressed on the cell surface, wherein the antigen recognition portion includes an affinity-binding domain specific to disease-associated antigens. In embodiments, the T cells are γδ T cells. In embodiments, the γδ T cells are δ1, δ2, δ3, or δ4 γδ T cells, preferably δ2. - γδ T cells, more preferably δ1 γδ T cells. In the embodiment, the T cells are αβ T cells.

[0025] In another aspect, the present invention provides a modified T cell with enhanced cytotoxicity, comprising disruption of the MED12 gene in the modified T cell and expression of membrane-bound IL-12 in the modified T cell. In an embodiment, the modified T cell further comprises an antigen recognition portion expressed on the cell surface, wherein the antigen recognition portion comprises an affinity-binding domain specific to disease-associated antigens. In an embodiment, the modified T cell further comprises disruption of the Fas gene or expression of dnFas in the modified T cell. In an embodiment, the modified T cell is a γδ T cell. In an embodiment, the γδ T cell is a δ1, δ2, δ3, or δ4 γδ T cell, preferably δ2. - γδ T cells, more preferably δ1 γδ T cells. In the embodiment, the modified T cells are αβ T cells.

[0026] Incorporate by reference

[0027] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference. Attached Figure Description

[0028] Figure 1 This demonstrates the in vitro cytotoxicity of PSMA CAR in the presence of TGFβ1.

[0029] Figure 2A This demonstrates that the expression of the CAR remains unchanged relative to the bare CAR in the PSMA CAR construct modified with "bolt-on". Figure 2B This demonstrates the expression of dominant-negative TGFβ receptor 2 (dnTGFβR2) in the "bolt-tightened" modified PSMA CAR construct compared to the unmodified naked CAR. Figure 2C This indicates that CD103 expression is reduced in the "bolt-tightened" modified PSMA CAR construct compared to the bare CAR. Figure 2D This demonstrates that, compared to the unmodified bare CAR, the expression of pSMAD2 / 3 is reduced in the "bolt-tightened" modified PSMA CAR construct after the addition of exogenous TGFβ.

[0030] Figure 3 This is a graph illustrating the gene knockout efficiency of two different guide RNAs containing SH2 protein (CISH) induced by targeted cytokines.

[0031] Figure 4A This is a chart illustrating that Vδ1 T cells with CISH knockout can be enriched after αβ T cell depletion.

[0032] Figure 4B This is a chart illustrating the viability of Vδ1 T cells with CISH knocked out.

[0033] Figures 5A to 5B This demonstrates that CISH KO enhances the in vitro cytotoxicity of Vδ1 T cells.

[0034] Figure 6 The design of the CAR-mbIL-12 construct is explained.

[0035] Figures 7A to 7D This demonstrates the amplification and expression of CAR-mbIL-12 in Vδ1 T cells.

[0036] Figure 8 This demonstrates the enhanced in vitro cytotoxicity of CAR-mbIL-12 in Vδ1 T cells.

[0037] Figure 9 This study demonstrates the in vivo therapeutic efficacy of CAR-mbIL-12 in Vδ1 T cells in a subcutaneous human xenograft Raji cell NSG mouse model.

[0038] Figures 10A to 10B This demonstrates that CBL-B KO enhances the in vitro cytotoxicity of Vδ1 T cells.

[0039] Figures 11A to 11B This demonstrates that Roquin KO enhances the in vitro cytotoxicity of Vδ1 T cells.

[0040] Figures 12A to 12B This demonstrates that CD58 or ICAM-1 KO enhances the in vitro cell survival of Vδ1 T cells in allogeneic MLR assays.

[0041] Figures 13A to 13C This study explains the TGFβRII KO in CAR Vδ1 T cells and its effect on TGFβ-mediated signal transduction.

[0042] Figure 14 This study demonstrated the in vitro cytotoxicity of PSMA CAR-transduced γδ T cells with gene-edited MED12 KO ± TGFβRII KO and mbIL-12 expression.

[0043] Figures 15A to 15B This study demonstrates the in vivo efficacy of PSMA CAR-transduced mbIL-12 with gene-edited MED12 KO ± TGFβRII KO in a subcutaneous human xenograft PC3-PSMA model in NSG mice.

[0044] Figures 16A to 16BThis study demonstrated that CD19 CAR γδ T cells armored with dnCD95 had improved survival in allogeneic mixed lymphocyte response (MLR). Detailed Implementation

[0045] definition

[0046] For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa. If any definition set forth conflicts with any reference incorporated herein by reference, the definition set forth herein shall prevail. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0047] When it comes to measurable values ​​such as quantity, duration, etc., the term “about” as used herein means to cover ±20% or ±10% of the specified value, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1%, because such variation is suitable for carrying out the disclosed method.

[0048] As used in this article, "w / v" refers to the weight of the component in a given volume of solution.

[0049] "Scope": Throughout this disclosure, various aspects of this disclosure may be presented in a scope format. It should be understood that the scope format is merely for convenience and brevity and should not be construed as an immutable limitation on the scope of this disclosure. Therefore, a scope description should be considered to have explicitly disclosed all possible sub-scopes and individual numerical values ​​within that scope. For example, a scope such as 1 to 6 should be considered to have explicitly disclosed sub-scopes such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and individual numbers within that scope, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the scope.

[0050] The terms “patient,” “subject,” “individual,” etc., are used interchangeably herein and refer to any animal that is compliant with the methods described herein. In some non-limiting embodiments, the patient, subject, or individual is a human being.

[0051] As used herein, the term "agent" refers to any protein, nucleic acid molecule (including chemically modified nucleic acids), compound, antibody, small molecule, organic compound, inorganic compound, other molecule of interest, or cell (e.g., cells engineered to express chimeric antigen receptors). Agents may include therapeutic agents, diagnostic agents, or pharmaceutical agents. A therapeutic agent or pharmaceutical agent, when administered to a subject (including to treat a subject with cancer or other diseases / conditions), alone or in combination with another agent, induces a desired response (such as inducing therapeutic or preventative effects).

[0052] The term "therapeutic effective amount," or simply "effective amount," refers to the amount of an agent or composition (e.g., a composition of inclusion agents) that researchers, veterinarians, MDs, or other clinicians seek to elicit a biological or medical response in an tissue, system, or subject. The term "therapeutic effective amount" includes, when administered, an amount of an agent or composition of inclusion agents sufficient, when applied, to prevent the development of one or more signs or symptoms of a condition or disease being treated (e.g., cancer), or to alleviate said signs or symptoms to some extent. Therapeutic effective amounts will vary depending on the composition, the disease and its severity, and the age, weight, etc., of the subject being treated.

[0053] The term "T lymphocyte" or "T cell" refers to an immune cell that expresses or has expressed CD3 (CD3+) and the T cell receptor (TCR+). T cells play a central role in cell-mediated immunity. T cells that "express" CD3 and TCR are engineered to eliminate CD3 and / or TCR expression on their cell surface. In some embodiments, the T cell is an αβ T cell. In some embodiments, the T cell is a γδ T cell. T cells as used herein include those used in adoptive cell therapy. In some embodiments, T cells used in adoptive cell therapy include tumor-infiltrating lymphocytes (TILs) (e.g., T cells containing a tumor cell recognition portion endogenously expressed by isolated wild-type T cells, such as those isolated from tumor-infiltrating lymphocytes from a tumor sample), virus-specific T cells (VSTs), TCR-T cells, CAR-T cells, CAR-NKT cells, etc.

[0054] As used herein, the term "γδ T cell (gamma-delta T cell)" refers to a subset of T cells that express different T cell receptors (TCRs) (i.e., γδ TCRs) consisting of one γ chain and one δ chain on their surface. The term "γδ T cell" specifically includes all subsets of γδ T cells, including but not limited to Vδ1 and Vδ2, Vδ3 γδ T cells, as well as initial, effector memory, central memory, and terminally differentiated γδ T cells. As another example, the term "γδ T cell" includes Vδ4, Vδ5, Vδ7, and Vδ8 γδ T cells, and Vγ2, Vγ3, Vγ5, Vγ8, Vγ9, Vγ10, and Vγ11 γδ T cells. In an embodiment, the γδ T cell is Vδ1 - Vδ2 - or Vδ1 - and Vδ2 - Compositions and methods for preparing and using engineered and non-engineered γδ T cells and / or their subtypes include, but are not limited to, those described in US 2016 / 0175358; WO 2017 / 197347; US 9499788; US 2018 / 0169147; US 9907820; US 2018 / 0125889 and US 2017 / 0196910, the contents of each of which are incorporated herein by reference for all purposes, including compositions and methods for preparing and using engineered and non-engineered γδ T cells and / or their subtypes. This application further contemplates T cells or other engineered leukocytes or lymphocytes expressing a γ chain or a δ chain (optionally combined with a second polypeptide to form a functional TCR). Such engineered leukocytes or lymphocytes expressing a γ chain or a δ chain can be used in the methods described herein or are present in the compositions described herein.

[0055] The term "αβ T cell" refers to a T cell that expresses the α and β chains of the TCR as part of a complex with the CD3 chain molecule. Each α and β chain contains a variable domain and a constant domain. αβ T cells primarily recognize peptide antigens presented by major histocompatibility complex (MHC) class I and II molecules, with most receptor diversity contained within the third complementarity-determining region (CDR3) of the TCR α and β chains.

[0056] The term "TCR" or "T cell receptor" refers to a dimer heterologous cell surface signaling protein that forms α-β or γ-δ receptors or combinations thereof. αβTCRs recognize antigens presented by MHC molecules, while γδTCRs can recognize antigens independently of MHC presentation.

[0057] The term "MHC" (major histocompatibility complex) refers to a subset of genes encoding cell surface antigen-presenting proteins. In humans, these genes are known as human leukocyte antigen (HLA) genes. In this document, the abbreviations MHC and HLA are used interchangeably.

[0058] The term "activation" refers to the state of T cells that have been adequately stimulated to induce detectable cell proliferation. Activation can also be associated with induced cytokine production and detectable effector function. The term "activated T cells" specifically refers to T cells that are undergoing cell division.

[0059] The term "chimeric antigen receptor (CAR)" refers to, for example, an artificial T-cell receptor, a T-body, a single-chain immune receptor, a chimeric T-cell receptor, or a chimeric immune receptor, and encompasses engineered receptors that transfer artificial specificity onto specific immune effector cells. CARs can be used to confer specificity to T-cell monoclonal antibodies, thereby allowing the generation of large numbers of specific T cells, for example, for adoptive cell therapy. In specific embodiments, CARs direct cell specificity to disease-associated antigens (e.g., tumor-associated antigens, autoimmune-associated antigens, or pathogenic antigens). In some embodiments, CARs comprise an intracellular activation domain (allowing T cells to activate upon binding of the targeting portion to target cells, such as target tumor cells), a transmembrane domain, and an extracellular domain of variable length that includes a disease or symptom-related region (e.g., a tumor antigen-binding region). In some embodiments, CARs comprise a fusion of a single-chain variable fragment (scFv) derived from a monoclonal antibody, fused to a CD3-ζ transmembrane domain and an endodomain. Other CAR designs may derive specificity from receptor ligands (e.g., peptides) or from pattern recognition receptors, such as Dectin. In some cases, the spacing of antigen recognition domains can be modified to reduce activation-induced cell death. In some cases, CARs contain domains for additional co-stimulatory signaling, such as CD3-ζ, FcR, CD27, CD28, CD137, DAP 10 / 12 and / or OX40, ICOS, TLR, etc. In some cases, molecules can be co-expressed with the CAR, including co-stimulatory molecules, reporter genes for imaging (e.g., for positron emission tomography), gene products that conditionally ablate T cells after prodrug administration, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors.

[0060] The "co-stimulatory domain" in the context of CAR enhances cell proliferation, cell survival, and memory cell development in CAR-expressing cytotoxic cells. CAR may include one or more co-stimulatory domains selected from proteins in the TNFR superfamily: CD28, CD137 (4-1BB), CD134 (OX40), Dapl0, CD27, CD2, CD7, CD5, ICAM-1, LFA-1 (CD11a / CD18), Lck, TNFR-I, PD-1, TNFR-II, Fas, CD30, CD40, ICOSLIGHT, NKG2C, B7-H3, or combinations thereof. If CAR includes more than one co-stimulatory domain, these domains may be arranged in tandem, optionally separated by a linker. As used herein, the term “co-stimulatory domain” also encompasses any modifications thereof, examples of which are described in U.S. Patent Application No. 20200129554; U.S. Patent Application No. 20200317777; WO2019010383; Li, W. et al., (2020) Immunity 53: 456-470; and Li, G. et al., (2017) JImmunol 198(1 Supplement): 198.4, the contents of each of which are incorporated herein by reference in their entirety.

[0061] In the context of CAR, the "intracellular signaling domain" transduces effector functional signals and directs cytotoxic cells to perform their specialized functions, such as damaging and / or destroying target cells. Examples of suitable intracellular signaling domains include, for example, the ζ chain of the T cell receptor complex or any homolog thereof, such as the η chain, FcsRI and β chains, MB1 (Iga) chain, B29 (Ig) chain, etc., the human CD3 ζ chain, CD3 polypeptides (Δ, δ, and ε), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.), and other molecules involved in T cell transduction, such as CD2, CD5, and CD28. In embodiments, the intracellular signaling domain of the chimeric receptor can be the human CD3 ζ chain, FcyRIII, FcsRI, the cytoplasmic tail of the Fc receptor, a cytoplasmic receptor carrying an immune receptor tyrosine activation motif (ITAM), and combinations thereof.

[0062] Intracellular signal transduction domains can include intracellular signal transduction domains of various other immune signal transduction receptors of several types, including but not limited to first-, second-, and third-generation T cell signal transduction proteins, including CD3, B7 family co-stimulatory receptors and tumor necrosis factor receptor (TNFR) superfamily receptors (Park et al., "Are allchimeric antigen receptors created equal?" J Clin Oncol., Vol. 33, pp. 651-653, 2015). Other intracellular signal transduction domains include those used by NK and NKT cells (Hermanson et al., "Utilizing chimeric antigen receptors to direct natural killer cell activity," Front Immunol., Vol. 6, p. 195, 2015), such as the signal transduction domain of NKp30 (Zhang et al., "An NKp30-based chimeric antigen receptor promotes T cell efficacy functions and antitumor efficacy in vivo," J Immunol., Vol. 189, pp. 2290-2299, 2012) and DAP12 (Topfer et al., "DAP12-based activating chimeric antigen receptor for NK cell tumor immunotherapy," J Immunol., Vol. 194, pp. 3201-3212, 2015), NKG2D, NKp44, NKp46, DAP10, and CD3z. In addition, intracellular signal transduction domains also include signal transduction domains of human immunoglobulin receptors containing immune receptor tyrosine activation motifs (ITAMs), such as FcγRI, FcγRIIA, FcγRIIC, FcγRIIIA, and FcRL5 (Gillis et al., "Contribution of Human Fc.gamma.Rs to Disease with Evidence from Human Polymorphisms and Transgenic Animal Studies," Front Immunol., Vol. 5, p. 254, 2014).

[0063] In some embodiments, the intracellular signal transduction domains include cytoplasmic signal transduction domains of TCR ζ, FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ε, CD5, CD22, CD79a, CD79b, or CD66d. In an exemplary embodiment, the intracellular signal transduction domains in the CAR include the cytoplasmic signal transduction domain of human CD3 ζ. As used herein, the term “intracellular signal transduction domain” also encompasses any modifications thereof, examples of which are described in U.S. Patent Application No. 2020 / 0317777 and in Roda-Navarro, P. and Reyburn, HT., (2009) J Biol Chem 284(24): 16463-16472; Giurisato, E. et al., (2007) Mol Cell Biol 27(24): 8583-8599; and Wu, J. et al., (2000) J Exp Med 192(7): 1059-1068, the contents of each of which are incorporated herein by reference in their entirety.

[0064] As used herein, the terms "affinity-binding domain" or "means for specific binding" refer to binding to a specific antigen with a higher affinity than that for nonspecific antigens and being conferred at least 10 -6 The affinity binding portion of M is determined by methods well-known in the art, including surface plasmon resonance (SPR). Depending on the specific embodiment, the affinity is 500 nM–0.01 nM, 100 nM–0.01 nM, 50 nM–0.01 nM, 10 nM–0.01 nM, or 5 nM–0.01 nM. In some embodiments, the affinity-binding domain or the means for specific binding recognizes tumor antigens. In some embodiments, the affinity-binding domain or the means for specific binding recognizes autoimmune disease-associated antigens. In some embodiments, the affinity-binding domain or the means for specific binding recognizes pathogenic antigens.

[0065] In the embodiments, the affinity-binding domain or the means for specific binding is an antibody. The term "antibody" is used in the broadest sense and specifically covers, for example, single monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, full-length or complete monoclonal antibodies), antibody compositions with multi-epitope specificity, polyclonal antibodies, multivalent antibodies, multispecific antibodies formed from at least two complete antibodies (e.g., bispecific antibodies, provided they exhibit the desired biological activity), single-chain antibodies, and fragments of antibodies (see below) (including Fab, Fab', F(ab')2, and Fv fragments), diabody antibodies, and single-domain antibodies (sdAbs), provided they exhibit the desired biological or immunological activity. Antibodies, and particularly fragments, also include antibody portions (and combinations of antibody portions, such as scFv) that can be used as a targeting arm against, for example, a target epitope in a chimeric antigen receptor of this disclosure (e.g., an epitope on a tumor antigen). Such fragments are not necessarily proteolytic fragments, but rather portions of a polypeptide sequence that can confer target affinity. The term "immunoglobulin" (Ig) is used interchangeably with antibody herein. Antibodies can be, for example, human antibodies, humanized antibodies, and / or antibodies with mature affinity.

[0066] Methods for preparing antibodies and antibody fragments are known in the art. (See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, which are incorporated herein by reference).

[0067] Antibodies can be generated by immunizing various animals (including mice, rats, rabbits, goats, primates, humans, and chickens) with a target antigen or a peptide fragment containing an epitope of this disclosure. Antibodies can also be generated using, for example, Clackson et al. Nature , 352: 624-8 (1991) and Marks et al., J. Mol. Biol The techniques described in ., 222: 581-97 (1991) are used to isolate antibodies from phage antibody libraries. The antibodies or antigen-binding fragments of the present invention can be purified by methods known in the art, such as gel filtration, ion exchange, affinity chromatography, etc. Affinity chromatography or any of many other techniques known in the art can be used to isolate polyclonal or monoclonal antibodies from, for example, serum, ascites, or hybridoma supernatant.

[0068] "Isolated antibody" is an antibody that has been identified, isolated, and / or recovered from components of its natural environment. Contaminant components of its natural environment are materials that will interfere with the therapeutic use of the antibody and may include enzymes, hormones, and other protein or non-protein solutes.

[0069] The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. In the case of IgG, the 4-chain unit is typically about 150,000 Daltons. Each L chain is linked to the H chain by a covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isoform. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at its N-terminus, followed by three constant domains (CH) of each of the α and γ chains, and four CH domains of the μ and ε isoforms. Each L chain has a variable domain (VL) at its N-terminus, followed by a constant domain (CL) at its other end. The VL is aligned with the VH, and the CL is aligned with the first constant domain (CH1) of the heavy chain. Specific amino acid residues are believed to form the interface between the light and heavy chain variable domains. The VH and VL pair together to form a single antigen-binding site. For the structure and properties of different classes of antibodies, see, for example... Basic and Clinical Immunology , 8th edition, edited by Daniel P. Stites, Abba I. Terr and Tristram G. Parslow, Appleton & Lange, Norwalk, CT, 1994, page 71 and chapter 6.

[0070] L-chains from any vertebrate species can be classified into one of two distinct types, called κ and λ, based on the amino acid sequence of their constant domain. Immunoglobulins can be classified into different classes or isotypes based on the amino acid sequence of their heavy chain (CH) constant domain. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains labeled α, δ, ε, γ, and μ, respectively. The γ and α classes are further subdivided into subclasses based on relatively minor differences in CH sequence and function; for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0071] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of either the heavy or light chain. The variable domain of the heavy chain can be referred to as "VH" or "V". H The variable structural domains of light chains can be referred to as "VL" or "V". L These domains are typically the most variable parts of an antibody and contain antigen-binding sites.

[0072] The term "variability" refers to the fact that certain segments of the variable domain differ significantly in sequence between antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its specific antigen. However, variability is not uniformly distributed across the 110 amino acid span of the variable domain. Instead, the V region consists of relatively invariant segments called framework regions (FRs), which are 15–30 amino acids long, separated by shorter, extremely variable regions called "hypervariates," each approximately 9–12 amino acids long. The variable domains of the natural heavy and light chains each contain four FRs, predominantly in a β-sheet configuration, and are linked by three hypervariates that form loops connecting the β-sheet structure and, in some cases, part of it. The hypervariates in each chain are tightly held together by the FRs and, together with hypervariates from the other chain, contribute to the formation of the antigen-binding site of the antibody (see Kabat et al.). Sequences of Proteins of Immunological Interest , 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD. (1991)).

[0073] An "intact" antibody is an antibody that contains an antigen-binding site, a CL domain, and at least heavy chain constant domains CH1, CH2, and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. Preferably, an intact antibody has one or more effector functions.

[0074] An "antibody fragment" comprises a portion of a complete antibody, preferably the antigen-binding region or one or more variable regions of the complete antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; biantibodies; linear antibodies (see U.S. Patent No. 5,641,870, Example 2; Zapata et al.). Protein Eng . 8(10): 1057-62 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. In one embodiment, the antibody fragment contains the antigen-binding site of the complete antibody, thus retaining the ability to bind antigens. The antibody fragment also includes an antibody portion (and combinations of antibody portions, such as scFv) that can be used as a targeting arm against an epitope in a chimeric antigen receptor of the present disclosure. Such fragments are not necessarily proteolytic fragments, but rather portions of a polypeptide sequence that can confer target affinity.

[0075] Papain digestion of the antibody yields two identical antigen-binding fragments (referred to as "Fab" fragments) and a residual "Fc" fragment (the name reflects its tendency to crystallize). The Fab fragment consists of the complete L chain, a variable region domain (VH) of the H chain, and a first constant domain (CH1) of a heavy chain. Each Fab fragment is monovalent in terms of antigen binding, meaning it has a single antigen-binding site. Pepsin treatment of the antibody yields a single large F(ab')2 fragment, which roughly corresponds to the two disulfide-linked Fab fragments with bivalent antigen-binding activity and is still capable of cross-linking the antigen. The Fab' fragment differs from the Fab fragment in that it has several additional residues at the carboxyl terminus of the CH1 domain, including one or more cysteine ​​residues from the antibody hinge region. Fab'-SH is the name used herein for Fab' fragments in which one or more cysteine ​​residues in the constant domain have a free thiol group. The F(ab')2 antibody fragment is initially produced as a pair of Fab' fragments with a hinge cysteine ​​residue between them. Other chemical conjugations of antibody fragments are also known.

[0076] The Fc fragment comprises the carboxyl-terminal portion of two H chains held together by disulfide bonds. The effector function of an antibody is determined by the sequence in the Fc region, which is also the portion recognized by Fc receptors (FcRs) present on certain types of cells.

[0077] "Fv" is the smallest antibody fragment containing a complete antigen recognition and binding site. This fragment consists of a dimer of a tightly non-covalently associated heavy chain variable region domain and a light chain variable region domain. In single-chain Fv (scFv) material, a heavy chain variable domain and a light chain variable domain are covalently linked by a flexible peptide linker, allowing the light and heavy chains to associate in a "dimeric" structure similar to that in double-chain Fv material. The folding of these two domains produces six hypervariable rings (three from the H chain and three from the L chain), which contribute amino acid residues to antigen binding and impart antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only the three CDRs specific to the antigen) can recognize and bind to the antigen, although with lower affinity than the entire binding site.

[0078] A "single-chain Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment comprising VH and VL antibody domains linked together as a single polypeptide chain. In embodiments, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the sFv to form the desired structure for antigen binding. For a review of sFv, see, for example, Pluckthun, published in [Journal Name]. The Pharmacology of Monoclonal AntibodiesVolume 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, see below. In one embodiment, the antibody-derived scFv is used as a targeting arm for modified T cells as disclosed herein. With respect to scFv antibody fragments in which certain sequences of the VH and VL regions in the binding domain are explicitly or implicitly described, this disclosure also includes alternative embodiments in which the order of the VH and VL regions is reversed (e.g., in scFvs or CARs containing scFv binding domains). Thus, for example in scFvs or CARs containing scFv binding domains, the description of the VH-VL sequence also describes an alternative VL-VH sequence. Furthermore, for example in scFvs or CARs containing scFv binding domains, the description of the VL-VH sequence also describes an alternative VH-VL sequence. The VH and VL regions can be directly connected or connected through a peptide-encoded linker, which connects the N-terminus of VH to the C-terminus of VL, or connects the C-terminus of VH to the N-terminus of VL.

[0079] scFv linkers are typically enriched with glycine to increase flexibility and with serine or threonine to increase solubility. Linkers can connect heavy chain variable regions and light chain variable regions of extracellular antigen-binding domains. Non-limiting examples of linkers are disclosed in Shen et al., Anal. Chem. 80(6): 1910-1917 (2008) and WO 2014 / 087010, the contents of which are hereby incorporated by reference in their entirety. Various linker sequences are known in the art, including but not limited to glycine-serine (GS) linkers, such as (GS)n, (GSGGS)n (SEQ ID NO: 174), (GGGS)n (SEQ ID NO: 175), and (GGGGS)n (SEQ ID NO: 176), where n represents an integer of at least 1. Exemplary adapter sequences may include, but are not limited to, the following amino acid sequences: GGSG (SEQ ID NO: 177), GGSGG (SEQ ID NO: 178), GGSG (SEQ ID NO: 179), GGSGG (SEQ ID NO: 180), GGGSG (SEQ ID NO: 181), GSSSG (SEQ ID NO: 182), GGGGS (SEQ ID NO: 183), GGGGSGGGGGGGSGGG (SEQ ID NO: 172), etc. Those skilled in the art will be able to select appropriate adapter sequences for use in this invention. In one embodiment, the affinity-binding domain of the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH and VL are separated by a linker sequence having the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 172), said linker sequence being encoded by the nucleic acid sequence GGAGGCGGAGGATCTGGTGGTGGTGGATCTGGCGCGGCGGAGGCTCT (SEQ ID NO: 173).

[0080] As used herein, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous group of antibodies, meaning that the individual antibodies constituting said group are identical except for the possibility of naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific, targeting a single antigenic site. Furthermore, unlike polyclonal antibody formulations which comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized without contamination by other antibodies. The modifier "monoclonal" should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies that can be used in this invention can be synthesized by methods first developed by Kohler et al. NatureMonoclonal antibodies can be prepared using the hybridoma method described in 256:495 (1975), or using recombinant DNA methods in bacterial, eukaryotic, or plant cells (e.g., U.S. Patent No. 4,816,567). "Monoclonal antibodies" can also be prepared using, for example, Clackson et al. Nature , 352: 624-8 (1991) and Marks et al., J. Mol. Biol The technique described in ., 222: 581-97 (1991) is used to isolate from a phage antibody library.

[0081] The terms “hypervariant region,” “HVR,” or “HV,” as used herein, refer to regions within the variable domains of an antibody that are sequence-hypervariant and / or form structurally defined loops. Typically, an antibody contains six hypervariant regions; three in the VH domain (H1, H2, H3) and three in the VL domain (L1, L2, L3). Many descriptions of hypervariant regions are used and are covered herein. The Kabat complementarity-determining region (CDR) is based on sequence variability and is the most commonly used (Kabat et al., ). Sequences of Proteins of Immunological Interest , 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Chothia's work concerns the location of structural rings (Chothia and Lesk). J. Mol. Biol. 196:901-917 (1987)). When numbered using the Kabat numbering convention, the ends of the Chothia CDR-H1 loop vary between H32 and H34, depending on the loop length (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDR and the Chothia structural loop and are used by the AbM antibody modeling software from Oxford Molecular. The “contact” hypervariable region is based on the analysis of the available complex crystal structures. The residues from each of these hypervariable regions are shown below.

[0082]

[0083] The hypervariable region may include the following “extended hypervariable regions”: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 (L3) in VL; and 26-35B (H1), 50-65, 47-65, or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in VH. For each of these definitions, the variable domain residues are numbered according to Kabat et al., ibid.

[0084] "Frame" or "FR" residues are those variable domain residues other than the hypervariable region residues defined in this paper.

[0085] The terms “variable domain residue numbering as in Kabat” or “amino acid position numbering as in Kabat” and their variations refer to the heavy chain or light chain variable domain numbering system used by Kabat et al., as described above, for antibody compilation. Using this numbering system, the actual linear amino acid sequence may contain shortened or inserted amino acids corresponding to the FR or CDR of the variable domain, or fewer or additional amino acids inserted into the FR or CDR of the variable domain. For example, the heavy chain variable domain may include a single amino acid insertion after residue 52 of H2 (according to residue 52a in Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc., according to Kabat). For a given antibody, the Kabat number of the residue can be determined by aligning the sequence in the homologous region of the antibody sequence with the sequence number of the “standard” Kabat number.

[0086] When referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain), the Kabat numbering system is typically used (e.g., Kabat et al., ibid.). When referring to residues in the constant region of the immunoglobulin heavy chain, the “EU numbering system” or “EU index” is typically used (e.g., Kabat et al., EU index reported above). “EU index as in Kabat” refers to the residue numbering of the human IgG1 EU antibody. Unless otherwise stated herein, references to residue numbers in the variable domain of an antibody mean residue numbers obtained using the Kabat numbering system.

[0087] An antibody that "binds" to an antigen or epitope of interest is an antibody that binds to the antigen or epitope with sufficient affinity that is measurably different from nonspecific interactions. Specific binding can be measured, for example, by determining the binding of a molecule by comparing it to the binding of a control molecule, which is typically a similarly structured molecule that does not have binding activity.

[0088] As used herein, the term "antigen" or "Ag" is defined as a molecule that elicits an immune response. This immune response may involve antibody production, or activation of specific immune-competent cells, or both. Those skilled in the art will understand that any macromolecule, including proteins or peptides, can be used as an antigen.

[0089] The term "epitope" includes any protein, lipid, or carbohydrate determinant capable of specifically binding to immunoglobulins or T-cell receptors. Epitope determinants typically consist of active surface groups of molecules (such as amino acid, lipid, or sugar side chains) and usually possess specific three-dimensional structural features as well as specific charge characteristics.

[0090] As used herein, the term "specific binding" refers to a receptor (which may include, but is not limited to, antibodies or antibody fragments) recognizing a specific molecule / ligand but not substantially recognizing or binding to other molecules in the sample. For example, a receptor that specifically binds to a molecule from one species may also bind to that molecule from one or more other species. However, this cross-species reactivity itself does not alter the specific classification. In another instance, a receptor that specifically binds to a molecule may also bind to different allelic forms of said molecule. However, this cross-reactivity itself does not alter the specific classification. In some cases, the terms "specific binding" or "specifically binding" may be used to refer to the interaction of a protein (or peptide) with a second chemical substance, meaning that the interaction depends on the presence of a specific structure on the chemical substance (e.g., an antigenic determinant or epitope); for example, the receptor recognizes and binds to the specific structure rather than the usual protein. If the receptor is specific for epitope "A," then in a reaction containing labeled "A" and the receptor, the presence of a molecule containing epitope A (or free unlabeled A) will reduce the amount of labeled A binding to the receptor.

[0091] In the implementation scheme, specific binding can be achieved through at least approximately 1 x 10 -8 Characterized by M or a smaller equilibrium dissociation constant (e.g., a smaller K). d (This indicates a tighter binding). Methods for determining whether two molecules are specifically bound are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc.

[0092] The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by uncontrolled cell growth. Cancer includes solid tumors and hematologic malignancies. Examples of cancers include acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphomas, anal cancer, appendiceal cancer, astrocytoma, neuroblastoma, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumors (such as cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, visual pathway and hypothalamic glioma), breast cancer, bronchial adenoma, Burkitt lymphoma, cancer of unknown primary origin, central nervous system lymphoma, cerebellar astrocytoma, cervical cancer, childhood cancers, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorder, colon cancer, cutaneous T-cell lymphoma, connective tissue proliferative small round cell tumors, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma. Sarcoma), germ cell tumors, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gliomas, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular carcinoma (liver cancer), Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell carcinoma, Kaposi's sarcoma, kidney cancer, laryngeal cancer, lip and oral cancer, liposarcoma, liver cancer, lung cancer (such as non-small cell lung cancer and small cell lung cancer), lymphoma, leukemia, macroglobulinemia, malignant fibrous histiocytoma / osteosarcoma of bone, medulloblastoma, melanoma, mesothelioma, metastatic squamous cell carcinoma of the neck with occult primary focus, oral cancer, multiple endocrine adenoma syndrome, myelodysplastic syndrome, myeloid leukemia, nasal and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma / osteosarcoma Fibrocytoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, pancreatic cancer, pancreatic islet cell cancer, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germ cell tumor, pituitary adenoma, pleural pulmonary blastoma, plasma cell tumor, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, cutaneous Merkel cell carcinoma, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, T-cell lymphoma, laryngeal cancer, thymoma, thymic carcinoma, thyroid cancer, trophoblastic tumor (pregnancy-related), cancer of unknown primary site, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, and Wilms tumor.

[0093] The term "autoimmune disease" refers to a condition caused by an autoimmune response. Autoimmune diseases result from an inappropriate overreaction to antigens associated with the autoimmune disease (e.g., self-antigens). Examples of autoimmune diseases include rheumatoid arthritis, rheumatic fever, multiple sclerosis, experimental autoimmune encephalomyelitis, psoriasis, uveitis, diabetes, systemic lupus erythematosus (SLE), lupus nephritis, eczema, scleroderma, polymyositis / scleroderma, polymyositis / dermatomyositis, ulcerative proctitis, ulcerative colitis, severe combined immunodeficiency disease (SCID), DiGeorge syndrome, ataxia-telangiectasia, seasonal allergies, perennial allergies, food allergies, anaphylactic reactions, mast cell hyperplasia, and anaphylaxis. Rhinitis, atopic dermatitis, Parkinson's disease, Alzheimer's disease, hypersplenism, leukocyte adhesion defect, X-linked lymphoproliferative disorders, X-linked agammaglobulinemia, selective immunoglobulin A deficiency, hyper-IgM syndrome, HIV, autoimmune lymphoproliferative syndrome, Wiskott-Aldrich syndrome, chronic granulomatous disease, common variant immunodeficiency disease (CVID), hyperimmune E syndrome Hashimoto's thyroiditis, acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham's chorea, myasthenia gravis, polyglandular syndrome, bullous pemphigoid, Henoch-Schonlein purpura, post-streptococcal nephritis, erythema nodosum, erythema multiforme, gA nephropathy, Takayasu's arteritis, Addison's disease, sarcoidosis. Ulcerative colitis, polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome, thromboangiitis obliterans, Sjogren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, chronic active hepatitis, polychondritis, pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant cell arteritis, polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis, psoriasis, and fibrotic alveolitis. In the implementation scheme, autoimmune diseases are selected from the group comprising or consisting of: lupus, lupus nephritis, systemic sclerosis, antineutrophilic cytoplasmic autoantibody-associated vasculitis, and autoimmune muscle diseases. In the implementation scheme, lupus is systemic lupus erythematosus. In the implementation plan, systemic lupus erythematosus is systemic lupus erythematosus with extrarenal involvement.In the implementation plan, autoimmune muscle disease is defined as idiopathic inflammatory myopathy or stiff-person syndrome.

[0094] The term “self” is intended to refer to any material derived from an individual that will subsequently be reintroduced into the same individual.

[0095] The term "alien" refers to material derived from an animal that was subsequently introduced into different animals of the same species.

[0096] As used herein, “modification” of an amino acid residue / position refers to a change in the primary amino acid sequence compared to the starting amino acid sequence, where such change is caused by a sequence alteration involving the amino acid residue / position. Typical modifications include, for example, the substitution of a residue (or at that position) with another amino acid (e.g., a conserved or non-conserved substitution), the insertion of one or more (typically fewer than 5 or 3) amino acids adjacent to that residue / position, and the deletion of that residue / position. “Amino acid substitution” or variations thereof refers to replacing an existing amino acid residue in a predetermined (starting) amino acid sequence with a different amino acid residue. Typically, modifications result in an alteration of at least one physicochemical activity of the variant peptide compared to a peptide containing the starting (or “wild-type”) amino acid sequence. For example, in the case of antibodies, the altered physicochemical activity could be binding affinity, binding capacity, and / or binding effect to a target molecule.

[0097] As used herein, the term "treatment or prevention" of a disease means reducing the frequency or severity of at least one sign or symptom of a disease or condition experienced by a subject. In one instance, a therapy (e.g., administration of a therapeutic agent of this disclosure) treats a disease or condition by reducing one or more signs or symptoms associated with the disease or condition, for example, compared to a response in the absence of a therapy. For example, administration of a therapeutic agent may provide an antitumor effect by reducing one or more signs or symptoms associated with cancer. Treatment or prevention may mean delaying the onset of symptoms, reducing the severity of symptoms, reducing the severity of acute attacks, reducing the number of symptoms, reducing the incidence of disease-related symptoms, reducing the latency of symptoms, improving symptoms, reducing secondary symptoms, reducing secondary infections, prolonging patient survival, preventing disease recurrence, reducing the number or frequency of recurrent attacks, increasing the latency between symptom attacks, increasing the duration of progression, accelerating remission, inducing remission, enhancing remission, accelerating recovery, or improving the efficacy of alternative therapeutic agents or reducing resistance to alternative therapeutic agents. In one embodiment, "treatment" means therapeutic treatment and preventive or preventive measures where the goal is to prevent or reduce the target pathological symptom or condition as described herein.

[0098] The term "administration" means the provision or administration of one or more agents to a subject by any effective route, such as agents for treating one or more signs or symptoms associated with a symptom / condition or disease, including but not limited to cancer (e.g., lymphoma), viral infection, bacterial infection, etc. Exemplary routes of administration include, but are not limited to, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, sublingual, rectal, transdermal, intranasal, vaginal, and inhalation routes. " Combined with one or more other therapeutic agents ” Application includes simultaneous (parallel) application and sequential application in any order.

[0099] As used in this article, terminology " Pharmaceutically acceptable ” This refers to a material (including, but not limited to, salts, carriers, or diluents) that does not eliminate the biological activity or properties of a compound and is relatively non-toxic, meaning the material can be administered to an individual without causing undesirable biological effects or interacting with any component contained in the composition in a harmful manner. Pharmaceutically acceptable carriers (mediators) that can be used in this disclosure are conventional. Remington's Pharmaceutical Sciences, edited by EWMartin, Mack Publishing Co., Easton, Pa., 19th edition (1995) describes compositions and formulations suitable for drug delivery of one or more agents (such as one or more modulators). Generally, the nature of the carrier will depend on the specific route of administration employed. For example, parenteral formulations may include injectable fluids, such as pharmaceutically and physiologically acceptable fluids (e.g., water, physiological saline, balanced salt solutions, dextran aqueous solutions, glycerol, etc.) as mediators. In addition to a biological neutral carrier, the agent to be administered may contain small amounts of non-toxic excipients, such as wetting agents or emulsifiers, preservatives, and pH buffers, for example, sodium acetate or sorbitan monolaurate, sodium lactate, potassium chloride, calcium chloride, and triethanolamine oleate. For example, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and one or more modified T cells. Examples of pharmaceutically acceptable carriers include buffers, excipients, stabilizers, or preservatives. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; peptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Cryopreservation solutions that can be used in the pharmaceutical compositions of the present invention include, for example, DMSO.

[0100] "Encoding" refers to the inherent characteristic and resulting biological property of a specific sequence of nucleotides in a polynucleotide (such as a gene, cDNA, or mRNA) serving as a template for the synthesis of other polymers and macromolecules having a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence in biological processes. Therefore, if the transcription and translation of mRNA corresponding to a gene produces a protein in a cell or other biological system, then the gene encodes that protein. Both the coding strand, which is identical to the mRNA sequence and is typically provided in the sequence listing, and the non-coding strand, which serves as a transcription template for a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0101] "Separated" means altered or removed from its natural state. For example, nucleic acids or peptides naturally present in living animals are not "separated," but the same nucleic acids or peptides that are partially or completely separated from their natural coexisting material are "separated." Separated nucleic acids or proteins can exist in substantially pure form or in non-natural environments (such as T cells).

[0102] Unless otherwise stated, "nucleotide sequences encoding amino acid sequences" includes all nucleotide sequences that are degenerate and encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNA may contain introns.

[0103] The terms “patient,” “subject,” “individual,” etc., are used interchangeably herein and refer to any animal that is compliant with the methods described herein. In some non-limiting embodiments, the patient, subject, or individual is a human being.

[0104] An "expression cassette" refers to a nucleic acid containing an expression control sequence operatively linked to a nucleic acid encoding a transcript or polypeptide to be expressed. The expression cassette contains sufficient cis-acting elements for expression; other elements for expression may be provided by T cells or in an in vitro expression system. The expression cassette may be a component of a vector such as a viscera, plasmid (e.g., naked or contained in liposomes), or a virus (e.g., lentivirus, retrovirus, adenovirus, and adeno-associated virus).

[0105] As used herein, the term “disruption” and its grammatical equivalents refer to the disturbance of gene expression. Gene disruption includes introducing one or more mutations into a gene, deleting a segment or the full length of a gene, inserting one or more nucleotides into a gene, repressing gene transcription, or any combination thereof.

[0106] T cells

[0107] The modified T cells described herein can be generated by engineering T cells, such as T cell lines or donor T cells (e.g., allogeneic, syngeneic). T cells can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors. T cells can be obtained from blood collected from a subject using any number of techniques known to those skilled in the art, such as Ficoll™ separation. For example, cells in an individual's circulating blood can be obtained via blood separation. In embodiments, T cells are separated from peripheral blood lymphocytes by lysing red blood cells and depleting mononuclear cells, for example, by PERCOLL™ gradient centrifugation or by countercurrent centrifugation. Specific T cell subsets can be further separated by positive or negative selection techniques. For example, T cells can be separated using a combination of antibodies targeting surface markers specific to positively selected cells, for example, by incubating with antibody-conjugated beads for a sufficient period of time to positively select the desired modified T cells. Alternatively, negative selection can be achieved by using a combination of antibodies targeting surface markers specific to negatively selected cells, thereby enriching the T cell population. Other specific methods of isolation and / or enrichment are disclosed in this article.

[0108] In the implementation scheme, the modified T cells exhibit in vitro or in vivo cytotoxic activity against disease cells (e.g., tumor cells, cells associated with autoimmune diseases, or pathogens or pathogen-infected cells) expressing disease-associated antigens on their cell surface. In some cases, the cytotoxic activity is innate. In some cases, the cytotoxicity is at least partially, significantly (> about 25%), or entirely due to the presence of a binding domain that specifically binds to disease-associated antigens expressed on the surface of disease cells and / or one or more modified CAR constructs described herein. In some cases, the modified T cells exhibit disease cell killing (e.g., tumor cell killing) activity greater than the innate level of in vitro and / or in vivo disease cell killing activity in control cells of the same cell type. In some cases, the control cells do not contain a CAR construct. In some cases, the control cells contain a CAR construct lacking the binding domain, hinge region, transmembrane domain, intracellular signal transduction domain, and / or co-stimulatory domain described herein. In some cases, the control cells do not contain one or more modifications described herein.

[0109] In some cases, cytotoxicity is at least partially, significantly (> about 25%), or entirely due to the presence of a binding domain having a specific binding antigen or epitope within the antigen and / or one or more modified CAR constructs as described herein.

[0110] In one embodiment, the modified T cells may exhibit HLA-restricted (e.g., HLA class I restricted) cytotoxicity. In other embodiments, most (>50%), substantially all (>90%), or all of the cytotoxic activity is not HLA-restricted (e.g., HLA class I restricted). HLA-restricted cytotoxic activity can be assessed by comparing in vitro cytotoxicity against HLA (e.g., HLA class I) tumor cell lines with that against HLA+ (e.g., HLA class I) tumor cell lines. + The HLA-restricted cytotoxic activity is assessed by in vitro cytotoxicity of tumor cell lines. In the implementation scheme, the HLA-restricted cytotoxic activity is provided at least partially, significantly (>25%), or completely by using a T-cell receptor-like binding domain. A T-cell receptor-like binding domain is a binding domain that specifically recognizes antigens presented on the cell surface when complexed with MHC molecules. T-cell receptor-like binding domains are further described, for example, in WO 2016 / 199141.

[0111] Modified T cells can exhibit robust and / or durable disease cell (e.g., tumor cell) killing activity. In some cases, disease cell (e.g., tumor cell) killing activity can persist for at least about 6 to 120 days, or at least about 6 to 180 days, from the first exposure to tumor cells. In other cases, disease cell (e.g., tumor cell) killing activity of the modified T cells or their progeny can persist for at least about 6 to 120 days, or at least about 6 to 180 days, from the first exposure to disease cells (e.g., tumor cells), or from the administration of the modified T cells. This sustained disease cell (e.g., tumor cell) killing activity can be exhibited in vitro, in vivo, or simultaneously in vitro and in vivo.

[0112] Modified T cells can proliferate in response to contact with cells that express or overexpress one or more disease-associated antigens on their cell surface. Cells expressing or overexpressing one or more disease-associated antigens on their cell surface can be tumor cells or non-tumor cells. In some cases, proliferation is an innate activity. In some cases, proliferation is at least partially, significantly (>about 20% or >about 25%), or entirely due to the presence of a binding domain that specifically binds to disease-associated antigens (e.g., tumor antigens, autoimmune disease-associated antigens, or pathogenic antigens) expressed on the surface of diseased cells and / or one or more modified CAR constructs described herein. In some cases, modified T cells exhibit higher levels of in vitro and / or in vivo proliferation compared to control cells of the same type. In some cases, control cells do not contain a CAR construct. In some cases, control cells contain a CAR construct lacking the binding domain, hinge region, transmembrane domain, intracellular signaling domain, and / or co-stimulatory domain described herein. In some cases, control cells do not contain one or more modifications described herein.

[0113] Modified T cells can exhibit robust and / or persistent proliferation in host organisms containing cells expressing or overexpressing one or more disease-associated antigens on their cell surface, such as tumor cells, cells of autoimmune diseases, pathogens, or pathogen-infected cells. In some cases, proliferation can persist for at least about 6 to 120 days, or at least about 6 to 180 days, from the first contact with disease-expressing cells or from the first administration of modified T cells to the host organism. In some cases, proliferation of the modified T cells described herein or their progeny in host organisms containing cells expressing or overexpressing one or more disease-associated antigens on their cell surface can persist for at least about 6 to 120 days, or at least about 6 to 180 days, from the first contact with cells expressing disease antigens or from the first administration of modified T cells to the host organism. In some cases, proliferation in the host organism is at least partially, significantly (> about 20% or > about 25%), or entirely due to the presence of a binding domain having a specific binding antigen or epitope within the antigen and / or one or more modified CAR constructs described herein.

[0114] In the embodiments, the modified T cells described herein express or sustainably express pro-inflammatory cytokines, such as tumor necrosis factor-α or interferon-γ, upon contact with cells expressing antigens. In the embodiments, the modified T cells described herein, or their progeny, express or sustainably express pro-inflammatory cytokines, such as tumor necrosis factor-α or interferon-γ, upon contact with cells expressing antigens (e.g., in a host organism containing cells expressing antigens).

[0115] In this embodiment, the modified T cells are γδ T cells. Specifically, the γδ T cells are δ1, δ2, δ3, or δ4 γδ T cells, preferably δ2. - γδ T cells, more preferably δ1 γδ T cells. In an embodiment, the modified T cells are δ1 γδ T cells. In an embodiment, the modified T cells are δ2 γδ T cells. In an embodiment, the modified T cells are δ3 γδ T cells. In an embodiment, the modified T cells are δ4 γδ T cells. In an embodiment, the modified T cells are δ2 γδ T cells. - γδ T cells. In the implementation scheme, the modified T cells are αβ T cells.

[0116] γδ T cells

[0117] In the implementation scheme, the modified T cells are modified γδ T cells. In the implementation scheme, when introduced into an allogeneic host, the modified γδ T cells or the pharmaceutical composition containing the modified γδ T cells substantially do not exhibit or do not exhibit graft-versus-host disease (GVHD). In the implementation scheme, when introduced into an allogeneic host, the modified γδ T cells or the pharmaceutical composition containing the modified γδ T cells exhibit a clinically acceptable level of GVHD. In the implementation scheme, a clinically acceptable level is the amount of GVHD that does not require discontinuation of modified γδ T cell therapy to achieve therapeutically effective treatment. In the implementation scheme, a clinically acceptable level of GVHD (GvHD) is an acute reaction less severe than Grade C, according to the applicable IBMTR grading scale. The severity of acute GVHD is determined by assessing the extent of involvement of the skin, liver, and gastrointestinal tract. The stages of involvement of each organ are combined to produce an overall grading, which has prognostic significance. Grade I(A) GvHD is characterized as mild, Grade II(B) as moderate, Grade III(C) as severe, and Grade IV(D) as life-threatening. The IBMTR grading system defines the severity of acute GvHD as follows (Rowlings et al., Br J Haematol 1997; 97:855): ●Grade A – Only stage 1 skin involvement (maculopapular rash on <25% of the body), without liver or gastrointestinal involvement. ●Grade B – Stage 2 skin involvement; Stage 1-2 intestinal or liver involvement ●Grade C – Stage 3 involving any organ system (generalized erythroderma; bilirubin 6.1 to 15.0 mg / dL; diarrhea 1500 to 2000 mL / day) ●Grade D – Stage 4 involving any organ system (generalized erythroderma with bullous formation; bilirubin >15 mg / dL; diarrhea >2000 mL / day or pain or intestinal obstruction).

[0118] See also Schoemans et al. Bone Marrow Transplantation Volume 53, pp. 1401–1415 (2018), for example in Tables 1 and 2, the literature discloses criteria for assessing and classifying acute GvHD.

[0119] In the implementation scheme, compared to graft-versus-host disease (GVHD) observed when control αβ T cells or control drug compositions containing control αβ T cells were administered to an allogeneic host, the modified γδ T cells or drug compositions containing modified γδ T cells exhibited reduced or significantly reduced GVHD when introduced into an allogeneic host. In some cases, the control αβ T cells are allogeneic, unengineered control αβ T cells. In some cases, the control αβ T cells do not contain a CAR or do not contain the same CAR as the reference γδ T cells.

[0120] The γδ T cells described herein can be δ1, δ2, δ3, or δ4 γδ T cells or combinations thereof. In some cases, the γδ T cells are predominantly (>50%), substantially (>90%), essentially all or entirely δ2 γδ T cells. In some cases, the γδ T cells are predominantly (>50%), substantially (>90%), essentially all or entirely δ1 γδ T cells. In some cases, the γδ T cells are predominantly (>50%), substantially (>90%), essentially all or entirely δ3 γδ T cells. In some embodiments, the γδ T cells are δ1 γδ T cells. In some embodiments, the γδ T cells are δ2 γδ T cells. In some embodiments, the γδ T cells are δ3 γδ T cells. In some embodiments, the γδ T cells are δ4 γδ T cells. In some embodiments, the γδ T cells are δ2 γδ T cells. - γδT cells.

[0121] The γδ T cells described herein may be obtained from an allogeneic donor. The γδ T cells may be partially or completely purified, or unpurified, and may be expanded in vitro. Methods and compositions for in vitro expansion include, but are not limited to, those described in WO 2017 / 197347. Expansion may be performed before or after, or before and after, the introduction of the CAR peptide and / or one or more modifications of this disclosure into γδ T cells. Other additional or alternative expansion methods include the use of, for example, artificial antigen-presenting cells (aAPCs), aminobisphosphonates, cytokine mixtures, and feeder cells (Cortés-Selva, D et al., (2021) Trends Pharmacol Sci. 42(1): 45-59).

[0122] According to one aspect, this document provides a plurality of modified γδ T cells as disclosed herein. In embodiments, the plurality of modified γδ T cells comprise a composition comprising at least 60%, 80%, 90%, or 95%, or about 60% or 80% to about 90% or 95% of δ1 γδ T cells. In embodiments, the plurality of modified γδ T cells comprise a composition comprising at least 60%, 80%, 90%, or 95%, or about 60% or 80% to about 90% or 95% of δ2 γδ T cells. In embodiments, the plurality of modified γδ T cells comprise a composition comprising at least 60%, 80%, 90%, or 95%, or about 60% or 80% to about 90% or 95% of δ3 γδ T cells. In embodiments, the plurality of modified γδ T cells comprise a composition comprising at least 60%, 80%, 90%, or 95%, or about 60% or 80% to about 90% or 95% of δ4 γδ T cells. In one embodiment, the plurality of modified γδ T cells comprise a composition comprising at least 60%, 80%, 90%, or 95%, or about 60% or 80% to about 90% or 95% δ1 or δ2 γδ T cells. In another embodiment, the plurality of modified γδ T cells comprise at least 60%, 80%, 90%, or 95%, or about 60% or 80% to about 90% or 95% δ2 γδ T cells. - A composition of γδ T cells.

[0123] In the implementation scheme, the modified γδ T cells include at least about 10 7 Modified γδ T cells, for example, at least 5 × 10⁶ 7 At least 10 8 At least 5×10 8 At least 10 9 At least 5×10 9 Or at least 10 10 Modified γδ T cells. In some instances, multiple modified γδ T cells comprise approximately 10 8Modified γδ T cells to approximately 10 11 Modified γδ T cells, for example, 10 7 Up to 10 8 5×10 7 Up to 5×10 8 10 8 Up to 10 9 5×10 8 Up to 5×10 9 , or 10 9 Up to 10 10 A modified γδ T cell.

[0124] Modified γδ T cells as described herein can be stored, for example, cryopreserved, for adoptive cell transfer. In one embodiment, the cells are stored before being engineered to express a CAR peptide and / or have one or more modifications as described herein. In another embodiment, γδ T cells are engineered to express a CAR peptide and / or have one or more modifications as described herein, and then the cells are stored.

[0125] Modification

[0126] In one aspect, the modified T cells of this invention comprise one or more modifications. These modifications include the expression of one or more exogenous proteins, the disruption of one or more endogenous genes, or a combination thereof. In embodiments, the modified T cells comprise a) at least one modification for regulating the disease microenvironment and / or at least one modification for regulating cytokine activity; and b) at least one allogeneic escape modification and / or at least one modification for regulating T cell metabolism.

[0127] In some instances, the modified T cells comprise at least one modification for regulating the disease microenvironment and at least one allogeneic escape modification. In some instances, the modified T cells comprise at least one modification for regulating the disease microenvironment and at least one modification for regulating the metabolism of the modified T cells. In some instances, the modified T cells comprise at least one modification for regulating cytokine activity and at least one allogeneic escape modification. In some instances, the modified T cells comprise at least one modification for regulating cytokine activity and at least one modification for regulating T cell metabolism. In some instances, the modified T cells comprise at least one modification for regulating the disease microenvironment, at least one modification for regulating cytokine activity, and at least one allogeneic escape modification. In some instances, the modified T cells comprise at least one modification for regulating the disease microenvironment, at least one modification for regulating cytokine activity, and at least one modification for regulating T cell metabolism. In some instances, the modified T cells comprise at least one modification for regulating the disease microenvironment, at least one allogeneic escape modification, and at least one modification for regulating the metabolism of the modified T cells. In some instances, the modified T cells comprise at least one modification for regulating cytokine activity and at least one allogeneic escape modification, as well as at least one modification for regulating the metabolism of the modified T cells. In some instances, the modified T cells comprise at least one modification for regulating the disease microenvironment, at least one modification for regulating cytokine activity, at least one allogeneic escape modification, and at least one modification for regulating the metabolism of the modified T cells.

[0128] One or more modifications for modulating the disease microenvironment can enhance the anti-disease activity of modified T cells in the disease microenvironment. In embodiments, at least one modification for modulating the disease microenvironment includes disruption of the CBL-B gene, disruption of the REGNASE-1 gene, disruption of the roquin gene, disruption of the TGF-β receptor type 2 (TGFβR2) gene, expression of dominant-negative TGF-β receptor type 2 (dnTGFβR2), or any combination thereof.

[0129] In some embodiments, the modified T cells contain at least one, at least two, at least three, at least four, or at least five modifications for modulating the disease microenvironment. In some embodiments, the modified T cells contain one modification for modulating the disease microenvironment. In some embodiments, the modified T cells contain two modifications for modulating the disease microenvironment. In some embodiments, the modified T cells contain three modifications for modulating the disease microenvironment. In some embodiments, the modified T cells contain four modifications for modulating the disease microenvironment. In some embodiments, the modified T cells contain five modifications for modulating the disease microenvironment.

[0130] In the implementation scheme, one or more modifications for modulating the disease microenvironment include disruption of the Cbl proto-oncogene B (CBL-B) gene. CBL-B is a negative regulator of T cell activation. Disruption of the CBL-B gene can provide a functional advantage in enhancing T cell activation relative to control cells with an intact CBL-B gene. In some instances, the CBL-B gene can be disrupted by methods described in Augustin R. et al., Targeting Cbl-b in cancer immunotherapy, J Immunother Cancer. Feb 2023; 11(2):e006007; Hooper K. et al., Knockout of CBLB Greatly Enhances Anti-Tumor Activity of CAR T Cells, Blood (2018) 132 (Supplement 1): 338; and Guo X. et al., CBLBablation with CRISPR / Cas9 enhances cytotoxicity of human placental stem cell-derived NK cells for cancer immunotherapy, J Immunother Cancer. Mar 2021; 9(3):e001975, each of which is incorporated herein by reference in its entirety. In one instance, the CBL-B gene was disrupted via gene editing using a CRISPR-Cas system comprising one or more guide RNAs containing any one of SEQ ID NO: 341-344.

[0131] In the implementation scheme, one or more modifications for modulating the disease microenvironment include disruption of the Regnase-1 gene. Regnase-1 (regulatory RNase 1), also known as ZC3H12A or MCPIP-1, is a ribonuclease that promotes the decay of target mRNA by recognizing a 3' UTR stem-loop motif. Disruption of the Regnase-1 gene can provide a functional advantage in enhancing T cell proliferation and persistence relative to control cells with an intact Regnase-1 gene. In one instance, disruption of the Regnase-1 gene can be described as in Jun Wei et al., Targeting Regnase-1 programs long-lived effector T cells for cancer therapy. Nature. Dec. 2019; 576(7787): 471–476, which is incorporated herein by reference in its entirety.

[0132] In the implementation scheme, one or more modifications for modulating the disease microenvironment include disruption of the Roquin (e.g., Roquin-1) gene. Disruption of the Roquin gene can provide a functional advantage relative to control cells with the intact Roquin gene in terms of increased T cell proliferation and enhanced antitumor activity. In some instances, the Roquin gene can be disrupted by the method described in Mai D et al., Combined disruption of T cell inflammatory regulators Regnase-1 and Roquin-1 enhances antitumor activity of engineered human T cells, Proc Natl Acad Sci US A. 2023 Mar 21;120(12):e2218632120, which is incorporated herein by reference in its entirety.

[0133] In embodiments, one or more modifications for modulating the disease microenvironment include expressing peptides that confer T cells resistance to disease-associated antigen-specific cellular immunity (e.g., cellular immunity mediated by transforming growth factor β (TGF-β)). For example, modifications for modulating the disease microenvironment may express a dominant-negative receptor for TGF-β (dnTGFβR2), as described, for example, in Foster et al., J Immunother. (2008); 31: 500-505, WO2019 / 173324A1, WO2020 / 183131A1, and WO2020042647A1. In the presence of disease cells (e.g., tumor cells) that secrete TGF-β, incorporation of such a dominant-negative TGF-β receptor may provide a functional advantage relative to control cells lacking such a dominant-negative TGF-β receptor, including enhanced anti-disease (e.g., anti-tumor) activity. In an embodiment, the modification for modulating the disease microenvironment includes expressing a polypeptide comprising or consisting of the amino acid sequence of the dominant-negative TGFβ receptor II (dnTGFβR2) as shown in SEQ ID NO: 241. In an embodiment, the polypeptide further comprises a signal peptide operatively linked to SEQ ID NO: 241 (e.g., comprising or consisting of the amino acid sequence shown in SEQ ID NO: 239).

[0134] In the implementation scheme, one or more modifications for modulating the disease microenvironment include disruption of the TGFβR2 gene. Disruption of the TGFβR2 gene can reduce the immunosuppressive effect of TGFβ signaling. In some instances, the TGFβR2 gene can be disrupted by the method described in Na Tang et al., TGF-β inhibition via CRISPR promotes the long-term efficacy of CAR T cells against solid tumors, JCI Insight. 27 Feb 2020; 5(4): e133977, which is incorporated herein by reference in its entirety.

[0135] One or more modifications for modulating cytokine activity can enhance the activation of modified T cells and / or their sensitivity to cytokines, and / or reduce the toxicity of cytokines produced by modified T cells. In embodiments, at least one modification for modulating cytokine activity includes the expression of membrane-bound IL-12, disruption of the CISH gene, expression of a cytokine switching receptor, or any combination thereof.

[0136] In one embodiment, the modified T cell contains at least one, at least two, or at least three modifications for regulating cytokine activity. In another embodiment, the modified T cell contains one modification for regulating cytokine activity. In yet another embodiment, the modified T cell contains two modifications for regulating cytokine activity. In yet another embodiment, the modified T cell contains three modifications for regulating cytokine activity.

[0137] In the implementation scheme, one or more modifications for modulating cytokine activity include expression of membrane-bound IL-12 (mbIL-12). Incorporation of this mbIL-12 into T cells can provide a functional advantage over control cells lacking this mbIL-12 in enhancing T cell effector function and / or limiting IL-12-related systemic toxicity. Examples of mbIL-12 include those described in Hu J. et al., Cell membrane-anchored and tumor-targeted IL-12 (attIL12)-T cell therapy for eliminating large and heterogeneous solid tumors, J Immunother Cancer. Jan 2022;10(1):e003633; Hombach A. et al., IL12 integrated into the CAR exodomain converts CD8+ T cells to poly-functional NK-like cells with superior killing of antigen-loss tumors, Mol Ther. Feb 2 2022;30(2):593-605; and Lee EH et al., Antigen-dependent IL-12 signaling in CART cells promotes regional to systemic disease targeting, bioRxiv. Jan 7 2023;2023.01.06.522784, each of which is incorporated herein by reference in its entirety.

[0138] In the implementation scheme, one or more modifications for regulating cytokine activity include disruption of a cytokine-induced SH2-containing protein (CISH) gene. CISH is a negative regulator of TCR signaling. Disruption of the CISH gene can provide functional advantages relative to control cells with an intact CISH gene in improving sensitivity to certain cytokines (e.g., IL-2 / IL-15), increasing T cell proliferation, and / or limiting T cell exhaustion. In some instances, the CISH gene can be disrupted by the method described in Daher M. et al., Targeting a cytokine checkpoint enhances the fitness of armored cordblood CAR-NK cells, Blood. 2021 Feb 4;137(5):624-636, which is incorporated herein by reference in its entirety. In some instances, the CISH gene is disrupted by gene editing using an RNA-guided nuclease system comprising one or more guide RNAs containing sequences of any one of SEQ ID NO: 198-201 and 339-340.

[0139] In embodiments, one or more modifications for modulating cytokine activity include expression of a cytokine conversion receptor, such as a chimeric conversion receptor, comprising an extracellular domain of a TGFβ receptor for binding TGFβ (e.g., TGFβRI and / or TGFβRII) and an intracellular domain of a cytokine receptor. The chimeric conversion receptor can convert TGFβ signaling into cytokine signaling that promotes cytotoxicity. Examples of such chimeric conversion receptors include those described in WO2012138858, WO2016122738, WO2018094244, WO2014172584, WO2019109980, and WO2022037562, each of which is incorporated herein by reference in its entirety.

[0140] One or more allogeneic escape modifications can reduce host resistance to graft allogeneic cytotoxicity. In embodiments, at least one allogeneic escape modification includes disruption of the ICAM-1 gene, disruption of the CD58 gene, disruption of the Fas gene, expression of a dominant-negative Fas receptor (dnFas), expression of a chimeric antigen receptor (CAR) that binds to CD70, or any combination thereof.

[0141] In some embodiments, the modified T cells contain at least one, at least two, at least three, at least four, or at least five allogeneic escape modifications. In some embodiments, the modified T cells contain one allogeneic escape modification. In some embodiments, the modified T cells contain two allogeneic escape modifications. In some embodiments, the modified T cells contain three allogeneic escape modifications. In some embodiments, the modified T cells contain four allogeneic escape modifications. In some embodiments, the modified T cells contain five allogeneic escape modifications.

[0142] In the implementation scheme, one or more allogeneic escape modifications include disruption of the ICAM gene. Disruption of the ICAM-1 gene can provide a functional advantage relative to control cells with an intact ICAM-1 gene in disrupting T cell adhesion and co-stimulatory interactions to reduce host resistance to graft allogeneic cytotoxicity. In one instance, the ICAM-1 gene can be disrupted as described in Teo HY et al. IL12 / 18 / 21 Preactivation Enhances the Antitumor Efficacy of Expanded γδTCells and Overcomes Resistance to Anti-PD-L1 Treatment, Cancer Immunol Res. 2023 July 5;11(7):978-999, which is incorporated herein by reference in its entirety. In one instance, the ICAM-1 gene is disrupted by gene editing using an RNA-guided nuclease system (e.g., a CRISPR-Cas or CRISPR-Mad7 system) comprising one or more guide RNAs containing sequences of any one of SEQ ID NO: 349-350.

[0143] In the implementation scheme, one or more allogeneic escape modifications include disruption of the CD58 gene. Disruption of the CD58 gene can provide a functional advantage relative to control cells with an intact CD58 gene in disrupting T cell adhesion and co-stimulatory interactions to reduce host resistance to graft allogeneic cytotoxicity. In one example, the CD58 gene is disrupted via gene editing using an RNA-guided nuclease system (e.g., a CRISPR-Cas or CRISPR-Mad7 system), said RNA-guided nuclease system comprising one or more guide RNAs containing sequences comprising any one of SEQ ID NO: 351-352.

[0144] In some instances, the modified T cells contain disruptions to both the CD58 and ICAM-1 genes.

[0145] In the implementation scheme, one or more allogeneic escape modifications include disruption of the Fas (also known as CD95) gene. Disruption of the Fas gene can provide a functional advantage in reducing T cell allogeneic cytotoxicity relative to control cells with an intact Fas gene. In one example, the ICAM-1 gene can be disrupted as described in Jiangtao Ren et al., A versatile system for rapid multiplex genome-edited CAR T cell generation, Oncotarget. 2017 Mar 7; 8(10): 17002–17011, which is incorporated herein by reference in its entirety.

[0146] In the implementation scheme, one or more allogeneic escape modifications include the expression of dominant-negative Fas (dnFas, also known as dnCD95). Incorporation of such dominant-negative Fas into T cells provides a functional advantage relative to control cells lacking such dominant-negative Fas in preventing Fas ligand-induced apoptosis and allowing T cell persistence and disease-fighting (e.g., antitumor) efficacy. Examples of dnFas include those described in Yamamoto TN et al., T cells genetically engineered to overcome death signaling enhance adoptive cancer immunotherapy, J Clin Invest. 2019 Feb 25;129(4):1551-1565, which are incorporated herein by reference in their entirety.

[0147] In the embodiments, one or more allogeneic escape modifications include the expression of an antibody or fragment thereof that binds to CD70. For example, the modification could be the expression of a CAR containing such an antibody or fragment that binds to CD70. Incorporation of such a CD70-binding molecule into T cells can provide a functional advantage over control cells lacking a CD70-binding molecule in reducing HvG allogeneic reactivity by targeting CD70+ activated T cells. Examples of such CD70-binding molecules include those described in PCT / US2023 / 29047, which is incorporated herein by reference in its entirety.

[0148] One or more modifications for regulating T cell metabolism can modulate the metabolism of molecules (e.g., nucleic acids, glucose, etc.) in T cells to enhance T cell activity, survival, and proliferation. In embodiments, at least one modification for regulating T cell metabolism includes disruption of the RASA2 gene, disruption of the MED12 gene, or any combination thereof. In embodiments, the modified T cells contain one or more modifications for regulating the disease microenvironment, as well as one or more modifications for regulating T cell metabolism that are different from the one or more modifications for regulating the disease microenvironment.

[0149] In one embodiment, the modified T cell contains at least one or at least two modifications for regulating T cell metabolism. In another embodiment, the modified T cell contains at least two modifications for regulating T cell metabolism.

[0150] In the implementation scheme, one or more modifications for regulating T cell metabolism include disruption of the RAS p21 protein activator 2 (RASA2) gene. RASA2 is a RAS GTPase activator protein (RasGAP) that serves as a signal transduction checkpoint in T cells; it is downregulated upon acute T cell receptor stimulation and gradually increases with chronic antigen exposure. Disruption of RASA2 enhances MAPK signaling in response to target antigens and the cytolytic activity of CAR T cells. Compared to control cells, RASA2-deficient T cells exhibit increased activation, cytokine production, and metabolic activity, and demonstrate a significant advantage in durable cancer cell killing. In some instances, the RASA2 gene can be disrupted by the method described in Julia Carnevale et al., RASA2 ablationin T cells boosts antigen sensitivity and long-term function. Nature. Sep 2022;609(7925):174-182, which is incorporated herein by reference in its entirety.

[0151] In the implementation scheme, one or more modifications for regulating T cell metabolism include disruption of the MED12 gene. Mediator complex subunit 12 (MED12) is a mediator subunit that modulates metabolic activity and adaptations, such as increasing glycolysis, oxidative phosphorylation, and spare respiratory capacity. Disruption of the MED12 gene can provide a functional advantage over control cells with an intact MED12 gene in enhancing T cell activation and effector function. In one instance, the MED12 gene can be disrupted as described in Katherine A Freitas et al., Enhanced T cell effector activity by targeting the Mediator kinase module, Science. 2022 Nov 11;378(6620):eabn5647, which is incorporated herein by reference in its entirety.

[0152] In some instances, the modified T cells contain at least one modification for regulating cytokine activity, including the expression of membrane-bound IL-12; and at least one modification for regulating T cell metabolism, including disruption of the MED12 gene. In some embodiments, the modified T cells also contain disruption of the Fas gene or expression of dnFas. In some embodiments, the modified T cells also contain disruption of the TGFβR2 gene or expression of dnTGFβR2.

[0153] In some instances, the modified T cells contain at least one modification for regulating the disease microenvironment, including disruption of the TGFβR2 gene or expression of dnTGFβR2; and at least one modification for regulating T cell metabolism, including disruption of the MED12 gene. In some embodiments, the modified T cells also contain disruption of the Fas gene or expression of dnFas. In some embodiments, the modified T cells contain at least one modification for regulating cytokine activity, including expression of membrane-bound IL-12.

[0154] In some instances, the modified T cells contain disruptions to the CISH gene, CBL-B gene, ICAM-1 gene, CD58 gene, Roquin-1 gene, Regnase-1 gene, TGFβR2 gene, RASA2 gene, MED12 gene, or combinations thereof.

[0155] In some instances, the modified T cells contain one or more heterologous cytokine receptors expressed on the cell surface, such as dnTGFβR2, cytokine switching receptor, or both.

[0156] Other modifications

[0157] The modified T cells described in this article may contain one or more modifications in addition to those described above.

[0158] In the implementation scheme, one or more additional modifications include disruption of the ZFP91 gene. Disruption of the ZFP91 gene can provide a functional advantage relative to control cells with an intact ZFP91 gene in terms of improving T cell glycolytic adaptation and effector function. In some instances, the ZFP91 gene can be disrupted by the method described in Wang F. et al., J Clin Invest. 1 Oct 2021;131(19):e144318, which is incorporated herein by reference in its entirety.

[0159] In the implementation scheme, one or more additional modifications include the expression of exogenous stem cell factor (SCF). The term “stem cell factor (SCF)” refers to a cytokine that exerts its biological function by binding to and activating the receptor tyrosine kinase c-Kit. Endogenous SCF is a stromal cell-derived cytokine synthesized by fibroblasts and other cell types. It is a glycoprotein that plays a key role in hematopoiesis as both a positive and negative regulator, often working synergistically with other cytokines. It also plays a role in mast cell development, gameteogenesis, and melanogenesis. SCF regulates cell viability, proliferation, and differentiation under physiological and pathological conditions through c-Kit interactions (see, for example, Mazzoldi, EL et al., Ajuxtacrine / paracrine loop between C-Kit and stem cell factor promotes cancerstem cell survival in epithelial ovarian cancer. Cell Death Dis 10, 412 (2019)). In the implementation scheme, the SCF includes a soluble form of SCF (e.g., containing EGICRNRVTNNVKDVTKLVANLPKDYMITLKYVPGMDVLPSHCWISEMVVQLSDSLTDLLDKFSNISEGLSNYSIIDKLVNIVDDLVECVKENSSKDLKKSFKSPEPRLFTPEEFFRIFNRSIDAFKDFVVASETSDCVVSSTLSPEKDSRVSVTKPFMLPPVAA (based on NP_000890, the first 165 aa of the mature peptide without the signal peptide, SEQ ID NO: 105) or composed of the same).In the implementation, the SCF includes or is composed of a transmembrane form of SCF (e.g., a mature peptide containing EGICRNRVTNNVKDVTKLVANLPKDYMITLKYVPGMDVLPSHCWISEMVVQLSDSLTDLLDKFSNISEGLSNYSIIDKLVNIVDDLVECVKENSSKDLKKSFKSPEPRLFTPEEFFRIFNRSIDAFKDFVVASETSDCVVSSTLSPEKDSRVSVTKPFMLPPVAASSLRNDSSSSNRKAKNPPGDSSLHWAAMALPALFSLIIGFAFGALYWKKRQPSLTRAVENIQINEEDNEISMLQEKEREFQEV (a human SCF based on NP_000890, without a signal peptide) or is also described in Aderson DM et al. Cell Growth Differ. Aug 1991;2(8):373-8), SEQ ID NO: 103), or containing EGICRNRVTNNVKDVTKLVANLPKDYMITLKYVPGMDVLPSHCWISEMVVQLSDSLTDLLDKFSNISEGLSNYSIIDKLVNIVDDLVECVKENSSKDLKKSFKSPEPRLFTPEEFFRIFNRSIDAFKDFVVASETSDCVVSSTLSPEKGKAKNPPGDSSLHWAAMALPALFSLIIGFAFGALYWKKRQPSLTRAVENIQINEEDNEISMLQEKEREFQEV (based on NP_NP_003985, a mature peptide without a signal peptide) or composed of it, see also Johan Lennartsson et al. Physiol Rev. 2012 Oct;92(4):1619-49, SEQ ID NO: 104).Examples of soluble and transmembrane forms of SCF also include those described in the following: J GFlanagan et al., Transmembrane form of the kit ligand growth factor is determined by alternative splicing and is missing in the Sld mutant Cell. March 8, 1991; 64(5):1025-35; DM Anderson et al., Alternate splicing of mRNAsencoding human mast cell growth factor and localization of the gene tochromosome 12q22-q24, Cell Growth Differ. August 1991; 2(8):373-8; and Johan Lennartsson et al., Stem cell factor receptor / c-Kit: from basic science to clinical implications, Physiol Rev. October 2012; 92(4):1619-49, each of which is incorporated herein by reference in its entirety.

[0160] In the implementation plan, the exogenous SCF can be a recombinant or synthetic stem cell factor, such as ancestim, a 166-amino acid protein produced by E. coli bacteria, in which inserts such as McNiece IK and Briddel RA, The Cytokine Handbook (4th edition), 2003; and MG da Silva et al. Ancestim (recombinant human stem cell factor, SCF) in association with filgrastim does not enhance chemotherapy and / or growth factor-induced peripheral blood progenitor cell (PBPC) mobilization in patients with a prior insufficient PBPC collection, Bone Marrow Transplant. The gene for soluble human stem cell factor (MEGICRNRVTNNVKDVTKLVANLPKDYMITLKYVPGMDVLPSHCWISEMVVQLSDSLTDLLDKFSNISEGLSNYSIIDKLVNIVDDLVECVKENSSKDLKKSFKSPEPRLFTPEEFFRIFNRSIDAFKDFVVASETSDCVVSSTLSPEKDSRVSVTKPFMLPPVAA, SEQ ID NO: 101) described in October 2004;34(8):683-91, each of which is incorporated herein by reference in its entirety.

[0161] Further examples of recombinant and synthetic stem cell factor molecules include those described in WO1991005795 (titled “Stemcell factor”), US6020469 (titled “Stem cell factor formulations and methods”), US6759215 (titled “Method of preparing human stem cell factor polypeptide”), and US6218148 (titled “DNS encoding stem cell factor”), each of which is incorporated herein by reference in its entirety. In embodiments, the exogenous SCF is an SCF analog peptide, for example, those described in US5885962 (titled “Stem cell factor analog compositions and method”); and Tilayov T. et al., Engineering Stem Cell Factor Ligands with Different c-Kit AgonisticPotencies, Molecules. 21 Oct 2020;25(20):4850, each of which is incorporated herein in its entirety.

[0162] In the implementation scheme, one or more additional modifications include disruption of the Arid1a gene. Arid1a regulates the acquisition of the terminal exhaustion epigenetic state. Disruption of the Arid1a gene can provide a functional advantage relative to control cells with the intact Arid1a gene in terms of improved tumor control and enhanced T cell persistence. In one instance, the Arid1a gene can be disrupted as described in Julia ABelk et al., Genome-wide CRISPR screens of T cell exhaustion identify chromatin remodeling factors that limit T cell persistence, Cancer Cell. 2022 July 11;40(7):768-786.e7.

[0163] In embodiments, one or more additional modifications include one or more tags or markers, such as those to facilitate the monitoring of expression levels of antigen recognition moieties (e.g., CARs), for use as internal controls, etc. In embodiments, one or more additional modifications include the expression of fluorescent proteins, examples of which include, but are not limited to, green fluorescent protein (GFP), red fluorescent protein (RFP), enhanced GFP (EGFP), enhanced cyan fluorescent protein (ECFP), enhanced yellow fluorescent protein (EYFP), etc. Other examples may include, but are not limited to, chloramphenicol acetyltransferase, β-galactosidase, β-glucuronidase, β-lactamase, luciferase, etc.

[0164] In embodiments, one or more additional modifications include expressing a protein on the cell surface to facilitate the detection and / or isolation of cells expressing that protein, e.g., via fluorescence activated cell sorting (FACS); or enriching the protein by positive selection using an antibody specific for the encoded protein, e.g., using an antibody to purify or enrich cell products on a column or device; or binding the antibody to the protein in vivo to enhance or eliminate its activity, e.g., to facilitate the removal of cells expressing that protein from a patient for safety reasons. Exemplary proteins that can be used for these purposes include, for example, CD19, CD20 (Rituxumab recognition domain), RQR8, LNGFR, truncated forms of human epidermal growth factor receptor (EGFRt), etc. By way of example, EGFRt can be targeted by a clinical-stage antibody, wherein such treatment of a patient with the antibody results in the elimination of isolated nucleic acids containing an antigen recognition portion (e.g., CAR) as disclosed herein. See, for example, Wang et al., A transgene-encoded cell surface polypeptide for selection, in vivo tracking, and ablation of engineered cells; Blood 2011 118(5):1255-63; Philip et al., A highly compact epitope-based marker / suicide gene for easier and safer T-cell therapy; Blood2014 124(8):1277-87; Smith J. et al., UCART19, an allogeneic “off-the-shelf” adoptive T-cell immunotherapy against CD19+ B-cell leukemias, DOI: 10.1200 / jco.2015.33.15_suppl.3069 Journal of Clinical Oncology 33, Issue 15_Supplement (May 20, 2015) 3069-3069; Gouble A. et al., InVivo Proof of Concept of Activity and Safety of UCART19, an Allogeneic “Off-the-Shelf” Adoptive T-Cell Immunotherapy Against CD19+ B-Cell Leukemias, Blood (2014) 124 (21): 4689, doi.org / 10.1182 / blood.V124.21.4689.4689, each of the references is incorporated in its entirety by reference.

[0165] In the implementation scheme, one or more additional modifications include the expression of proteins that have the function of increasing resistance to exhaustion and activation-induced apoptosis and / or upregulating one or more pro-inflammatory cytokines, co-stimulatory molecules, and / or antigen presentation mechanisms. Representative examples include, but are not limited to, the lymphotoxin β receptor (LTBR). LTBR is typically expressed in a subset of myeloid cells but is absent in lymphocytes. When expressed in T cells, LTBR can induce transcriptional remodeling, thereby conferring one or more of the aforementioned beneficial functions to T cells (Legut et al., Blood. (2021); 138(1): 1726).

[0166] In embodiments, the signal peptide is operatively linked to an expressed protein to facilitate the directing of one or more proteins expressed in the modified T cell to a secretory pathway. Such proteins may be those residing within certain organelles, secreted from the modified T cell, or inserted into the cell membrane. In embodiments, the signal peptide comprises or is composed of the amino acid sequence shown in SEQ ID NO: 170. In embodiments, the signal peptide comprises or is composed of the amino acid sequence shown in SEQ ID NO: 196. In embodiments, the signal peptide comprises or is composed of the amino acid sequence shown in SEQ ID NO: 235. In embodiments, the signal peptide comprises or is composed of the amino acid sequence shown in SEQ ID NO: 239. In embodiments, the signal peptide comprises or is composed of the amino acid sequence shown in SEQ ID NO: 243. In embodiments, the signal peptide comprises or is composed of the amino acid sequence shown in SEQ ID NO: 247.

[0167] In embodiments, one or more additional modifications include the expression of a polypeptide comprising or consisting of the EGFRt amino acid sequence as shown in SEQ ID NO: 237. In embodiments, one or more additional modifications include the expression of a polypeptide comprising or consisting of the full-length LTBR amino acid sequence as shown in SEQ ID NO: 245. In embodiments, a signal peptide comprising or consisting of the amino acid sequence as shown in SEQ ID NO: 243 is operatively linked to SEQ ID NO: 245. In embodiments, one or more additional modifications include the expression of a polypeptide comprising or consisting of the LNGFR amino acid sequence as shown in SEQ ID NO: 249. In embodiments, a signal peptide comprising or consisting of the amino acid sequence as shown in SEQ ID NO: 247 is operatively linked to SEQ ID NO: 249. In embodiments, one or more additional modifications include the expression of a polypeptide comprising or consisting of the sIL-15 amino acid sequence as shown in SEQ ID NO: 197. In embodiments, a signal peptide comprising or consisting of the amino acid sequence as shown in SEQ ID NO: 196 is operatively linked to SEQ ID NO: 197. In the implementation, a signal peptide comprising the amino acid sequence MKKTQTWILTCIYLQLLLFNPLVKT or composed thereof is operatively linked to an SCF sequence, for example, SEQ ID NO: 101, 103, 104 or 101.

[0168] In embodiments, one or more additional modifications include the expression of a chimeric DAP10 adaptor polypeptide capable of associating with the chimeric antigen receptor of this disclosure, for example, the CAR of this disclosure may contain a DAP10 interacting domain. In such instances, the chimeric DAP10 adaptor polypeptide may also associate with one or more other endogenous or exogenous polypeptides (e.g., endogenous or exogenous NKG2D). In embodiments, the chimeric DAP10 adaptor polypeptide may not associate with the CAR of this disclosure. For example, the modified T cells of this invention may also contain DAP10 and / or DAP12 interacting domains in the absence of a chimeric antigen receptor, and alternatively may interact with endogenous or exogenous polypeptides (e.g., NKG2D) containing a DAP10 interacting domain. Examples of chimeric DAP10 adaptor peptides and encoding nucleic acid sequences include those described in PCT Application Publication No. WO 2023 / 076523 and PCT Application Publication No. WO 2023 / 235440, the contents of each of which are hereby expressly incorporated herein by reference in their entirety. Examples of chimeric DAP10 and DAP12 adaptors include those described in WO 2024 / 209084 and Obajdin J et al., Solid tumor immunotherapy using NKG2D-based adaptor CAR Tcells, Cell Rep Med. 2024 Nov 19;5(11):101827, each of which is incorporated herein by reference in its entirety.

[0169] In the implementation scheme, one or more additional modifications include the disruption of one or more endogenous genes in addition to those described above. For example, one or more additional modifications include disruption of one or more of the following: adenosine A2a receptor (ADORA), CD276, V-domain-containing T-cell activation inhibitor 1 (VTCN1), B and T lymphocyte-associated protein (BTLA), cytotoxic T-lymphocyte-associated protein 4 (CTLA4), indoleamine 2,3-dioxygenase 1 (IDO1), killer cell immunoglobulin-like receptor, three domains, long cytoplasmic tail, 1 (KIR3DL1), lymphocyte activation gene 3 (LAG3), programmed cell death 1 (PD-1), hepatitis A virus cell receptor 2 (HAVCR2), T-cell activation V-domain immunoglobulin inhibitor (VISTA), natural killer cell receptor 2B4 (CD244), hypoxanthine phosphoribosyltransferase 1 (HPRT), and adeno-associated virus integration site (AAVSSITE). (e.g., AAVS1, AAVS2, etc.)), or chemokine (CC motif) receptor 5 (gene / pseudogene) (CCR5), CD160 molecule (CD160), T cell immune receptor with Ig and ITIM domains (TIGIT), CD96 molecule (CD96), cytotoxic and regulatory T cell molecule (CRTAM), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), sialic acid-binding Ig-like lectin 7 (SIGLEC7), sialic acid-binding Ig-like lectin 9 (SIGLEC9), tumor necrosis factor receptor superfamily member 10b (TNFRSF10B), tumor necrosis factor receptor superfamily member 10a (TNFRSF10A), caspase 8 (CASP8), caspase 10 (CASP10), caspase 3 (CASP3), caspase 6 (CASP6), caspase 7 (CASP7), Fas-associated via death domain protein.FADD), Fas cell surface death receptor (FAS), transforming growth factor β receptor II (TGFBRII), transforming growth factor β receptor I (TGFBR1), SMAD family member 2 (SMAD2), SMAD family member 3 (SMAD3), SMAD family member 4 (SMAD4), SKI proto-oncogene (SKI), SKI-like proto-oncogene (SKIL), TGFB inducible factor homeobox 1 (TGIF1), interleukin 10 receptor subunit α (IL10RA), interleukin 10 receptor subunit β (IL10RB), heme oxygenase 2 (HMOX2), interleukin 6 receptor (IL6R), interleukin 6 signal transducer (IL6ST), c-src tyrosine kinase (CSK), phosphoprotein membrane anchor 1 with glycosphingolipid microdomain (PAG1), signal transduction threshold regulating transmembrane adaptor 1 (SIT1), forkhead box P3 (FOXP3), PR domain 1 (PRDM1), ATF-like basic leucine zipper transcription factor (BATF), soluble guanylate cyclase 1 α2 (GUCY1A2), soluble guanylate cyclase 1 α3 (GUCY1A3), soluble guanylate cyclase 1 β2 (GUCY1B2), soluble guanylate cyclase 1 β3 (GUCY1B3), prolyl hydroxylase domains (PHD1, PHD2, PHD3) protein families, or any combination thereof.

[0170] In the implementation scheme, one or more additional modifications include disruption of the ZFP91 gene. Disruption of the ZFP91 gene can provide a functional advantage relative to control cells with an intact ZFP91 gene in terms of improving T cell glycolytic adaptation and effector function. In some instances, the ZFP91 gene can be disrupted by the method described in Wang F. et al., J Clin Invest. 1 Oct 2021;131(19):e144318, which is incorporated herein by reference in its entirety.

[0171] In the implementation scheme, one or more additional modifications include the expression of exogenous stem cell factor (SCF). The term “stem cell factor (SCF)” refers to a cytokine that exerts its biological function by binding to and activating the receptor tyrosine kinase c-Kit. Endogenous SCF is a stromal cell-derived cytokine synthesized by fibroblasts and other cell types. It is a glycoprotein that plays a key role in hematopoiesis as both a positive and negative regulator, often working synergistically with other cytokines. It also plays a role in mast cell development, gameteogenesis, and melanogenesis. SCF regulates cell viability, proliferation, and differentiation under physiological and pathological conditions through c-Kit interactions (see, for example, Mazzoldi, EL et al., Ajuxtacrine / paracrine loop between C-Kit and stem cell factor promotes cancerstem cell survival in epithelial ovarian cancer. Cell Death Dis 10, 412 (2019)). In the implementation scheme, the SCF includes a soluble form of SCF (e.g., containing EGICRNRVTNNVKDVTKLVANLPKDYMITLKYVPGMDVLPSHCWISEMVVQLSDSLTDLLDKFSNISEGLSNYSIIDKLVNIVDDLVECVKENSSKDLKKSFKSPEPRLFTPEEFFRIFNRSIDAFKDFVVASETSDCVVSSTLSPEKDSRVSVTKPFMLPPVAA (based on NP_000890, the first 165 aa of the mature peptide without the signal peptide, SEQ ID NO: 105) or composed of the same).In the implementation, the SCF includes or is composed of a transmembrane form of SCF (e.g., a mature peptide containing EGICRNRVTNNVKDVTKLVANLPKDYMITLKYVPGMDVLPSHCWISEMVVQLSDSLTDLLDKFSNISEGLSNYSIIDKLVNIVDDLVECVKENSSKDLKKSFKSPEPRLFTPEEFFRIFNRSIDAFKDFVVASETSDCVVSSTLSPEKDSRVSVTKPFMLPPVAASSLRNDSSSSNRKAKNPPGDSSLHWAAMALPALFSLIIGFAFGALYWKKRQPSLTRAVENIQINEEDNEISMLQEKEREFQEV (a human SCF based on NP_000890, without a signal peptide) or is also described in Aderson DM et al. Cell Growth Differ. Aug 1991;2(8):373-8), SEQ ID NO: 103), or containing EGICRNRVTNNVKDVTKLVANLPKDYMITLKYVPGMDVLPSHCWISEMVVQLSDSLTDLLDKFSNISEGLSNYSIIDKLVNIVDDLVECVKENSSKDLKKSFKSPEPRLFTPEEFFRIFNRSIDAFKDFVVASETSDCVVSSTLSPEKGKAKNPPGDSSLHWAAMALPALFSLIIGFAFGALYWKKRQPSLTRAVENIQINEEDNEISMLQEKEREFQEV (based on NP_NP_003985, a mature peptide without a signal peptide) or composed of it, see also Johan Lennartsson et al. Physiol Rev. 2012 Oct;92(4):1619-49, SEQ ID NO: 104).Examples of soluble and transmembrane forms of SCF also include those described in the following: J GFlanagan et al., Transmembrane form of the kit ligand growth factor is determined by alternative splicing and is missing in the Sld mutant Cell. March 8, 1991; 64(5):1025-35; DM Anderson et al., Alternate splicing of mRNAsencoding human mast cell growth factor and localization of the gene tochromosome 12q22-q24, Cell Growth Differ. August 1991; 2(8):373-8; and Johan Lennartsson et al., Stem cell factor receptor / c-Kit: from basic science to clinical implications, Physiol Rev. October 2012; 92(4):1619-49, each of which is incorporated herein by reference in its entirety.

[0172] In the implementation plan, the exogenous SCF can be a recombinant or synthetic stem cell factor, such as ancestim, a 166-amino acid protein produced by E. coli bacteria, in which inserts such as McNiece IK and Briddel RA, The Cytokine Handbook (4th edition), 2003; and MG da Silva et al. Ancestim (recombinant human stem cell factor, SCF) in association with filgrastim does not enhance chemotherapy and / or growth factor-induced peripheral blood progenitor cell (PBPC) mobilization in patients with a prior insufficient PBPC collection, Bone Marrow Transplant. The gene for soluble human stem cell factor (MEGICRNRVTNNVKDVTKLVANLPKDYMITLKYVPGMDVLPSHCWISEMVVQLSDSLTDLLDKFSNISEGLSNYSIIDKLVNIVDDLVECVKENSSKDLKKSFKSPEPRLFTPEEFFRIFNRSIDAFKDFVVASETSDCVVSSTLSPEKDSRVSVTKPFMLPPVAA, SEQ ID NO: 101) described in October 2004;34(8):683-91, each of which is incorporated herein by reference in its entirety.

[0173] Further examples of recombinant and synthetic stem cell factor molecules include those described in WO1991005795 (titled “Stemcell factor”), US6020469 (titled “Stem cell factor formulations and methods”), US6759215 (titled “Method of preparing human stem cell factor polypeptide”), and US6218148 (titled “DNS encoding stem cell factor”), each of which is incorporated herein by reference in its entirety. In embodiments, the exogenous SCF is an SCF analog peptide, for example, those described in US5885962 (titled “Stem cell factor analog compositions and method”); and Tilayov T. et al., Engineering Stem Cell Factor Ligands with Different c-Kit AgonisticPotencies, Molecules. 21 Oct 2020;25(20):4850, each of which is incorporated herein in its entirety.

[0174] In the implementation scheme, one or more additional modifications include disruption of the Arid1a gene. Arid1a regulates the acquisition of the terminal exhaustion epigenetic state. Disruption of the Arid1a gene can provide a functional advantage relative to control cells with the intact Arid1a gene in terms of improved tumor control and enhanced T cell persistence. In one instance, the Arid1a gene can be disrupted as described in Julia ABelk et al., Genome-wide CRISPR screens of T cell exhaustion identify chromatin remodeling factors that limit T cell persistence, Cancer Cell. 2022 July 11;40(7):768-786.e7.

[0175] In embodiments, one or more modifications described herein increase the activity and / or function of the modified T cells (e.g., increased cytotoxicity and / or potency when used as T-cell therapy) compared to corresponding T cells that do not contain one or more such modifications. In embodiments, the modified T cells comprise disruption of the MED12 gene and expression of membrane-bound IL-12, and this document demonstrates that such modifications significantly increase the cytotoxicity of the modified T cells. In embodiments, the modified T cells comprise disruption of the MED12 gene, expression of membrane-bound IL-12, and disruption of the Fas gene or expression of dnFas in the modified T cells, and such modifications increase the cytotoxicity of the modified T cells.

[0176] Antigen recognition part

[0177] The modified T cells described herein may include (e.g., stably expressed) one or more antigen recognition portions that recognize disease-associated antigens, such as tumor-associated antigens (also known as tumor antigens), autoimmune disease-associated antigens, or pathogen-associated antigens (also known as pathogenic antigens). In embodiments, the modified T cells contain antigen recognition portions expressed on the cell surface, and the antigen recognition portions include affinity-binding domains that are specific to disease-associated antigens. Examples of antigen recognition moieties include TCRs, αβ TCRs, γδ TCRs, chimeric antigen receptors (CARs), whole antibodies or their antigen-binding fragments, single-chain variable fragments (scFvs), heavy-chain or light-chain single-domain antibodies (sdAbs), Fab, F(ab)2, or any combination thereof, wherein the antigen recognition moieties bind to: (i) cell surface tumor antigens, (ii) peptides derived from tumor antigens expressed on the cell surface as MHC complexes (peptide-MHC complexes), (iii) cell surface antigens associated with autoimmune diseases or pathogens, or (iv) peptides derived from antigens associated with autoimmune diseases or pathogens expressed on the cell surface as MHC complexes (peptide-MHC complexes).

[0178] The modified T cells described herein may express one or more antigen recognition moieties. In some embodiments, the modified T cells express two or more antigen recognition moieties. In some instances, each antigen recognition moiety recognizes a different epitope of the same antigen. In some instances, each antigen recognition moiety recognizes a different epitope of a different antigen.

[0179] Two or more antigen recognition moieties can be expressed in modified T cells by genetically different, substantially different, or substantially identical TCR polynucleotides, either by the modified T cells themselves or by genetically different TCR polynucleotides stably incorporated into the modified T cells. In the case of one or more genetically different TCRs, one or more TCRs recognizing different antigens associated with the same symptom can be utilized. In a preferred embodiment, the modified T cells are engineered to express different TCRs derived from human or mouse origin, from one or more expression cassettes recognizing the same antigen in different MHC haplotype backgrounds. In another preferred embodiment, the modified T cells are engineered to express one TCR and two or more antibodies targeting the same or different peptides from a given antigen complexed with different MHC haplotypes. In some cases, T cell expression of a single TCR promotes correct TCR pairing. Modified T cells expressing different TCRs can provide universal allogeneic modified T cells. In a second preferred embodiment, the modified T cells are engineered to express one or more different antibodies against the peptide-MHC complex, each antibody targeting the same or different peptides complexed with the same or different MHC haplotypes. In some cases, the antigen recognition portion may be an antibody that binds to the peptide-MHC complex.

[0180] Modified T cells can be engineered to express TCRs derived from one or more expression cassettes that recognize the same antigen in different MHC haplotype backgrounds. In some cases, modified T cells are engineered to express a single TCR or a combination of a TCR and a CAR to minimize the possibility of TCR mismatch within the engineered cells. The antigen recognition moieties expressed from two or more expression cassettes preferably have different polynucleotide sequences and encode antigen recognition moieties that recognize different epitopes of the same target, for example, in different HLA haplotype backgrounds. Modified T cells expressing such different TCRs or CARs can provide universal allogeneic modified T cells.

[0181] In some cases, modified T cells are engineered to express two or more antigen recognition moieties. These two or more antigen recognition moieties can be expressed by genetically identical or substantially identical antigen-specific chimeric (CAR) polynucleotides engineered in the modified T cells. Alternatively, they can be expressed by genetically distinct CAR polynucleotides engineered in the modified T cells. One or more genetically distinct CARs can be designed to recognize different antigens associated with the same condition.

[0182] The antigen recognition component can be engineered to recognize antigens with a certain affinity. For example, the antigen recognition portion encoded by a TCR or CAR construct can be at least 10 fM, at least 100 fM, at least 1 picomolar (pM), at least 10 pM, at least 20 pM, at least 30 pM, at least 40 pM, at least 50 pM, at least 60 pM, at least 7 pM, at least 80 pM, at least 90 pM, at least 100 pM, at least 200 pM, at least 300 pM, at least 400 pM, at least 500 pM, at least 600 pM, at least 700 pM, at least 800 pM, at least 900 pM, at least 1 nanomolar (nM), at least 2 nM, at least 3 nM, at least 4 nM, at least 5 nM, at least 6 nM, at least 7 nM, at least 8 nM, at least 9 nM, at least 10 nM, at least 20 nM, at least 30 nM, at least 40 nM, at least 50 nm, at least 60 nM, at least 70 nM, at least 80 nm, at least 1 nanomolar (nM), at least 2 nM, at least 3 nM, at least 4 nM, at least 50 nm, at least 60 nM, at least 70 nM, at least 80 nm, at least 10 nM, at least 20 nM, at least 30 nM, at least 40 nM, at least 50 nm, at least 60 nM, at least 70 nM, at least 80 nm, at least 10 nm, at least 20 nM, at least 30 nM, at least 40 nM, at least 50 nm, at least 60 nM, at least 70 nM, at least 80 The dissociation constants of at least nM, at least 90 nM, at least 100 nM, at least 200 nM, at least 300 nM, at least 400 nM, at least 500 nM, at least 600 nM, at least 700 nM, at least 800 nM, at least 900 nM, at least 1 mM, at least 2 mM, at least 3 pM, at least 4 pM, at least 5 pM, at least 6 pM, at least 7 pM, at least 8 pM, at least 9 pM, at least 10 pM, at least 20 pM, at least 30 pM, at least 40 pM, at least 50 pM, at least 60 pM, at least 70 pM, at least 80 pM, at least 90 pM, or at least 100 pM identify antigens.

[0183] In some cases, the antigen recognition portion can be engineered to accommodate quantities up to 10 fM, up to 100 fM, up to 1 picomol (pM), up to 10 pM, up to 20 pM, up to 30 pM, up to 40 pM, up to 50 pM, up to 60 pM, up to 7 pM, up to 80 pM, up to 90 pM, up to 100 pM, up to 200 pM, up to 300 pM, up to 400 pM, up to 500 pM, up to 600 pM, up to 700 pM, up to 800 pM, up to 900 pM, up to 1 nanomolar (nM), up to 2 nM, up to 3 nM, up to 4 nM, up to 5 nM, up to 6 nM, up to 7 nM, up to 8 nM, up to 9 nM, up to 10 nM, up to 20 nM, up to 30 nM, up to 40 nM, up to 50 nm, up to 60 nM, up to 70 nM, up to 80 nM, up to 90 nM, up to 100 nM, up to 200 nM, up to 300 nM, up to 400 nM, up to 500 nM, up to 600 nM, up to 700 nM, up to 800 nM, up to 900 nM, up to 1 mM, up to 2 mM, up to 3 pM, up to 4 pM, up to 5 pM, up to 6 pM, up to 7 pM, up to 8 pM, up to 9 pM, up to 10 pM, up to 20 pM, up to 30 pM, up to 40 pM, up to 50 pM, up to 60 pM, up to 70 pM, up to 80 pM, up to 90 pM or up to 100 The dissociation constant of pM identifies antigens.

[0184] The antigen recognition component can identify tumor-associated antigens. The term "tumor-associated antigen" or "tumor antigen" includes any molecule that promotes tumorigenic characteristics of tumor cells and is expressed on (or associated with) tumor cell development. Numerous tumor antigens are known in the art. Whether a molecule is a tumor antigen can also be determined using techniques and assays well known to those skilled in the art, such as clonogenic assays, transformation assays, in vitro or in vivo tumorigenesis assays, gel migration assays, gene knockout analyses, etc. Examples of suitable tumor antigens include, but are not limited to, CD19, CD20, CD30, CD22, CD37, CD38, CD56, CD33, CD138, CD123, CD79b, CD70, CD75, CA6, GD2, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), RON, CEACAM5, CA-125, MUC-16, 5T4, NaPi2b, ROR1, ROR2, PLIF, Her2 / Neu, EGFRvIII, GPMNB, LIV-1, glycolipid F77, fibroblast activation protein (FAP), PSMA, STEAP-1, STEAP-2, mesothelin, c-Met, CSPG4, PVRL-4, VEGFR2, PSCA, CLEC12a, and LI. CAM, GPC2, GPC3, folate-binding protein / receptor, SLC44A4, Cripto, CTAG1B, AXL, IL-13R, IL-3Ra2, SLTRK6, gp100, MARTI, tyrosinase, SSX2, SSX4, NYESO-1, WT-1, PRAME, epithelial tumor antigen (ETA), MAGEA family genes (such as MAGEA3, MAGEA4), KKLC1, mutant ras, VRaf, p53, MHC class I chain-associated molecule A (MICA) or MHC class I chain-associated molecule B (MICB), or one or more antigens of HPV, CMV, or EBV, BCMA, GPC3, TyrD, B7H6, CD70, or PSMA.

[0185] The antigen recognition component identifies antigens associated with autoimmune diseases. These antigens include endogenous antigens that stimulate the production of autoimmune responses, such as the production of autoantibodies. Antigens associated with autoimmune diseases also include antigens from normal tissues that are targets of cell-mediated or antibody-mediated immune responses that may contribute to the development of autoimmune diseases. Examples of antigens associated with autoimmune diseases include agglutinin, alanyl-tRNA synthetase (PL-12), αβ crystallin, α-cytosine (Sptan 1), α-actin, α1-antichymotrypsin, α1-antitrypsin, α1-microglobulin, aldolase, aminoacyl-tRNA synthetase, amyloid, annexin, apolipoprotein, aquaporin, bactericidal / permeability-enhancing protein (BPI), β-globulin precursor BP1, β-actin, β-lactoglobulin A, β-2-glycoprotein I, β2-microglobulin, blood group antigens, C-reactive protein (CRP), calmodulin, calreticulin, cardiolipin, catalytic enzymes, cathepsin B, centromere, chondroitin sulfate, chromatin, collagen, complement components, cytochrome C, cytochrome P450 2D6, cytokeratin, core proteoglycan, dermatan sulfate, DNA topoisomerase I, elastin, and Epstein-Barr nuclear antigen 1. (EBNA1), elastin, nestin, extractable nuclear antigen, factor I, factor P, factor B, factor D, factor H, factor X, fibrinogen, fibronectin, imine methyltransferase cyclization deaminase (LC-1), gp210 nuclear envelope protein, GP2 (major zymogen membrane glycoprotein), glutenin, glycoprotein gpIIb / IIIa, collagen fibrillary acidic protein (GFAP), glycated albumin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), haptoglobulin A2, heat shock protein, hemocyanin, heparin, histone, histidine-tRNA synthetase (Jo-1), barley protein, hyaluronidase, immunoglobulins, integrins, interstitial retinol-binding protein 3, endogenous factors, Ku (p70 / p80), lactate dehydrogenase, laminin, type 1 hepatic cytoplasmic fluid antigen (LC1), liver / kidney microsomal antigen 1 (LKM1), lysozyme, melanoma differentiation-associated protein 5 (MDAS), Mi-2 (chromatin domain helicase DNA-binding protein 4), mitochondrial proteins, muscarinic receptors, myelin-associated glycoproteins, myosin, myelin basic proteins, myelin lipoproteins, myelin oligodendrocyte glycoproteins, myeloperoxidase (MPO), rheumatoid factor (IgM anti-IgG), neuron-specific enolase, nicotinic acid acetylcholine receptor A chain, nucleolar protein, nucleoporin, nucleosome antigen, PM / Sc1100, PM / Scl 75, pancreatic β-cell antigen, pepsinogen, peroxidase 1, phosphoglucose isomerase, phospholipids, phosphatidylinositol, platelet-derived growth factor, polymerase β(POLB), potassium channel KIR4.1, proliferating cell nuclear antigen (PCNA), protease-3, protein lipoprotein, proteoglycan, prothrombin, recovery protein, rhodopsin, ribonuclease, ribonucleoprotein, ribosome, ribosomal phosphoprotein, RNA, Sm protein, Sp100 nucleoprotein, SRP54 (signal recognition particle 54 kDa), selectin, smooth muscle protein, sphingomyelin, streptococcal antigen, superoxide dismutase, synovial arthrin, T1F1 γ collagen, threonyl-tRNA synthetase (PL-7), tissue transglutaminase, thyroid peroxidase, thyroglobulin, thyroid-stimulating hormone receptor, transferrin, triose phosphate isomerase, tubulin, tumor necrosis factor α, topoisomerase, U1-dnRNP 68 / 70 kDa, U1-snRNP A, U1-snRNP C, U-snRNP B / B′, ubiquitin, vascular endothelial growth factor, vimentin, and vitrin.

[0186] In the implementation scheme, the antigen recognition portion recognizes disease-associated antigens expressed on immune cells. In some instances, such immune cells are B cells, encompassing all stages of development, including those that eventually differentiate into plasma cells, such as proB cells, preB cells, transitional / immature B cells, naïve B cells, GC B cells, memory B cells, plasmablasts, and plasma cells. In some instances, such immune cells are T cells, such as Th1 / Th17 T cells, CD4+ T cells, keratinocytes, and cells described in Lee et al., B cell depletion therapies in autoimmune disease: advances and mechanical insights, Nat Rev Drug Discov. 2021 Mar;20(3):179-199, which is incorporated herein by reference in its entirety.

[0187] In the implementation scheme, the antigen recognition portion binds to one or more of CD19, CD20, CD22, CD37, CD38, CD40, CD40L, CD52, CD79b, CD123, CD138, BAFF-R, BCMA, FcRL5, GPRC50, TAC1, FcgRIIB, IL-36, IL-36R, IL-6, IL-6R, α4 integrin, CXCR6, DSG3, PD1, VISTA, BTLA, LAG3, ICOS, and ICOS-L. In the implementation scheme, the antigen recognition portion binds to B-cell antigens selected from or comprising the group consisting of: CD19, CD20, CD22, CD37, CD38, CD40, CD52, CD79b, CD123, CD138, BAFF-R, BCMA, FcRL5, GPRC50, TAC1, and FcgRIIB. In one embodiment, the antigen recognition portion binds to plasma cell antigens selected from or comprising the group consisting of: CD38, CD138, BCMA, FcRL5, GPRC5D, TACI, and FcgRIIB. In another embodiment, the antigen recognition portion binds to T cell antigens selected from or comprising the group consisting of: CD52, CD40L, α4 integrin, CXCR6, PD1, VISTA, BTLA, LAG3, ICOS, and ICOS-L.

[0188] The antigen recognition portion can recognize pathogenic antigens. Pathogenic antigens can be bacterial, viral, or fungal molecules, such as bacterial, viral, or fungal proteins. In embodiments, antigen-presenting cells can internalize pathogenic molecules (e.g., pathogenic proteins, nucleic acids, lipids, or fragments produced by pathogenic organisms such as bacteria or viruses), for example, through phagocytosis or receptor-mediated endocytosis, and display antigen fragments that bind to appropriate MHC molecules. For example, various 9-mer fragments of pathogenic proteins can be displayed by APCs. The engineered, enriched, modified T cell populations of this disclosure can be programmed to recognize various antigens and antigen fragments of pathogenic bacteria or viruses.

[0189] Examples of pathogenic bacteria can be found in the following: a) *Bordetella* species, such as *Bordetella pertussis*; b) *Borrelia* species, such as *Borrelia burgdorferi*; c) *Brucelia* species, such as *Brucella abortus*, *Brucella canis*, *Brucella muttonis*, and / or *Brucella suis*; d) *Campylobacter* species, such as *Campylobacter jejuni*; e) *Chlamydia* and *Chlamydophil* species, such as *Chlamydia pneumoniae*, *Chlamydia trachomatis*, and / or *Chlamydophila psittaci*. f) Clostridium species, such as *Clostridium botulinum*, *Clostridium difficile*, *Clostridium perfringens*, and *Clostridium tetani*; g) Corynebacterium species, such as *Corynebacterium diphtheria*; h) Enterococcus species, such as *Enterococcus faecalis* and / or *Enterococcus faecium*; i) Escherichia species, such as *Escherichia coli*; j) Francisella species, such as *Francisella tularensis*. tularensis) species; k) Haemophilus, such as Haemophilus influenzae species; 1) Helicobacter, such as Helicobacter pylori species; m) Legionella, such as Legionella pneumophila species;n) *Leptospira* species, such as *Leptospira interrogans*; o) *Listeria* species, such as *Listeria monocytogenes*; p) *Mycobacterium* species, such as *Mycobacterium leprae*, *Mycobacterium tuberculosis*, and / or *Mycobacterium ulcerans*; q) *Mycoplasma* species, such as *Mycoplasma pneumoniae*; r) *Neisseria* species, such as *Neisseria gonorrhoeae* and / or *Neisseria meningitidis*. s) *Pseudomonas* species, such as *Pseudomonas aeruginosa*; t) *Rickettsia* species, such as *Rickettsia rickettsii*; u) *Salmonella* species, such as *Salmonella typhi* and / or *Salmonella typhimurium*; v) *Shigella* species, such as *Shigella sonnei*; w) *Staphylococcus* species, such as *Staphylococcus aureus*, *Staphylococcus epidermidis*, and / or *Staphylococcus saprophyticus*. (x) *Streptococcus*, such as *Streptococcus agalactiae*, *Streptococcus pneumoniae*, and / or *Streptococcus pyogenes*; (y) *Treponema*, such as *Treponema pallidum*; (z) *Vibrio*, such as *Vibrio cholera*.And / or aa) species of Yersinia, such as Yersinia pestis.

[0190] Examples of pathogenic viruses can be found in the following virologic families and are illustrated by example: a) Adenoviridae, such as adenovirus species; b) Herpesviridae, such as herpes simplex virus type 1, herpes simplex virus type 2, varicella-zoster virus, Epstein-Barr virus, human cytomegalovirus, and human herpesvirus type 8; c) Papillomaviridae, such as human papillomavirus species; d) Polyomaviridae, such as BK virus and JC virus species; e) Poxviridae, such as smallpox species; f) Hepatoviridae, such as hepatitis B virus species; g) Parvoviridae, such as human bocavirus and parvovirus B19 species; h) Astroviridae, such as human astrovirus species; i) Caliciviridae, such as norovirus species; j) Flaviviridae, such as hepatitis C virus (HCV), yellow fever virus, dengue virus, and West Nile virus species; k) Cloaked viruses. The family is divided into several families, including: 1) Hepatitis E Viridae, including hepatitis E virus species; m) Retroviridae, including human immunodeficiency virus (HIV) species; n) Orthomyxoviridae, including influenza virus species; o) Arenaviridae, including Guanarito virus, Junin virus, Lassa virus, Machupo virus, and / or Sabya virus species; p) Bunyaviridae, including Crimean-Congo hemorrhagic fever virus species; q) Filoviridae, including Ebola virus and / or Marburg virus species; Paramyxoviridae, including measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, human metapneumovirus, Hendra virus, and / or Nipah virus species; r) Rhabdoviridae, including rabies virus species; s) Reoviridae, including rotavirus, circovirus, Colorado tick fever virus (Coltivirus), and / or Banna virus species. In some instances, viruses are not classified into a Viridae family, such as hepatitis D virus.

[0191] In the embodiments, one or more antigen recognition moieties in the modified T cells are or include monoclonal antibodies, polyclonal antibodies, synthetic antibodies, human antibodies, humanized antibodies, non-human antibodies, bispecific antibodies, and any fragments thereof. In the embodiments, the antigen recognition moieties are or include antibodies, antigen-binding fragments (Fab), single-chain variable fragments (scFv), heavy-chain or light-chain single-domain antibodies (sdAb), F(ab)2, or any combination thereof. In the embodiments, one or more antigen recognition moieties include mammalian antibodies or fragments thereof. The selection of further antigen recognition moieties may depend on the type and quantity of antigens present on the surface of the target cells.

[0192] Other examples of antigen-binding domains include those described in PCT / US2019 / 054132, PCT / US2019 / 054144, PCT / US2023 / 034227, PCT / US2023 / 029047, and PCT / US2023 / 030115, each of which is incorporated herein by reference in its entirety.

[0193] Polyclonal antibodies

[0194] Polyclonal antibodies can be generated in animals through multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant. Conjugating the relevant antigen (especially when using synthetic peptides) to a protein that is immunogenic in the species to be immunized can be useful. For example, using bifunctional or derivatizing agents such as maleimide benzoyl sulfosuccinimide (conjugated via cysteine ​​residues), N-hydroxysuccinimide (conjugated via lysine residues), glutaraldehyde, succinic anhydride, SOCl2, or R′N═C═NR, where R and R1 are distinct alkyl groups, can conjugate the antigen to keyhole hemocyanin (KLH), serum albumin, bovine thyroglobulin, or soybean trypsin inhibitors.

[0195] Animals are immunized against antigens, immunogenic conjugates, or derivatives by combining, for example, 100 μg or 5 μg of protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, the animal is immunized with the solution containing Freund's complete adjuvant by subcutaneous injection at multiple sites. Animals are boosted with up to 1 / 10 of the original amount of the peptide or conjugate. Seven to 14 days later, the animals are bled, and serum antibody titers are measured. Boosted immunizations are then administered until the titers reach stable levels. Conjugates can also be prepared as protein fusions in recombinant cell cultures. Additionally, aggregaters such as alum can be appropriately used to enhance the immune response.

[0196] Monoclonal antibodies

[0197] Monoclonal antibodies (mAbs) against antigens of interest can be prepared using any technique known in the art. These techniques include, but are not limited to, hybridoma techniques originally described by Kohler and Milstein (1975, Nature 256, 495-497), and human B-cell hybridoma techniques (Kozbor et al., 1983, ...). Immunology Today 4:72) and EBV-hybridoma technology (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy(Alan R. Liss, Inc., pp. 77-96). Selective Lymphocyte Antibody Approach (SLAM) (Babcook, JS et al., A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined specificities). Proc Natl Acad Sci USA , 1996. 93 (15): pp. 7843-8.) and (McLean G et al., 2005, J Immunol .174(8): 4768-78. These antibodies can belong to any immunoglobulin class, including IgG, IgM, IgE, IgA, and IgD, and any of their subclasses. Hybridomas for the mAbs used in this invention can be cultured in vitro or in vivo.

[0198] Monoclonal antibodies can be used by Kohler et al. Nature The hybridoma method first described in 256:495 (1975) can be used to prepare the hybridoma, or it can be prepared by a recombinant DNA method (US Patent No. 4,816,567).

[0199] In the hybridoma approach, mice or other suitable host animals, such as hamsters, are immunized as described above to obtain lymphocytes that produce or are capable of producing antibodies that specifically bind to proteins used for immunization. Alternatively, the lymphocytes can be immunized in vitro. Following immunization, the lymphocytes are isolated and then fused with a myeloma cell line using a suitable fusion agent (such as polyethylene glycol) to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[0200] Hybridoma cells thus prepared are seeded in a suitable culture medium and grown, which may contain one or more substances that inhibit the growth or survival of unfused parental myeloma cells (also known as fusion couples). For example, if the parental myeloma cells lack the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), the selective culture medium for hybridomas will typically include hypoxanthine, aminopterin, and thymidine (HAT medium), which prevent the growth of HGPRT-deficient cells.

[0201] Preferred fusion-couple myeloma cells are those that fuse efficiently, support stable high-level antibody production by selected antibody-producing cells, and are sensitive to selective media selected against the unfused parental cells. Preferred myeloma cell lines are mouse myeloma cell lines, such as those derived from MOPC-21 and MPC-11 mouse tumors, available from the Salk Institute Cell Distribution Center, San Diego, Calif., USA; and SP-2 and its derivatives, such as X63-Ag8-653 cells available from the American Type Culture Collection, Manassas, Va., USA. Human myeloma and mouse-human xenologous myeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol ., 133: 3001 (1984); and Brodeur et al., Monoclonal Antibody Production Techniques and Applications Pages 51-63 (Marcel Dekker, Inc., New York, 1987).

[0202] The production of monoclonal antibodies against antigens in the culture medium for growing hybridoma cells is measured. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0203] The binding affinity of monoclonal antibodies can be assessed, for example, by Munson et al. Anal. Biochem The Scatchard analysis described in . 107: 220 (1980) is used to determine this.

[0204] Once hybridoma cells that produce antibodies with the desired specificity, affinity, and / or activity are identified, the clones can be subcloned using a limiting dilution procedure and grown using standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable media for this purpose include, for example, D-MEM or RPMI-1640. Furthermore, hybridoma cells can be grown in vivo as ascites tumors in animals, for example, by intraperitoneal injection of cells into mice.

[0205] Subclonal secreted monoclonal antibodies can be appropriately separated from culture medium, ascites, or serum using conventional antibody purification procedures such as affinity chromatography (e.g., using protein A or protein G agarose gel) or ion exchange chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, etc.

[0206] DNA encoding monoclonal antibodies can be readily isolated and sequenced using standard procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of mouse antibodies). Hybridoma cells serve as a preferred source of this DNA. Once isolated, the DNA can be placed into an expression vector and then transfected into host cells, such as *E. coli* cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not additionally produce antibody proteins, to obtain monoclonal antibody synthesis in recombinant host cells. Review articles on the recombinant expression of antibody-encoding DNA in bacteria include those by Skerra et al. Curr. Opinion in Immunol . 5: 256-62 (1993) and Plückthun, Immunol. Rev 130: 151-88 (1992).

[0207] In a further embodiment, the monoclonal antibody or antibody fragment may be derived from McCafferty et al. Nature Antibodies were isolated from phage libraries produced using the techniques described in Clackson et al., 348: 552-54 (1990). Nature , 352: 624-28 (1991) and Marks et al., J. Mol. Biol. , 222: 581-97 (1991) described the isolation of mouse and human antibodies using phage libraries, respectively. Subsequent publications described the isolation of antibodies via chain tamponade (Marks et al., 222: 581-97). Bio / Technology , 10: 779-83 (1992)) and combined infection and in vivo recombination to produce high-affinity (nM range) human antibodies as a strategy for constructing very large phage libraries (Waterhouse et al., , 10: 779-83 (1992)). Nuc. Acids. Res 21: 2265-6 (1993)). Therefore, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques used for isolating monoclonal antibodies.

[0208] The DNA encoding antibodies can be modified to produce chimeric or fusion antibody peptides, for example by substituting the human heavy and light chain constant domains (CH and CO sequences) for homologous mouse sequences (US Patent No. 4,816,567; and Morrison et al.). Proc. Natl. Acad. Sci. USA, 81: 6851 (1984)), or by fusing immunoglobulin-coding sequences with all or part of the coding sequences of non-immunoglobulin polypeptides (heterologous polypeptides). Non-immunoglobulin polypeptide sequences may replace constant domains of antibodies, or they may replace variable domains of an antigen-binding site of an antibody, to produce chimeric bivalent antibodies containing one antigen-specific antigen-binding site and another antigen-binding site specific to a different antigen.

[0209] Chimeric antibodies, humanized antibodies and human antibodies

[0210] In the implementation, the antigen recognition portion is or includes a chimeric antibody. Certain examples of chimeric antibody designs are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al. Proc. Natl. Acad. Sci. USA In 81:6851-5 (1984)). In one instance, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate such as a monkey) and a human constant region. In another instance, a chimeric antibody is a "class-switched" antibody, wherein the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include their antigen-binding fragment.

[0211] In this embodiment, the chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce its immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Typically, a humanized antibody contains one or more variable domains, wherein the HVR, such as the CDR (or a portion thereof), is derived from the non-human antibody, and the FR (or a portion thereof) is derived from the human antibody sequence. Optionally, the humanized antibody will also contain at least a portion of a human constant region. In this embodiment, some FR residues in the humanized antibody are substituted with corresponding residues from the non-human antibody (e.g., the antibody from which the CDR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0212] The antigen recognition portion of this document may include humanized antibodies or human antibodies. Humanized forms of non-human (e.g., mouse or rabbit) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding sequences of the antibody) containing a minimal sequence derived from a non-human immunoglobulin. Humanized antibodies include human immunoglobulins (receptor antibodies) in which residues from the complementarity-determining region (CDR) of the receptor are replaced by residues from the CDR of a non-human species (donor antibody) with the desired specificity, affinity, and capability, such as mouse, rat, or rabbit. In some cases, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also contain residues not found in either the receptor antibody or the introduced CDR or framework sequence. Generally, humanized antibodies will contain substantially all at least one, typically two, variable domains, where all or substantially all of the CDR regions correspond to the CDR regions of the non-human immunoglobulin, and all or substantially all of the FR regions are FR regions of the human immunoglobulin common sequence. Humanized antibodies may optionally also include at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the constant region (Fc) of human immunoglobulins (Jones et al.). Nature , 321: 522-5 (1986); Riechmann et al., Nature , 332: 323-9 (1988); and Presta, Curr. Op. Struct. Biol ., 2: 593-6 (1992)).

[0213] The humanized antibody of the present invention may include one or more human- and / or human-shared non-hypervariable regions (e.g., frames) sequences in its heavy chain and / or light chain variable domains. In embodiments, one or more additional modifications are present in the human- and / or human-shared non-hypervariable regions. In one embodiment, the heavy chain variable domain of the antibody of the present invention includes a human-shared frame sequence, which in one embodiment is a subgroup III shared frame sequence. In one embodiment, the antibody of the present invention includes a variant subgroup III shared frame sequence modified at at least one amino acid position.

[0214] As is known in the art, the amino acid positions / boundaries defining the hypervariable region of an antibody can vary depending on the context and various definitions known in the art. Some positions within a variable domain can be considered hybridization hypervariable positions because these positions can be considered within a hypervariable region under one set of criteria and outside a hypervariable region under another set of criteria. One or more of these positions are also visible in extended hypervariable regions (as further defined below). The present invention provides antibodies comprising modifications at these hybridization hypervariable positions. In one embodiment, these hypervariable positions include one or more positions 26-30, 33-35B, 47-49, 57-65, 93, 94, and 101-102 in the heavy chain variable domain. In one embodiment, these hybridization hypervariable positions include one or more positions 24-29, 35-36, 46-49, 56, and 97 in the light chain variable domain. In one embodiment, the antibody of the present invention comprises a human variant human subgroup common framework sequence modified at one or more hybridization hypervariable positions.

[0215] The antigen recognition portion of this document may contain any suitable human or human common light chain framework sequence, provided that the antibody exhibits the desired biological characteristics (e.g., desired binding affinity). In one embodiment, the antibody of the present invention contains at least a portion (or all) of the framework sequence of the human κ light chain. In one embodiment, the antibody of the present invention contains at least a portion (or all) of the human κ subgroup I framework common sequence.

[0216] The method of humanizing nonhuman antibodies is well known in the art. As discussed, humanized antibodies typically have one or more amino acid residues introduced from a nonhuman source. These nonhuman amino acid residues are often referred to as “introduced” residues and are typically derived from an “introduced” variable domain. Humanization can be performed essentially as Winter et al. (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)) by replacing the corresponding sequence of a human antibody with a rodent CDR or CDR sequence. Thus, such “humanized” antibodies are chimeric antibodies (US Patent No. 4,816,567), in which substantially less than the complete human variable domain has been replaced by the corresponding sequence from a nonhuman species. In practice, humanized antibodies are usually human antibodies, with some CDR residues and possibly some FR residues replaced by residues from similar sites in rodent antibodies.

[0217] When antibodies are intended for human therapeutic use, the selection of the human variable domains (both light and heavy chains) used to prepare humanized antibodies is crucial for reducing antigenicity and HAMA reactions (human anti-mouse antibodies). Reduction or elimination of HAMA reactions is an important aspect of the clinical development of suitable therapeutic agents (see, for example, Khaxzaeli et al.). J. Natl. Cancer Inst (1988), 80:937; Jaffers et al., Transplantation (1986), 41:572; Shawler et al., J. Immunol (1985), 135:1530; Sears et al., J. Biol. Response Mod (1984), 3:138; Miller et al., Blood (1983), 62:988; Hakimi et al., J. Immunol (1991), 147:1352; Reichmann et al., Nature (1988), 332: 323; Junghans et al., Cancer Res (1990), 50: 1495). As described herein, the present invention provides humanized antibodies that reduce or eliminate the HAMA response. Variants of these antibodies can be further obtained using conventional methods known in the art, some of which are further described below. According to the so-called “best fit” method, sequences of variable domains for rodent antibodies were screened against an entire library of known human variable domain sequences. The human V domain sequence most closely related to rodents was identified, and the human frame region (FR) within it was accepted for use in humanized antibodies (Sims et al., 1990, 50: 1495). J. Immunol 151: 2296 (1993); Chothia et al., J. Mol. Biol ., 196: 901 (1987)). Another approach uses specific frame regions from the shared sequences of all human antibodies derived from specific light or heavy chain subgroups. The same frame can be used for several different humanized antibodies (Carter et al., 196: 901 (1987)). Proc. Natl. Acad. Sci. USA , 89: 4285 (1992); Presta et al., J. Immunol . 151: 2623 (1993)).

[0218] For example, amino acid sequences from antibodies as described herein can be used as diverse starting (parental) sequences for frameworks and / or hypervariable sequences. The selected framework sequence linked to the starting hypervariable sequence is referred to herein as the recipient human framework. While recipient human frameworks may be derived from or derived from human immunoglobulins (their VL and / or VH regions), it is preferred that recipient human frameworks be derived from or derived from human common framework sequences, as such frameworks have been shown to have minimal or no immunogenicity in human patients.

[0219] When the receptor is derived from human immunoglobulin, a human frame sequence may be optionally selected. This selection is based on the homology between the donor frame sequence and the various human frame sequences in the human frame sequence set, and the most homologous frame sequence is selected as the receptor.

[0220] In one implementation, the shared frames in this paper are derived from or derived from shared frame sequences of VH subgroup III and / or VLκ subgroup I.

[0221] Although the receptor may be sequence-identical to a selected human frame sequence, whether derived from human immunoglobulin or human common frame, the present invention anticipates that the receptor sequence may contain pre-existing amino acid substitutions relative to the human immunoglobulin sequence or human common frame sequence. These pre-existing substitutions are preferably minimal; typically differing by only four, three, two, or one amino acid relative to the human immunoglobulin sequence or common frame sequence.

[0222] The hypervariable residues of the nonhuman antibody are incorporated into the human frame of the VL and / or VH receptor. For example, residues corresponding to Kabat CDR residues, Chothia hypervariable loop residues, Abm residues, and / or contact residues may be incorporated. Optionally, extended hypervariable residues such as 24-34 (L1), 50-56 (L2), and 89-97 (L3), 26-35B (H1), 50-65, 47-65, or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) may be incorporated.

[0223] Although this article discusses “incorporation” of hypervariable residues, it will be understood that this can be achieved in various ways, such as by mutating the nucleic acid encoding the mouse variable domain sequence to produce a nucleic acid encoding the desired amino acid sequence, thereby changing its framework residues to recipient human framework residues, or by mutating the nucleic acid encoding the human variable domain sequence to change the hypervariable domain residues to non-human residues, or by synthesizing a nucleic acid encoding the desired sequence, etc.

[0224] As described in this paper, hypervariable transplant variants can be generated through Kunkel mutagenesis of nucleic acids encoding human receptor sequences, using individual oligonucleotides for each hypervariable region. Kunkel et al., Methods Enzymol. 154:367-382 (1987). Appropriate alterations can be introduced within the framework and / or hypervariable regions using conventional techniques to correct and reconstruct proper hypervariable-antigen interactions.

[0225] Phage display (also referred to as phage display in some contexts herein) is a convenient and rapid method for generating and screening antibodies against many different potential variants from libraries generated through sequence randomization. However, methods for preparing and screening altered antibodies are not readily available to those skilled in the art.

[0226] Phage display technology provides a powerful tool for generating and selecting novel proteins that bind to ligands such as antigens. Phage display technology allows for the generation of large libraries of protein variants whose sequences bind with high affinity to target molecules, enabling rapid sorting. Typically, the nucleic acid encoding the variant polypeptide is fused with a nucleic acid sequence encoding a viral capsid protein, such as gene III or gene VIII. Monovalent phage display systems have been developed in which a nucleic acid sequence encoding a protein or polypeptide is fused with a nucleic acid sequence encoding a portion of gene III protein (Bass, S., Proteins, 8:309 (1990); Lowman and Wells, Methods: A Companion to Methods in Enzymology, 3:205 (1991)). In monovalent phage display systems, the gene fusion is expressed at low levels and also expresses the wild-type gene III protein, thus preserving the infectivity of the particle. Methods for generating peptide libraries and screening such libraries have been disclosed in numerous patents (e.g., U.S. Patent No. 5,723,286, U.S. Patent No. 5,432,018, U.S. Patent No. 5,580,717, U.S. Patent No. 5,427,908, and U.S. Patent No. 5,498,530).

[0227] Libraries of antibodies or antigen-binding peptides have been prepared in various ways, including by altering single genes through the insertion of random DNA sequences or by cloning related gene families. Methods for displaying antibody or antigen-binding fragments using phage display have been described in U.S. Patents 5,750,373, 5,733,743, 5,837,242, 5,969,108, 6,172,197, 5,580,717, and 5,658,727. Libraries expressing antibodies or antigen-binding proteins with the desired characteristics are then screened.

[0228] Methods for substituting selected amino acids into template nucleic acids are well established in the art, and some of them are described herein. For example, methods for introducing modifications into nucleic acid sequences may include the use of various commercially available kits (e.g., QuickChange site-directed mutagenesis kit, Agilent, Santa Clara, CA). As another example, the Kunkel method can be used to substitute hypervariable residues (e.g., Kunkel et al., Methods Enzymol. 154:367-382 (1987)).

[0229] Importantly, antibody humanization preserves high binding affinity for antigens and other advantageous biological properties. To achieve this, humanized antibodies are prepared according to preferred methods by analyzing parental sequences and various conceptual humanization products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are widely available and familiar to those skilled in the art. Computer programs are available that illustrate and visualize possible three-dimensional conformations of selected candidate immunoglobulin sequences. Examination of these visualizations allows analysis of the possible roles of residues in the function of the candidate immunoglobulin sequences, i.e., analysis of residues affecting the ability of candidate immunoglobulins to bind their antigens. In this way, FR residues can be selected and combined from receptor and introduction sequences to obtain desired antibody characteristics, such as achieving increased affinity for target antigens. Generally, hypervariable region residues are directly and most substantially involved in influencing antigen binding.

[0230] Humanized antibodies in various forms are anticipated. For example, a humanized antibody can be an antibody fragment, such as Fab. Alternatively, a humanized antibody can be a complete antibody, such as a complete IgG1 antibody.

[0231] As an alternative to humanization, human antibodies can be generated. For example, it is now possible to generate transgenic animals (e.g., mice) that, upon immunization, can produce a complete human antibody library without the production of endogenous immunoglobulins. For instance, homozygous deletion of the antibody heavy chain linker (JH) gene in chimeric and germline mutant mice has been described as leading to complete suppression of endogenous antibody production. Transferring a human germline immunoglobulin gene array into such germline mutant mice would result in the production of human antibodies upon antigen challenge (see, for example, Jakobovits et al.). Proc. Natl. Acad. Sci. USA , 90:2551 (1993); Jakobovits et al., Nature , 362: 255-8 (1993); Bruggemann et al., Year in Immuno7:33 (1993); U.S. Patent Nos. 5,545,806, 5,569,825, 5,591,669, and 5,545,807; and WO 97 / 17852).

[0232] Alternatively, phage display technology (McCafferty et al., Nature 348: 552-53 (1990)) can be used to generate human antibodies and antibody fragments in vitro from a gene library of immunoglobulin variable (V) domains from non-immunized donors. According to this technology, antibody V domain genes are in-frame cloned into the major or minor coat protein genes of filamentous phages (such as M13 or fd) and displayed as functional antibody fragments on the surface of phage particles. Because filamentous particles contain a single-stranded DNA copy of the phage genome, selection based on the functional properties of the antibody also leads to the selection of genes encoding antibodies that express those properties. Thus, phages mimic some of the characteristics of B cells. Phage display can be performed in various forms, as reviewed in, for example, Johnson, Kevin S, and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). V gene segments from several sources can be used for phage display. Clackson et al., Nature, 352:624-628 (1991) isolated a variety of anti-oxazolone antibodies from a small randomized library of the V gene derived from the spleen of immunized mice. A V gene library from non-immunized human donors can be constructed, and the process can be largely followed according to Marks et al. J. Mol. Biol 222:581-97 (1991) or Griffith et al., EMBO J The techniques described in 12:725-34 (1993) (see also U.S. Patent Nos. 5,565,332 and 5,573,905) isolate antibodies against a variety of antigens, including autoantigens.

[0233] Human antibodies can also be generated from in vitro activated B cells (see, for example, U.S. Patent Nos. 5,567,610 and 5,229,275).

[0234] Therefore, in the implementation plan, transgenic or transchromosomal mice carrying a portion of the human immune system instead of the mouse system can be used to generate human monoclonal antibodies.

[0235] HuMAb Mouse™ (Medarex, Inc.) contains a small locus of the human immunoglobulin gene encoding unrearranged human heavy chain (µ and γ) and κ light chain immunoglobulin sequences, as well as targeted mutations that inactivate the endogenous µ and κ chain loci (see, for example, Lonberg et al., (1994)). Nature 368(6474): 856-9). Therefore, mice exhibited reduced expression of mouse IgM or κ, and in response to immunity, introduced human heavy and light chain transgenes underwent class switching and somatic mutations to produce high-affinity human IgGκ monoclonal antibodies (Lonberg, N. et al. (1994), ibid.; in Lonberg, N. (1994)). Handbook of Experimental Pharmacology A review in 113: 49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol 13: 65-93; and Harding, F. and Lonberg, N. (1995) Ann. NY Acad. Sci . 764: 536-46). In Taylor, L. et al. (1992) Nucleic Acids Research 20:6287-6295; Chen, J. et al. (1993) International Immunology 5: 647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA 90: 3720-4; Choi et al. (1993) Nature Genetics 4:117-23; Chen, J. et al. (1993) EMBO J 12: 21-830; Tuaillon et al., (1994) J. Immunol 152: 2912-20; Taylor, L. et al. (1994) International Immunology 6: 579-91; and Fishwild, D. et al. (1996) Nature BiotechnologyThe preparation and use of HuMAb Mouse™, as well as the genomic modifications carried by such mice, are further described in 14: 845-51, and the contents of all these documents are hereby explicitly incorporated in their entirety by reference. See also U.S. Patent Nos. 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,789,650, 5,877,397, 5,661,016, 5,814,318, 5,874,299, and 5,770,429; U.S. Patent No. 5,545,807; PCT Publications WO 92 / 03918, WO 93 / 12227, WO 94 / 25585, WO 97 / 13852, WO 98 / 24884, and WO 99 / 45962; and PCT Publication WO 01 / 14424.

[0236] In another embodiment, a mouse known as “KM Mouse™” can be used to generate the human antibodies of this disclosure, the mouse carrying human immunoglobulin sequences on transgenes and transchromosomes, as described in detail in PCT Publication WO 02 / 43478.

[0237] In another embodiment, an alternative transgenic system called Xenomouse (Abgenix, Inc.) may be used; such mice are described, for example, in U.S. Patent Nos. 5,939,598, 6,075,181, 6,114,598, 6,150,584 and 6,162,963.

[0238] Other related transchromosomal animal systems expressing human immunoglobulin genes are available in the art and can be used to generate antibodies. For example, mice carrying both human heavy chain and human light chain transchromosomals, referred to as "TC mice," can be used; such mice were described by Tomizuka et al. (2000). Proc. Natl. Acad. Sci. USA 97: 722-7. As another example, cows carrying human heavy and light chain transchromosomals have been described in the art (e.g., Kuroiwa et al. (2002)). Nature Biotechnology20: 889-94 and PCT application No. WO 2002 / 092812), and can be used to generate antibodies of the present disclosure. Other examples of transgenic animals that can be used to generate antibodies include OmniRat™ and OmniMouse™ (see, for example, Osborn M. et al. (2013) Journal of Immunology 190: 1481-90; Ma B. et al. (2013) Journal of Immunological Methods 400-401: 78-86; Geurts A. et al. (2009) Science 325: 433, U.S. Patent No. 8,907,157; ​​European Patent No. 2,152,880B1; European Patent No. 2,336,329B1). Another example involves the use of VELOCIMMUNE® technology (see, for example, U.S. Patent No. 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®). In short, VELOCIMMUNE® technology involves generating transgenic mice with a genome containing human heavy and light chain variable regions operatively linked to endogenous mouse constant region loci, such that the mice respond to antigen stimulation by producing antigen-binding proteins, such as antibodies, containing both human variable regions and mouse constant regions. DNA encoding the variable regions of the heavy and light chains of the antibody is isolated and operatively linked to DNA encoding the human heavy and light chain constant regions. The DNA is then expressed in cells capable of expressing fully human antibodies.

[0239] antibody fragments

[0240] In the implementation scheme, the antigen recognition portion of this document is or includes antibody fragments. Various techniques have been developed for generating antibody fragments. Traditionally, these fragments are obtained via proteolytic digestion of intact antibodies (see, for example, Morimoto et al.). Journal of Biochemical and Biophysical Methods 24:107-7 (1992); and Brennan et al., Science , 229: 81 (1985)). However, these fragments can now be produced directly from recombinant host cells. Fab, Fv, and scFv antibody fragments can all be expressed and secreted from E. coli, thus allowing for the easy production of large quantities of these fragments. Antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab′-SH fragments can be recovered directly from E. coli and chemically coupled to form F(ab′)2 fragments (Carter et al., 229: 81 (1985)). Bio / Technology10:163-7 (1992)). According to another method, the F(ab′)2 fragment can be isolated directly from recombinant host cell cultures. Fab and F(ab′)2 fragments with increased in vivo half-lives are described in U.S. Patent No. 5,869,046, which contain salvage receptor-binding epitope residues. Other techniques for generating antibody fragments will be readily apparent to those skilled in the art. In other embodiments, the antibody of choice is a single-chain Fv fragment (scFv) (see WO 93 / 16185; U.S. Patent No. 5,571,894; and U.S. Patent No. 5,587,458). Fv and sFv are the only species with complete binding sites lacking constant regions; therefore, they are suited for reduced nonspecific binding during in vivo use. sFv fusion proteins can be constructed to produce fusions of effector proteins at the amino or carboxyl termini of sFv (see...). Antibody Engineering (Borrebaeck, ed., ibid.). The antibody fragment may also be a “linear antibody,” for example, as described in, for example, U.S. Patent No. 5,641,870.

[0241] In one embodiment, an antibody-derived scFv is used in the CAR of this disclosure. The antibody fragment includes an antibody portion (and combinations of antibody portions, such as scFv) that can be used as a targeting arm against an epitope. Such fragments are not necessarily proteolytic fragments, but rather portions of the polypeptide sequence that can confer target affinity.

[0242] Multispecific antibodies

[0243] In implementations, the antigen recognition portion is or includes a multispecific antibody, such as a bispecific antibody. A bispecific antibody is an antibody that has binding specificity to at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of a protein as described herein. Other such antibodies may combine an antigen-binding site with a binding site of another protein. In some instances, the antigen-binding arm may be combined with an arm that binds to triggering molecules such as T-cell receptor molecules (e.g., CD3) or Fc receptors (FcγRs) of IgG such as FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16) to concentrate and localize cellular defense mechanisms to cells expressing antigens. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing antigens. These antibodies have an antigen-binding arm and an arm that binds to cytotoxic agents (e.g., saponins, interferon-α, vinca alkaloids, ricin A chain, methotrexate, or radioisotope haptens). Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibody).

[0244] Methods for preparing bispecific antibodies are known in the art. Conventional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where the two chains have different specificities (Millstein et al., Nature 305: 537-9 (1983)). Due to the random assignment of the heavy and light chains of immunoglobulins, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule, usually accomplished by affinity chromatography, is quite cumbersome and yields low product yields. A similar procedure is disclosed in WO93 / 08829 and in Traunecker et al., EMBO J. 10:3655-3659 (1991).

[0245] Other methods for preparing bispecific antibodies are known. One method is the "pestle-in-a-pot" or "protrusion-in-a-cavity" method (see, for example, U.S. Patent No. 5,731,168). In this method, two immunoglobulin polypeptides (e.g., heavy chain polypeptides) each contain an interface. The interface of one immunoglobulin polypeptide interacts with a corresponding interface on the other immunoglobulin polypeptide, thereby causing the two immunoglobulin polypeptides to associate. These interfaces can be engineered such that a "pestle" or "protrusion" (these terms are used interchangeably herein) located at the interface of one immunoglobulin polypeptide corresponds to a "pot" or "cavity" (these terms are used interchangeably herein) located at the interface of the other immunoglobulin polypeptide. In embodiments, the pot is the same size as or similar to the pestle and is appropriately positioned such that when the two interfaces interact, the pestle of one interface can be positioned in the corresponding pore of the other interface. Without being bound by theory, this is considered to stabilize heteropolymers and facilitate the formation of heteropolymers relative to other substances (e.g., homopolymers). In this implementation, the method can be used to promote heteropolymerization of two different immunoglobulin peptides, thereby generating a bispecific antibody comprising two immunoglobulin peptides that have binding specificity to different epitopes.

[0246] Depending on the method, an antibody variable domain (antibody-antigen combination site) with desired binding specificity is fused to an immunoglobulin constant domain sequence. The fusion is preferably performed with the immunoglobulin heavy chain constant domain, including at least a portion of the hinge region, CH2 region, and CH3 region. Typically, the first heavy chain constant region (CH1) contains the site necessary for light chain binding and is present in at least one of the fusion compounds. DNA encoding the immunoglobulin heavy chain fusion compound and (if desired) the immunoglobulin light chain is inserted into separate expression vectors and co-transfected into a suitable host organism. This provides great flexibility in adjusting the ratio of the three polypeptide fragments in the embodiment when using unequal ratios of the three polypeptide chains in the construction provides optimal yield. However, when expressing at least two polypeptide chains in equal ratios yields high yields or when the ratios are not particularly significant, the coding sequences of two or all three polypeptide chains can be inserted into a single expression vector.

[0247] In one embodiment of this method, the bispecific antibody consists of a hybrid immunoglobulin heavy chain with first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing second binding specificity) in the other arm. Studies have found that this asymmetric structure facilitates the separation of the desired bispecific compound from the unwanted immunoglobulin chain combination, as only half of the bispecific molecule contains the immunoglobulin light chain, providing a simple separation method. This method is disclosed in WO 94 / 04690. For further details on the generation of bispecific antibodies, see, for example, Suresh et al. Methods in Enzymology 121:210 (1986).

[0248] According to another method described in WO96 / 27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell cultures. One interface comprises the C0 of the antibody constant domain. H At least a portion of the 3-domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine), a compensating “cavity” of the same or similar size as the large side chain is created at the interface of the second antibody molecule. This provides a mechanism to increase the yield of heterodimers relative to other unwanted end products (such as homodimers).

[0249] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one antibody in a heteroconjugate may be conjugated to avidin, and the other to biotin. Such antibodies have been proposed for targeting unwanted cells with immune system cells (US Patent No. 4,676,980) and for treating HIV infection (WO 91 / 00360, WO 92 / 200373, and EP 03089). Heteroconjugate antibodies can be prepared using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed in US Patent No. 4,676,980, along with numerous cross-linking techniques.

[0250] Techniques for generating bispecific antibodies from antibody fragments are also described in the literature. For example, chemical bonding can be used to prepare bispecific antibodies. (Brennan et al., ...) Science 229:81 (1985) describes a procedure in which an intact antibody is cleaved by proteolytic hydrolysis to produce F(ab′)2 fragments. These fragments are reduced in the presence of a dithiol chelating agent, sodium arsenite, to stabilize the ortho-dithiol and prevent intermolecular disulfide formation. The resulting Fab′ fragments are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives is then reconverted to Fab′-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of another Fab′-TNB derivative to form a bispecific antibody. The resulting bispecific antibody can be used as a selective immobilization agent for enzymes. Shalaby et al., J. Exp. Med., 175:217-225 (1992) describes the production of the fully humanized bispecific antibody F(ab′)2 molecule. Each Fab′ fragment was individually derived from *E. coli*. (E. coli) It is secreted in the body and undergoes directed chemical coupling in vitro to form bispecific antibodies.

[0251] Various techniques for preparing and isolating bispecific antibody fragments directly from recombinant cell cultures are also described. For example, bispecific antibodies have been produced using leucine zippers. (Kostelny et al.) J. Immunol., 148(5):1547-1553 (1992). Leucine zipper peptides from Fos and Jun proteins are linked to the Fab′ region of two different antibodies via gene fusion. Antibody homodimers are reduced to monomers in the hinge region and then re-oxidized to form antibody heterodimers. This method can also be used to generate antibody homodimers. Hollinger et al., Proc. Natl. Acad. Sci. USA, The “dual antibody” technique described in 90:6444-6448 (1993) provides an alternative mechanism for preparing bispecific antibody fragments. These fragments contain components connected via a linker to a light chain variable domain (V...).L ) connected heavy chain variable structural domain (V H The joint is too short for two structural domains on the same chain to pair. Therefore, a segment of V... H and V L The structural domain is forced to interact with the complementary V of another segment. L and V H Domain pairing forms two antigen-binding sites. Another strategy for preparing bispecific antibody fragments using single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol, 152:5368 (1994).

[0252] Another technique for preparing bispecific antibody fragments is the “bispecific T-cell adaptor” or BiTE® method (see, for example, WO2004 / 106381, WO2005 / 061547, WO2007 / 042261 and WO2008 / 119567). This method utilizes two antibody variable domains arranged on a single polypeptide. For example, a single polypeptide chain comprises two single-chain Fv (scFv) fragments, each with a variable heavy chain (V... H ) and variable light chains (V L The two scFv fragments are separated by a polypeptide linker of sufficient length to enable intramolecular association between them. This single polypeptide further includes a polypeptide spacer sequence between the two scFv fragments. Each scFv recognizes a different epitope, and these epitopes may be specific to different cell types, so that when each scFv binds to its homologous epitope, cells of two different cell types will approach or tether together. A particular embodiment of this method involves an scFv that recognizes a cell surface antigen expressed by immune cells (e.g., the CD3 polypeptide on T cells) linked to another scFv that recognizes a cell surface antigen expressed by target cells (such as malignant tumor cells or tumor cells).

[0253] Because it is a single polypeptide, the bispecific T-cell adaptor can be expressed using any prokaryotic or eukaryotic cell expression system known in the art (e.g., CHO cell lines). However, specific purification techniques (see, for example, EP1691833) may be required to separate the monomeric bispecific T-cell adaptor from other multimers that may possess biological activities beyond the monomer's intended activity. In one exemplary purification protocol, a solution containing the secreted polypeptide is first subjected to metal affinity chromatography, followed by elution of the polypeptide with an imidazole concentration gradient. This eluent is further purified using anion exchange chromatography, followed by elution of the polypeptide with a sodium chloride concentration gradient. Finally, this eluent is subjected to size exclusion chromatography to separate the monomer from the multimers.

[0254] Other related bispecific antibody fragment types include, but are not limited to, biaffinity-based targeted proteins (DART) and tandem biantibodies (TandAbs). DART consists of two Fv fragments, which heterodimerize to form two unique antigen-binding sites (Holliger et al., ...). Proc. Natl. Acad. Sci. USA. 90:6444–6448 (1993). Specifically, Fv1 consists of a VH from antibody “A” and a VL from antibody “B”, while Fv2 consists of a VH from antibody “B” and a VL from antibody “A”. Unlike BiTE antibodies linked via peptide linkers, this combination allows DART to mimic the natural interactions within the IgG molecule. Adding another cysteine ​​residue to the end of each heavy chain can improve stability by forming a C-terminal disulfide bridge. TandAbs are tetravalent bispecific antibodies that provide two binding sites for each antigen to maintain the affinity of the natural bivalent antibody. Furthermore, the molecular weight of TandAbs (approximately 105 kDa) exceeds the first-pass renal clearance threshold, thus resulting in a longer half-life compared to smaller antibody constructs (Reusch et al., ). Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. 22:5829–5838 (2016); Reusch et al., MAbs. 6:728–739 (2014); Compte et al., Oncoimmunology. 3:e28810 (2014). For the latest review of common types of bispecific antibodies (including scFv-based antibodies and full-length IgG-like asymmetric antibodies, including their generation methods), see Wang et al., Antibodies (Basel), 8(3): 43 (2019).

[0255] Antibody variants and modifications

[0256] Replacement, insertion, and missing variants

[0257] In the implementation, the antigen recognition portion is or includes an antibody variant. Antibody variants can be prepared by introducing appropriate nucleotide changes into encoding DNA and / or by synthesizing the desired antibody or peptide. Those skilled in the art will understand that amino acid changes can alter the post-translational processing of the antibody, such as changing the number or location of glycosylation sites or altering membrane anchoring characteristics.

[0258] The antibody variations described herein can be performed, for example, using any techniques and guidelines for conserved and non-conserved mutations as set forth in, for example, U.S. Patent No. 5,364,934. Variations can be substitutions, deletions, or insertions of one or more codons encoding an antibody or polypeptide, resulting in a change in the amino acid sequence compared to the native sequence of the antibody or polypeptide. Optionally, variation is achieved by substituting at least one amino acid in one or more domains of the antibody with any other amino acid. By comparing the sequence of the antibody with the sequence of a known homologous protein molecule and minimizing the number of amino acid sequence changes performed in highly homologous regions, guidelines can be established to determine which amino acid residues can be inserted, substituted, or deleted without adversely affecting the desired activity. Amino acid substitution can be the result of replacing one amino acid with another amino acid having similar structural and / or chemical properties, such as replacing leucine with serine, i.e., a conserved amino acid substitution. Insertions or deletions can optionally range from about 1 to 5 amino acids. Permissible variations can be determined by systematically inserting, deleting, or substituting amino acids in the sequence and testing the activity exhibited by the resulting variants by the full-length or mature native sequence.

[0259] This article provides antibody fragments. For example, such fragments may be truncated at the N-terminus or C-terminus, or may lack internal residues, when compared to full-length natural antibodies or proteins. Some fragments lack amino acid residues that are not essential for the desired biological activity of the antibody.

[0260] Antibody fragments can be prepared using any of a variety of conventional techniques. The desired peptide fragment can be chemically synthesized. Alternative methods involve generating antibody or polypeptide fragments via enzymatic digestion, such as by treating proteins with an enzyme known to cleave proteins at sites defined by specific amino acid residues, or by digesting DNA with a suitable restriction enzyme and isolating the desired fragment. Another suitable technique involves isolating and amplifying DNA fragments encoding the desired antibody or polypeptide fragment via polymerase chain reaction (PCR). Oligonucleotides defining the desired ends of the DNA fragment are used at the 5' and 3' primers in the PCR. Preferably, the antibody fragment shares at least one biological and / or immunological activity with the natural antibodies disclosed herein.

[0261] In certain embodiments, the conservative substitutions of interest are shown under the headings of preferred substitutions in Table 1. If such substitutions result in a change in biological activity, more substantial changes are introduced, namely the exemplary substitutions named in Table 1, or as further described below with reference to the amino acid categories, and products are screened.

[0262] Table 1

[0263] Substantial modification of antibody function or immunological properties is achieved through selective substitution, where the substitutions vary significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the substituted region, such as folded or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the volume of the side chains. Naturally occurring residues are grouped into several groups based on common side chain characteristics: (1) Hydrophobic: Leucine, met, ala, val, leu, ile; (2) Neutral and hydrophilic: cys, ser, thr; (3) Acidic: asp, glu; (4) Alkaline: asn, gln, his, lys, arg; (5) Residues that affect chain orientation: gly, pro; and (6) Fang ethnic group: trp, tyr, phe.

[0264] Non-conservative substitutions will require replacing one of these categories with a member of another category. Such substituted residues may also be introduced into conserved substitution sites, or more preferably, into the remaining (non-conservative) sites.

[0265] Mutations can be generated using methods known in the art, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (Carter et al., Nucl. Acids Res ., 13: 4331 (1986); Zoller et al., Nucl. Acids Res ., 10: 6487 (1987)), box-type mutagenesis (Wells et al., 10: 6487 (1987)), Gene , 34:315 (1985)), restrictive selection mutagenesis (Wells et al., , Philos. Trans. R. Soc. London SerA (317: 415 (1986)) or other known techniques can be used to process cloned DNA to produce antibody variant DNA.

[0266] Scanning amino acid analysis can also be used to identify one or more amino acids along a continuous sequence. Preferred scanning amino acids include relatively small numbers of neutral amino acids. These include alanine, glycine, serine, and cysteine. Alanine is often a preferred scanning amino acid in this group because it eliminates the side chain except for the β-carbon and is unlikely to alter the main chain conformation of variants (Cunningham and Wells, ...). Science, 244: 1081-5 (1989)). Alanine is generally preferred because it is the most common amino acid. Furthermore, it is frequently found in both buried and exposed locations (Creighton, The Proteins, (WH Freeman & Co., NY); Chothia, 244: 1081-5 (1989)). J. Mol. Biol ., 150: 1(1976)). If alanine substitution does not produce a sufficient amount of variants, isomorphic amino acids may be used.

[0267] Any cysteine ​​residue that does not participate in maintaining the correct conformation of the antibody can be substituted, usually with serine, to improve the oxidative stability of the molecule and prevent undesirable cross-linking. Conversely, cysteine ​​bonds can be added to antibodies to improve their stability (especially when the antibody is an antibody fragment such as an Fv fragment).

[0268] Particularly preferred types of substitution variants involve replacing one or more hypervariable residues of the parent antibody (e.g., a humanized antibody or a human antibody). Typically, the resulting variants selected for further development exhibit improved biological properties relative to the parent antibody from which they originated. A convenient method for generating such substitution variants involves maturation using phage-displayed affinity. In short, several hypervariable sites (e.g., 6-7 sites) are mutated to produce all possible amino substitutions at each site. The resulting antibody variant is displayed monovalently from filamentous phage particles as a fusion with the M13 gene III product packaged within each particle. As disclosed herein, the biological activity (e.g., binding affinity) of the phage-displayed variants is then screened. To identify candidate hypervariable sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable residues that significantly contribute to antigen binding. Alternatively or additionally, it may be beneficial to analyze the crystal structure of the antigen-antibody complex to identify contact sites between the antibody and the antigenic peptide. Such contact residues and adjacent residues are candidates for substitution according to the techniques detailed herein. Once such variants are generated, the variant group is screened as described herein, and antibodies that exhibit superior properties in one or more relevant assays can be selected for further development.

[0269] Nucleic acid molecules encoding amino acid sequence variants of antibodies can be prepared using a variety of methods known in the art. These methods include, but are not limited to, preparation by isolating from natural sources (in the case of naturally occurring amino acid sequence variants) or by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of variant or non-variant forms of previously prepared antibodies.

[0270] Modification

[0271] Covalent modification of antibodies is included within the scope of this invention. One type of covalent modification involves reacting the target amino acid residues of the antibody with an organic derivatizing agent capable of reacting with selected side chains or N-terminal or C-terminal residues of the antibody. Derivatization with bifunctional reagents is useful, for example, for crosslinking antibodies with water-insoluble supporting matrices or surfaces for use in antibody purification methods, and vice versa. Commonly used crosslinking agents include, for example, 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters (e.g., esters with 4-azidosalicylic acid), homodifunctional imino esters (including disuccinimide esters, such as 3,3'-dithiobis(succinimide propionate)), bifunctional maleimides (such as bis-N-maleimide-1,8-octane), and agents such as methyl-3-[(p-azidophenyl)dithio]propionimide ester.

[0272] Other modifications include deamidation of glutamine and asparagine residues to the corresponding glutamyl and asparagine residues, hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of serine or threonyl residues, methylation of the α-amino groups of the lysine, arginine and histidine side chains (TE Creighton, Proteins: Structure and Molecular Properties, WH Freeman & Co., San Francisco, pp. 79-86 (1983)), acetylation of N-terminal amines, and amidation of any C-terminal carboxyl group.

[0273] Another type of covalent modification of antibodies, including within the scope of this invention, involves altering the native glycosylation pattern of the antibody or peptide. For the purposes of this document, “altering the native glycosylation pattern” is intended to mean the deletion of one or more carbohydrate moieties present in the native sequence antibody (by removing potential glycosylation sites or by chemical and / or enzymatic means of glycosylation deletion) and / or the addition of one or more glycosylation sites not present in the native sequence antibody. Furthermore, this phrase encompasses qualitative changes in the glycosylation of native proteins, involving changes in the nature and proportions of the various carbohydrate moieties present.

[0274] Glycosylation of antibodies and other peptides is typically N-linked or O-linked. N-linking refers to the attachment of the carbohydrate moiety to the asparagine residue as a side chain. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid other than proline) are recognition sequences used for the enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Therefore, the presence of either of these tripeptide sequences in a peptide creates a potential glycosylation site. O-linked glycosylation involves attaching one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyl amino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

[0275] Adding glycosylation sites to antibodies can be conveniently accomplished by altering their amino acid sequences, resulting in the presence of one or more of the aforementioned tripeptide sequences (for N-linked glycosylation sites). Alterations can also be made by adding one or more serine or threonine residues to the original antibody sequence, or by substituting one or more serine or threonine residues into the original antibody sequence (for O-linked glycosylation sites). Optionally, the antibody amino acid sequence can be altered by changes at the DNA level, particularly by mutating the DNA encoding the antibody at pre-selected bases, resulting in codons that will translate into the desired amino acids.

[0276] Another method to increase the amount of carbohydrate moiety on an antibody is through chemical or enzymatic coupling of glycosides with peptides. Such methods are described in the art, for example in WO 87 / 05330, published on 11 September 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

[0277] The removal of the carbohydrate moiety present on an antibody can be accomplished chemically or enzymatically, or by mutating and substituting codons encoding amino acid residues that act as glycosylation targets. Chemical deglycosylation techniques are known in the art and are described, for example, by Hakimuddin et al., Arch. Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem., 118:131 (1981). Enzymatic cleavage of the carbohydrate moiety on a polypeptide can be achieved using various endonucleases and exonucleases, as described by Thotakura et al., Meth. Enzymol., 138:350 (1987).

[0278] Fc region variants

[0279] It may be desirable to modify the antibodies of the present invention in terms of effector function, for example, to enhance the antigen-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) of the antibody. This can be achieved by introducing one or more amino acid substitutions into the Fc region of the antibody. Alternatively or additionally, cysteine ​​residues may be introduced into the Fc region, thereby allowing the formation of interchain disulfide bonds in this region. The resulting homodimeric antibodies may have improved internalization capacity and / or increased complement-mediated cell killing and antibody-dependent cytotoxicity (ADCC) (see Caron et al., J. Exp Med . 176:1191-5 (1992); Shopes, B. J. Immunol . 148: 2918-22 (1992)). Alternatively, Wolff et al. may be used. Cancer Research The heterobifunctional cross-linking agents described in 53: 2560-5 (1993) are used to prepare homodimeric antibodies with enhanced antitumor activity. Alternatively, the antibodies can be engineered to have dual Fc regions, thereby exhibiting enhanced complement cleavage and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design 3: 219-30 (1989). To increase the serum half-life of an antibody, a salvage receptor-binding epitope can be incorporated into the antibody (particularly an antibody fragment), as described, for example, in U.S. Patent 5,739,277. As used herein, the term "salvage receptor-binding epitope" refers to an epitope in the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.

[0280] Cysteine-engineered antibody variants

[0281] In some embodiments, it may be desirable to create cysteine-engineered antibodies, such as “thioMAb”, wherein one or more residues of the antibody are replaced with cysteine ​​residues. In specific embodiments, the substituted residues are located at accessible sites on the antibody. By replacing those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites on the antibody and can be used to conjugate the antibody with other parts (such as pharmaceutical parts or linker-pharmaceutical parts) to create immunoconjugates, as further described herein. Cysteine-engineered antibodies can be produced, for example, as described in U.S. Patent No. 7,521,541.

[0282] Chimeric antigen receptor (CAR)

[0283] In some embodiments, the antigen recognition portion of the modified T cell is a CAR. The CAR may be expressed on the surface of the modified T cell and contains an affinity-binding domain specific to disease-associated antigens (e.g., tumor antigens, autoimmune disease-associated antigens, or pathogenic antigens), or means for specifically binding to disease-associated antigens. This disclosure also provides nucleic acids encoding CARs, as well as constructs and vectors containing said nucleic acids. This disclosure also provides an isolated polynucleotide (which may be used interchangeably with "nucleic acid" herein) comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR) and a nucleic acid sequence encoding at least one c-kit agonist. In some cases, the nucleic acid is a heterologous component, for example, an expression cassette. In some embodiments, the nucleic acid is a heterologous component, for example, a retroviral vector. In some embodiments, the nucleic acid is a heterologous component, for example, a modified T cell. In some embodiments, the nucleic acid is a heterologous component, for example, a modified γδ T cell. In some embodiments, the nucleic acid is a heterologous component, for example, a γδ T cell. + T cells and / or delta cells + Heterogeneous components of T cells.

[0284] A CAR may include an affinity-binding domain or a means for specific binding operatively connected to another domain of the CAR, such as a transmembrane domain, a co-stimulatory domain, and / or an intracellular signaling domain, as described herein. The affinity-binding domain or means for specific binding described herein may be combined with any of the transmembrane domains, co-stimulatory domains, and / or intracellular signaling domains described herein and / or any other domains described herein that may be included in the CAR herein. A CAR may also include a hinge domain as described herein. A CAR may also include at least one spacer domain as described herein.

[0285] The present invention includes nucleic acids encoding CARs, and constructs and vectors containing such nucleic acids. In some cases, the nucleic acid is a heterologous component, for example, an expression cassette. In embodiments, the nucleic acid is a heterologous component, for example, a retroviral vector. In embodiments, the nucleic acid is a heterologous component, for example, a T cell. In embodiments, the nucleic acid is a heterologous component, for example, a γδ T cell. In embodiments, the nucleic acid is, for example, a γδ T cell. + T cells and / or delta cells + Heterogeneous components of T cells. In the implementation scheme, nucleic acids are, for example, α-cells. - T cells and / or beta cells - Heterogeneous components of T cells.

[0286] Affinity-binding domain / means for specific binding

[0287] The affinity-binding domain may include any domain that binds to disease-associated antigens (e.g., antigens associated with tumors and autoimmune diseases, as well as pathogenic antigens described above). Similarly, the means for specific binding may interact with any disease-associated antigen, such as antigens associated with tumors and autoimmune diseases, as well as pathogenic antigens. In embodiments, the affinity-binding domain or the means for specific binding binds to tumor antigens. For example, the affinity-binding domain or the means for specific binding may bind to CD20, BCMA, GPC3, TyrD, B7H6, CD70, or PSMA.

[0288] The affinity-binding domain or means for specific binding may include the antibodies described above, fragments thereof, or variants thereof. In embodiments, the affinity-binding domain or means for specific binding includes monoclonal antibodies, polyclonal antibodies, synthetic antibodies, human antibodies, humanized antibodies, non-human antibodies, bispecific antibodies, and any fragments thereof. In embodiments, the affinity-binding domain or means for specific binding may be an antibody, an antigen-binding fragment (Fab), a single-chain variable fragment (scFv), a heavy-chain or light-chain single-domain antibody (sdAb), F(ab)2, or any combination thereof. In embodiments, the affinity-binding domain or means for specific binding includes mammalian antibodies or fragments thereof. The selection of the affinity-binding domain or means for specific binding may depend on the type and quantity of antigen present on the surface of the target cell.

[0289] This disclosure provides antibodies and CARs that have “substantial identity” or “substantial similarity” to the sequences provided herein in the CDR or frame region. When referring to nucleic acids or fragments thereof, the terms “substantial identity” or “substantial similarity” indicate, when best aligned with another nucleic acid (or its complementary strand), the presence of a percentage of nucleotide sequence identity, such as at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, at least 99.5%, or 100% of the nucleotide bases, as measured by any well-known sequence identity algorithm (such as FASTA, BLAST, or GAP), as discussed below. In some cases, nucleic acid molecules that have substantial identity with a reference nucleic acid molecule may encode a polypeptide with the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0290] When applied to peptides, the terms "substantial similarity" or "substantial resemblance" mean that, in the case of optimal alignment (such as using procedures like GAP or BESTFIT with default vacancy weights), two peptide sequences share at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, at least 99.5%, or 100% sequence identity. In some respects, dissimilar residue positions differ due to conserved amino acid substitutions. A "conserved amino acid substitution" is a substitution in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conserved amino acid substitutions will not substantially alter the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conserved substitutions, the percentage or degree of similarity can be adjusted upwards to correct for the conservatism of the substitution. The means of making such adjustments are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331, which is incorporated herein by reference. Examples of amino acid groups with side chains having similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxy side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conserved amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change with a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443 45 (incorporated herein by reference). A “moderately conservative” substitution is any change with a non-negative value in the PAM250 log-likelihood matrix.

[0291] Sequence identity and / or similarity of peptides are typically measured using sequence analysis software. Protein analysis software uses similarity measures assigned to various substitutions, deletions, and other modifications, including conserved amino acid substitutions, to match similar sequences. For example, GCG software contains programs such as GAP and BESTFIT, which can determine sequence homology or sequence identity between closely related peptides (such as homologous peptides from different biological species) or between wild-type proteins and their mutant counterparts using default parameters. See, for example, GCG version 6.1. Peptide sequences can also be compared using FASTA (a program within GCG version 6.1) with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignment of the best overlapping regions between the query and retrieved sequences and a percentage of sequence identity (Pearson (2000) ibid.). Sequences can also be compared using the Smith-Waterman homology retrieval algorithm with an affine gap retrieval having a gap open penalty of 12, a gap extension penalty of 2, and a BLOSUM matrix of 62. Another preferred algorithm when comparing the sequences disclosed herein with databases containing a large number of sequences from different organisms is the computer program BLAST using default parameters, particularly BLASTP or TBLASTN. See, for example, Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and (1997) Nucleic Acids Res. 25:3389-3402, each of which is incorporated herein by reference.

[0292] Transmembrane domain

[0293] A CAR may include a transmembrane domain that couples the CAR's affinity-binding domain to one or more intracellular domains of the CAR. The transmembrane domain of the CAR disclosed herein is a region capable of crossing the plasma membrane of a cell (e.g., a T cell). In embodiments, the transmembrane domain is dispersed between the CAR's affinity-binding domain / means for specific binding and one or more intracellular domains.

[0294] In the implementation scheme, the transmembrane domain naturally associates with one or more domains in the CAR. In the implementation scheme, the transmembrane domain can be selected or modified by one or more amino acid substitutions to prevent such domains from binding to transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex.

[0295] For example, but not limited to, transmembrane domains can be derived from natural or synthetic sources. In the case of natural sources, the domains can be derived from any membrane-binding or transmembrane protein. The transmembrane regions particularly used in this invention can be derived from (i.e., include at least the following transmembrane regions): 4-1BB / CD137, activated NK cell receptor, immunoglobulin, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276. (B7-H3), CD28, CD29, CD3δ, CD3ε, CD3γ, CD3ζ, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8α, CD8β, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, cytokine receptors, DAP10, DNAM1 (CD226), Fcγ receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Ig α (CD79a), IL-2R β, IL-2R γ, IL-7R α, Inducible T cell co-stimulatory factor (ICOS), integrins, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITG1, KIRDS2, LAT, LFA-1, CD83-specific ligands, LIGHT, LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1; CD11a / CD18), MHC class 1 molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed cell death-1 (PD-1), PSGL1, SELPLG (CD162), signal transduction lymphocyte activation molecules (SLAM proteins), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A;Lyl08), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1 or VLA-6 or fragments, truncated forms or combinations thereof.Alternatively, the transmembrane domain can be synthetic, in which case it will primarily contain hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan, and valine will be present at each end of the synthetic transmembrane domain.

[0296] In some embodiments, the transmembrane domain includes a transmembrane domain of CD8. In some embodiments, the transmembrane domain of CD8 is a transmembrane domain of CD8α. In some embodiments, the transmembrane domain of CD8 contains the amino acid sequence shown in SEQ ID NO: 211. In some embodiments, the transmembrane domain includes a transmembrane domain of CD28. In some embodiments, the transmembrane domain of CD28 contains the amino acid sequence shown in SEQ ID NO: 215. In some embodiments, the transmembrane domain includes a transmembrane domain of ICOS. In some embodiments, the transmembrane domain of ICOS contains the amino acid sequence shown in SEQ ID NO: 216.

[0297] The transmembrane domains described herein may be combined with any affinity-binding domains described herein, any intracellular domains described herein, or any other domains described herein that may be included in the subject CAR.

[0298] In embodiments, the transmembrane domain further includes a hinge region. The subject matter of the present invention, the CAR, may also include a hinge region. The hinge region of a CAR is a hydrophilic region located between the affinity-binding domain / means for specific binding and the transmembrane domain. In embodiments, this domain facilitates proper protein folding of the CAR. The hinge region is an optional component of the CAR. The hinge region may include domains selected from: the Fc fragment of an antibody, the hinge region of an antibody, the CH2 region of an antibody, the CH3 region of an antibody, artificial hinge sequences, or combinations thereof. Examples of hinge regions include, but are not limited to, CD8α hinges, CD8β hinges, CD28 hinges, 4-1BB hinges, CD7 hinges, artificial hinges prepared from peptides that may be as small as three glycine residues (Gly), and the CHI and CH3 domains of IgG (such as human IgG4). Naturally occurring hinge domains may be used as wild-type hinge regions, or the molecule may be modified.

[0299] In embodiments, the subject matter CAR of this disclosure includes a hinge region that couples an affinity-binding domain / means for specific binding to a transmembrane domain, which in turn couples to one or more intracellular domains. The hinge region is preferably capable of supporting the affinity-binding domain / means for specific binding to recognize and bind to target antigens on target cells (see, for example, Hudecek et al., Cancer Immunol. Res. (2015) 3(2): 125-135). In embodiments, the hinge region is a flexible domain, thereby allowing the affinity-binding domain / means for specific binding to have a specific structure and density that optimally recognizes target antigens on cells (such as tumor cells) (Hudecek et al., ibid.). The flexibility of the hinge region allows it to adopt many different conformations. In embodiments, the hinge region is an immunoglobulin heavy chain hinge region. In embodiments, the hinge region is a receptor-derived hinge region polypeptide (e.g., a CD8-derived hinge region).

[0300] The length of the hinge region can be from about 4 amino acids to about 50 amino acids, for example, from about 4 aa to about 10 aa, from about 10 aa to about 15 aa, from about 15 aa to about 20 aa, from about 20 aa to about 25 aa, from about 25 aa to about 30 aa, from about 30 aa to about 40 aa, or from about 40 aa to about 50 aa. In embodiments, the length of the hinge region can be greater than 5 aa, greater than 10 aa, greater than 15 aa, greater than 20 aa, greater than 25 aa, greater than 30 aa, greater than 35 aa, greater than 40 aa, greater than 45 aa, greater than 50 aa, greater than 55 aa, or greater.

[0301] Suitable hinge regions can be readily selected and can be any of a number of suitable lengths, such as 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids. Suitable hinge regions can have a length greater than 20 amino acids (e.g., 30, 40, 50, 60, or more amino acids).

[0302] For example, the hinge region includes glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n (SEQ ID NO: 174) and (GGGS)n (SEQ ID NO: 175), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible connectors known in the art. Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured and can therefore be used as neutral tethers between components. Glycine polymers can be used; glycine can enter significantly more phi-psi spaces than even alanine and is much less restricted than residues with longer side chains (see, for example, Scheraga, Rev. Computational. Chem. (1992)2: 73-142). The exemplary hinge region may contain amino acid sequences, including but not limited to GGSG (SEQ ID NO: 177), GGSGG (SEQ ID NO: 178), GGSG (SEQ ID NO: 179), GGSGG (SEQ ID NO: 180), GGGSG (SEQ ID NO: 181), GSSSG (SEQ ID NO: 182), etc.

[0303] In the implementation, the hinge region is the immunoglobulin heavy chain hinge region. The amino acid sequence of the immunoglobulin hinge region is known in the art; see, for example, Tan et al., Proc. Natl. Acad. Sci. USA (1990) 87(1):162-166; and Huck et al., Nucleic Acids Res. (1986) 14(4): 1779-1789. As a non-limiting example, the immunoglobulin hinge region may include one of the following amino acid sequences: DKTHT (SEQ ID NO: 217); CPC (SEQ ID NO: 218); CPEPKSCDTPPPCPR (SEQ ID NO: 219) (see, for example, Glaser et al., J. Biol. Chem. (2005) 280:41494-41503); ELKTPLGDTTHT (SEQ ID NO: 220); KSCDKTHTCP (SEQ ID NO: 221); KCCVDCP (SEQ ID NO: 222); KYGPPCP (SEQ ID NO: 223); EPKSCDKTHTCPPCP (SEQ ID NO: 224) (human IgG1 hinge); ERKCCVECPPCP (SEQ ID NO: 225) (human IgG2 hinge); ELKTPLGDTTHTCPRCP (SEQ ID NO: 223); 226)(human IgG3 hinge); SPNMVPHAHHAQ (SEQ ID NO: 227)(human IgG4 hinge); etc.

[0304] The hinge region may contain the amino acid sequence of a human IgG1, IgG2, IgG3, or IgG4 hinge region. In one embodiment, the hinge region may include one or more amino acid substitutions and / or insertions and / or deletions compared to a wild-type (naturally occurring) hinge region. For example, His229 of the human IgG1 hinge may be substituted with Tyr, such that the hinge region contains the sequence EPKSCDKTYTCPPCP (SEQ ID NO: 228); see, for example, Yan et al., J. Biol. Chem. (2012) 287:5891-5897.

[0305] In some embodiments, the hinge region may comprise an amino acid sequence derived from human CD8 or a variant thereof. In some embodiments, the CAR comprises a CD8α hinge sequence comprising the amino acid sequence shown in SEQ ID NO: 209. In some embodiments, the CAR comprises a hinge and a transmembrane domain sequence comprising the amino acid sequence shown in SEQ ID NO: 213.

[0306] Co-stimulatory domain

[0307] In an embodiment, the CAR includes at least one co-stimulatory domain, wherein the co-stimulatory domain includes a functional co-stimulatory signaling domain derived from, for example, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, etc. For example, within the scope of this disclosure, the CAR may include 2, 3, 4, or more co-stimulatory domains. Also within the scope of this disclosure, when more than one co-stimulatory domain is included, the co-stimulatory domains may be the same or they may be different.In the implementation scheme, the co-stimulatory domain is derived from one or more of the following: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, B7-H3, CEACAM1, CRTAM, CD2, CD3C, CD4, CD7, CD8α, CD8β, CD11a, CD11b, CD11c, CD11d, IL2Rβ, IL2γ, IL7Rα, IL4R, IL7R, IL15R, IL21R, CD18, CD19, CD19a, CD27, CD28, CD29, CD30, CD40, CDS, CD49a, CD49D, CD49f, CD54 (ICAM), CD69, CD70, CD80, CD83, CD84, CD86, CD96 (Tactile), CD100 (SEMA4D), CD103, CD134 (OX40), CD137 (4-1BB), CD152 (CTLA-4), CD160 (BY55), CD162 (SELPLG), CD244 (2B4), CD270 (HVEM), CD226 (DNAM1), CD229 (Ly9), CD278(ICOS), ICAM-1, LFA-1 (CD11a / CD18), FcR, FcγRI, FcγRII, FcγRIII, LAT, NKG2C, SLP76, TRIM, ZAP70, GITR, BAFFR, LTBR, ​​LAT, GADS, LIGHT, HVEM (LIGHTR), KIRDS2, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, NKG2C, NKG2D, IA4, VLA-1, VLA-6, SLAM (SLAMF1, CD150, IPO-3), SLAMF4, SLAMF6 (NTB-A, Ly108), SLAMF7, SLAMF8 (BLAME), SLP-76, PAG / Cbp, NKp80 (KLRF1), NKp44, NKp30, NKp46, BTLA, JAML, CD150, PSGL1, TSLP, TNFR2, and TRANCE / RANKL or parts thereof and combinations thereof.

[0308] In one embodiment, the nucleic acid encoding the CAR encodes at least one 4-1BB co-stimulatory domain and optionally encodes a second co-stimulatory domain selected from 4-1BB, 2B4, ICOS, CD28, OX40, and CD27 co-stimulatory domains or any of the aforementioned co-stimulatory domains. In another embodiment, the nucleic acid encodes at least two 4-1BB co-stimulatory domains, or at least two 4-1BB co-stimulatory domains combined with one, two, three, or four or more co-stimulatory domains selected from 4-1BB, ICOS, CD28, OX40, and CD27 or any of the aforementioned co-stimulatory domains. In another embodiment, the 4-1BB co-stimulatory domain comprises the amino acid sequence shown in SEQ ID NO: 202. In another embodiment, the 4-1BB co-stimulatory domain comprises at least one, at least two, or at least three or more modified amino acid sequences having the amino acid sequence of SEQ ID NO: 202. In another embodiment, the 4-1BB co-stimulatory domain is substantially similar to the 4-1BB co-stimulatory domain comprising SEQ ID NO: 202.

[0309] In one embodiment, the nucleic acid encoding the CAR encodes at least one CD27 co-stimulatory domain and optionally at least one second co-stimulatory domain selected from 4-1BB, ICOS, CD28, OX40, 2B4, and CD27 co-stimulatory domains, or any of the aforementioned co-stimulatory domains. In another embodiment, the nucleic acid encodes at least one CD27 co-stimulatory domain and a 4-IBB co-stimulatory domain. In yet another embodiment, the nucleic acid encodes two CD27 co-stimulatory domains and at least one second co-stimulatory domain selected from 4-1BB, ICOS, CD28, and CD27. In one embodiment, the CD27 co-stimulatory domain comprises SEQ ID NO: 208. In another embodiment, the CD27 co-stimulatory domain comprises at least one, at least two, at least three, or more modified amino acid sequences having the amino acid sequence of SEQ ID NO: 208. In yet another embodiment, the CD27 co-stimulatory domain is substantially similar to the CD27 co-stimulatory domain comprising SEQ ID NO: 208.

[0310] In one embodiment, the nucleic acid encoding CAR encodes at least one CD28 co-stimulatory domain and optionally encodes a second co-stimulatory domain selected from 4-1BB, 2B4, ICOS, CD28, OX40, and CD27 co-stimulatory domains, or any of the aforementioned co-stimulatory domains. In another embodiment, the nucleic acid encodes at least two CD28 co-stimulatory domains, or at least two CD28 co-stimulatory domains combined with one, two, three, or four or more co-stimulatory domains selected from 4-1BB, ICOS, CD28, OX40, and CD27, or any of the aforementioned co-stimulatory domains. In one embodiment, the CD28 co-stimulatory domain comprises SEQ ID NO: 204. In another embodiment, the CD28 co-stimulatory domain comprises SEQ ID NO: 205. SEQ ID NO: 204 and SEQ ID NO: 205 include three subdomains, YMNM, PRRP, and PYAP, capable of modulating signal transduction pathways. In the embodiments, the disclosed CAR includes mutations or deletions in one or more of the subdomains (see, for example, WO2019010383). In the embodiments, the CD28 co-stimulatory domain includes at least one, at least two, at least three or more modified amino acid sequences having the amino acid sequence of SEQ ID NO: 204 or the amino acid sequence of SEQ ID NO: 205. In the embodiments, the CD28 co-stimulatory domain is substantially similar to the CD28 co-stimulatory domain containing SEQ ID NO: 204. In the embodiments, the CD28 co-stimulatory domain is substantially similar to the CD28 co-stimulatory domain containing SEQ ID NO: 205.

[0311] In one embodiment, the nucleic acid encoding CAR encodes at least one ICOS co-stimulatory domain and optionally encodes a second co-stimulatory domain selected from 4-1BB, 2B4, ICOS, CD28, OX40, and CD27 co-stimulatory domains, or any of the aforementioned co-stimulatory domains. In another embodiment, the nucleic acid encodes at least two ICOS co-stimulatory domains, or at least two ICOS co-stimulatory domains combined with one, two, three, or four or more co-stimulatory domains selected from 4-1BB, ICOS, CD28, OX40, and CD27, or any of the aforementioned co-stimulatory domains. In another embodiment, the ICOS co-stimulatory domain comprises SEQ ID NO: 206. In yet another embodiment, the ICOS co-stimulatory domain comprises at least one, at least two, at least three, or more modified amino acid sequences having the amino acid sequence of SEQ ID NO: 206 (see, for example, US20170209492). In yet another embodiment, the ICOS co-stimulatory domain is substantially similar to the ICOS co-stimulatory domain comprising SEQ ID NO: 206.

[0312] In one embodiment, the nucleic acid encoding the CAR encodes at least one OX40 co-stimulatory domain and optionally encodes a second co-stimulatory domain selected from 4-1BB, 2B4, ICOS, CD28, OX40, and CD27 co-stimulatory domains, or any of the aforementioned co-stimulatory domains. In another embodiment, the nucleic acid encodes at least two OX40 co-stimulatory domains, or at least two OX40 co-stimulatory domains combined with one, two, three, or four or more co-stimulatory domains selected from 4-1BB, ICOS, CD28, OX40, and CD27, or any of the aforementioned co-stimulatory domains. In another embodiment, the OX40 co-stimulatory domain comprises SEQ ID NO: 207. In another embodiment, the OX40 co-stimulatory domain comprises at least one, at least two, at least three, or more modified amino acid sequences having the amino acid sequence of SEQ ID NO: 207. In another embodiment, the OX40 co-stimulatory domain is substantially similar to the OX40 co-stimulatory domain comprising SEQ ID NO: 207.

[0313] Intracellular signal transduction domains

[0314] In embodiments, the CAR includes at least one intracellular signaling domain. In embodiments, the at least one intracellular signaling domain is in addition to one or more co-stimulatory domains. In embodiments, one or more intracellular signaling domains are included to increase the proliferation, persistence, and / or cytotoxic activity of γδ cells modified with CARs disclosed herein. For example, in some embodiments, one or more intracellular signaling domains include CD3ζ, repeated (e.g., 2-5) DAP10 YINM motifs, signaling domains derived from LFA-1, DAP12, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD79a, CD79b, CD5, CD22, FcεRI, CD66d, etc. Within the scope of this disclosure, the intracellular domains of the disclosed CAR may include multiple (e.g., 2, 3, 4, or more) intracellular signaling domains. In cases where more than one intracellular signaling domain is included, the intracellular signaling domains may be the same or may be different.

[0315] In the embodiments, the intracellular signal transduction domain of the disclosed CAR is or includes a CD3ζ signal transduction domain. In the embodiments, the CD3ζ signal transduction domain is or includes an amino acid sequence as shown in SEQ ID NO: 229, 231 or 232.

[0316] Other components of CAR

[0317] In embodiments, the isolated nucleic acid encoding the CAR may also encode one or more polycistronic linker regions configured to facilitate the translation of the CAR peptide and one or more additional peptides. In embodiments, the nucleic acid encoding one or more additional peptides and the associated linker region may be located at the 3' end of the isolated nucleic acid, or at the 5' end of the isolated nucleic acid, or in some instances at both the 5' and 3' ends of the isolated nucleic acid. In some embodiments, the linker region may encode a self-cleaving and / or cleaving peptide sequence. In some instances, the self-cleaving sequence is a 2A self-cleaving sequence (e.g., T2A, P2A, E2A, F2A), which can induce ribosome jumping during CAR translation. In embodiments, the cleaving sequence is a furin sequence. In some instances, the cleaving sequence (e.g., a furin cleaving sequence as shown in SEQ ID NO: 190) is the N-terminus of a self-cleaving sequence such as furin-P2A (FP2A). In embodiments, the polycistronic linker region encodes an internal ribosome entry site. In the implementation scheme, the addition of optional linkers such as "GSG" or "SGSG" can improve cleavage efficiency. In this way, one or more additional peptides can be released from the CAR and directed to the secretion pathway.

[0318] In some embodiments, the cleavage sequence is the FP2A amino acid sequence as shown in SEQ ID NO: 184. In some embodiments, the cleavage sequence is the P2A amino acid sequence as shown in SEQ ID NO: 186 or SEQ ID NO: 188-189. In some embodiments, the cleavage sequence is the furin amino acid sequence as shown in SEQ ID NO: 190. In some embodiments, the cleavage sequence is the F2A amino acid sequence as shown in SEQ ID NO: 191. In some embodiments, the cleavage sequence is the E2A amino acid sequence as shown in SEQ ID NO: 192. In some embodiments, the cleavage sequence is the T2A amino acid sequence as shown in SEQ ID NO: 193. In some aspects, multiple cleavage and / or autocleavage sequences may be encoded at the C-terminus of one or more signal transduction and / or co-stimulatory domains and the N-terminus of one or more additional polypeptides. In some aspects, the construct described herein encodes one or more autocleavage sequences and one or more sequences cleaved by endogenous proteases. In some embodiments, the endogenous protease recognition site is encoded at the N-terminus of the autocleavage sequence.

[0319] In the implementation, the polycistronic linker region encodes the internal ribosome entry site. An exemplary internal ribosome entry site is encoded by the nucleotide sequence shown in SEQ ID NO:194. Another exemplary internal ribosome entry site is encoded by the nucleotide sequence shown in SEQ ID NO:195. Other suitable internal ribosome entry sites include, but are not limited to, those disclosed in Nucleic Acids Res. Jan 2010;38(Database Special Issue): D131-6. doi: 10.1093 / nar / gkp981. Epub November 16, 2009, those described on iresite.org, those described in WO 2018 / 215787, the sequences described in GenBank accession number KP019382.1, and the IRES element disclosed in GenBank accession number LT727339.1. Additional multicis-tie junction regions, including self-cutting cutters and IRES elements, are disclosed in US 2018 / 0360992 and US 8,865,467.

[0320] In some instances, the modified T cells described in this paper contain chimeric antigen receptors (CARs) expressed on the cell surface that contain affinity-binding domains specific to tumor antigens and express exogenous stem cell factors.

[0321] In some instances, the modified T cells described herein comprise chimeric antigen receptors (CARs) expressed on the cell surface and containing affinity-binding domains specific to disease-associated antigens (e.g., CD20, CD70, or PSMA), with disruption of the CISH gene, CBL-B gene, ICAM-1 gene, CD58 gene, Roquin-1 gene, Regnase-1 gene, TGFβR2 gene, RASA2 gene, MED12 gene, and optionally expressing dnTGFβR2 and dnFas.

[0322] carrier

[0323] In the implementation scheme, the modified T cells described herein comprise one or more nucleic acid constructs encoding one or more modifications (e.g., foreign proteins) and / or antigen recognition portions (e.g., CARs).

[0324] The nucleic acid sequence encoding the desired molecule can be obtained using recombination methods known in the art, such as screening a library from cells expressing the gene, obtaining the gene from a vector known to contain the gene, or directly isolating the gene from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest can be synthesized rather than cloned.

[0325] This invention provides vectors in which the DNA of this invention is inserted. Vectors derived from retroviruses (such as lentiviruses) are suitable tools for achieving long-term gene transfer because they allow for the long-term stable integration of transgenes and their proliferation in daughter cells. Lentiviral vectors offer additional advantages over vectors derived from oncogenic retroviruses (such as murine leukemia virus) because they can transduce non-proliferating cells, such as hepatocytes. They also have the advantage of increased low immunogenicity.

[0326] In another embodiment, the vector containing nucleic acid is an adenovirus vector (A5 / 35). In yet another embodiment, transposons (such as Sleeping Beauty, Crisper, CAS9, and zinc finger nucleases) can be used to achieve nucleic acid expression.

[0327] In simple terms, the expression of natural or synthetic nucleic acids is typically achieved by operatively linking the nucleic acid or a portion thereof to a promoter and incorporating the construct into an expression vector. Vectors are suitable for replication and integration in eukaryotic cells. A typical cloning vector contains transcription and translation terminators, a start sequence, and a promoter that can be used to regulate the expression of the desired nucleic acid sequence.

[0328] The expression constructs of this invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods for gene delivery are known in the art (e.g., U.S. Patents 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entirety). In another embodiment, this invention provides a gene therapy vector.

[0329] Nucleic acids can be cloned into various types of vectors. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phage particles, phage derivatives, animal viruses, and entrapments. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0330] Furthermore, the expression vector can be delivered to cells in the form of a viral vector. Viral vector technology is well known in the art and has been described, for example, by Sambrook et al. (2001). Molecular Cloning: A Laboratory Manual, (Cold Spring Harbor Laboratory, New York) and other virology and molecular biology handbooks. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, a suitable vector contains a replication origin that functions in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more optional markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193). Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene transfer systems. Selected genes can be inserted into vectors and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered in vivo or in vitro to cells of a subject. Many retroviral systems are known in the art. In one embodiment, an adenoviral vector is used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

[0331] Other promoter elements, such as enhancers, regulate the frequency of transcription initiation. These are typically located in regions 30–110 bp upstream of the start site, although recent studies have shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is generally flexible, allowing promoter function to be preserved when elements are inverted or moved relative to each other. In the thymidine kinase (TK) promoter, the spacing between promoter elements can be increased to 50 bp before activity begins to decline. Depending on the promoter, it appears that individual elements can act synergistically or independently to activate transcription.

[0332] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strongly constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operatively linked to it. Another example of a suitable promoter is extension growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Ebola virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as, but not limited to, actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, the invention is not limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of inducible promoters provides a molecular switch that can turn on the expression of an operablely linked polynucleotide sequence when such expression is desired, or turn off the expression when it is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0333] To assess the expression of a peptide or its fraction, the expression vector to be introduced into cells may also contain a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a population of cells attempting to be transfected or infected via a viral vector. Alternatively, the selectable marker may be carried on a separate DNA fragment and used in co-transfection procedures. Both the selectable marker and the reporter gene may be side-linked with appropriate regulatory sequences to enable expression in modified T cells. Available selectable markers include, for example, antibiotic resistance genes, such as neo.

[0334] Reporter genes are used to identify potential transfected cells and to assess the functionality of regulatory sequences. Generally, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue, and that encodes a polypeptide whose expression is indicated by some easily detectable property (e.g., enzyme activity). Reporter gene expression is measured at an appropriate time after DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000). FEBS Letters479: 79-82). Suitable expression systems are well-known and can be prepared using known techniques or are commercially available. Generally, constructs with minimal 5' flanking regions that exhibit the highest expression levels of the reporter gene are identified as promoters. These promoter regions can be linked to reporter genes and used to evaluate the ability of drugs to regulate promoter-driven transcription.

[0335] Methods for introducing and expressing genes into cells are known in the art. In the context of expression vectors, vectors can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0336] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection, and electroporation. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Laboratory, New York). One method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0337] Biological approaches to introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian cells (e.g., human cells). Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses. See, for example, U.S. Patents 5,350,674 and 5,585,362.

[0338] Chemical methods for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as a delivery medium in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0339] In the case of using non-viral delivery systems, an exemplary delivery medium is liposomes. Liposome formulations are intended to introduce nucleic acids into host cells (in vitro, ex vivo, or in vivo). Alternatively, nucleic acids can associate with lipids. Liposome-associated nucleic acids can be encapsulated within the aqueous interior of liposomes, dispersed within the lipid bilayer of liposomes, attached to liposomes via linker molecules associated with both liposomes and oligonucleotides, retained within liposomes, complexed with liposomes, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, contained in lipids as a suspension, contained in micelles, complexed with micelles, or otherwise associated with lipids. Lipids, lipid / DNA, or lipid / expression vector-related compositions are not limited to any particular structure in solution. For example, they can exist in a bilayer structure, as micelles, or have a “collapsed” structure. They can also simply be dispersed in solution, possibly forming aggregates of non-uniform size or shape. Lipids are fatty substances that can be naturally occurring or synthetic. For example, lipids include fat droplets that are naturally present in the cytoplasm, as well as a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives (such as fatty acids, alcohols, amines, amino alcohols, and aldehydes).

[0340] Suitable lipids are available from commercial sources. For example, dimyristicophosphatidylcholine (“DMPC”) is available from Sigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) is available from K & K Laboratories (Plainview, NY); cholesterol (“Choi”) is available from Calbiochem-Behring; dimyristicophosphatidylglycerol (“DMPG”) and other lipids are available from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at approximately -20°C. Chloroform is used as the sole solvent because it evaporates more readily than methanol. “Liposomes” is a general term encompassing a variety of monolayer and multilayer lipid mediators formed by the production of closed lipid bilayers or aggregates. Liposomes are characterized by a vesicular structure having a phospholipid bilayer membrane and an internal aqueous medium. Multilayer liposomes have multiple lipid layers separated by an aqueous medium. When phospholipids are suspended in excess aqueous solution, they spontaneously form. The lipid components undergo self-rearrangement before forming a closed structure, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991). Glycobiology 5: 505-10). However, this also includes compositions that have structures in solution that differ from normal vesicle structures. For example, lipids may exhibit micellar structures or exist simply as heterogeneous aggregates of lipid molecules. Cationic lipofectamine-nucleic acid complexes are also anticipated.

[0341] Regardless of the method used to introduce exogenous nucleic acids into host cells or to expose cells to the inhibitors of this invention, various assays can be performed to confirm the presence of recombinant DNA sequences in host cells. Such assays include, for example, "molecular biology" assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR, and PCR; and "biochemical" assays, such as detecting the presence or absence of specific peptides, for example by immunological means (ELISA and Western blotting) or by assays described herein to identify agents falling within the scope of this invention.

[0342] Gene editing

[0343] In the implementation scheme, gene editing is used to introduce one or more modifications and / or one or more antigen recognition moieties into modified T cells. In some instances, gene editing can disrupt one or more endogenous genes in the modified T cells. In some instances, gene editing can introduce one or more exogenous genes encoding one or more proteins into the modified T cells. In some instances, gene editing can be used to simultaneously introduce one or more exogenous genes and disrupt one or more genes in the modified T cells.

[0344] Gene editing is a type of genetic engineering in which one or more nucleotides / nucleic acids are inserted, deleted, and / or substituted in a DNA sequence, such as the genome of a modified T cell. Targeted gene editing enables insertion, deletion, and / or substitution at pre-selected sites in the genome of a target cell. When an endogenous gene sequence is edited, for example by deleting, inserting, or substituting nucleotides / nucleic acids, the endogenous gene containing the affected sequence can be knocked out or reduced due to the sequence alteration. Therefore, targeted editing can be used to disrupt the expression of endogenous genes. The term "disrupted gene" discussed herein refers to a gene containing insertions, deletions, or substitutions relative to an endogenous gene that reduce or suppress the expression of a functional protein derived from the endogenous gene. As used herein, "disrupted gene" refers to a method of inserting, deleting, or substituting at least one nucleotide / nucleic acid in an endogenous gene that reduces or suppresses the expression of a functional protein derived from the endogenous gene. Methods of disrupting genes are known to those skilled in the art and are described, for example, in U.S. Patent No. 11,254,912, which is incorporated herein by reference in its entirety.

[0345] In the implementation scheme, a nuclease-dependent approach can be used to perform targeted gene editing on T cells. This nuclease-dependent approach achieves targeted editing by specifically introducing double-strand breaks (DSBs) through a particular nuclease. This nuclease-dependent targeted editing utilizes DNA repair mechanisms, such as non-homologous end joining (NHEJ), which occurs in response to DSBs. DNA repair by NHEJ typically results in a small number of random insertions or deletions (indels) of endogenous nucleotides. In contrast to NHEJ-mediated repair, repair can also occur via homology-directed repair (HDR). When a donor template containing exogenous genetic material and flanked by a pair of homologous arms is present, exogenous genetic material can be introduced into the genome via HDR, leading to targeted integration of the exogenous genetic material. Available nucleases capable of introducing specific and targeted DSBs include, but are not limited to, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-directed CRISPR-Cas9 nucleases (CRISPR / Cas9; clustered regularly spaced short palindromic repeats related to 9). This article discusses how CRISPR systems or CRISPR nuclease systems may include DNA-binding non-coding RNA molecules (e.g., guide RNA) and Cas proteins with nuclease functions (e.g., Cas9) (Sander et al., Nature Biotechnology (2014); 32:347-355; Hsu et al., Cell (2014);157(6):1262-1278).

[0346] Pharmaceutical Composition

[0347] The modified T cells of this disclosure can be administered to a subject or in the form of a pharmaceutical composition mixed with a suitable carrier or excipient. In another aspect, this disclosure provides pharmaceutical compositions comprising a plurality of the modified T cells described herein and one or more pharmaceutically acceptable carriers. In embodiments, the pharmaceutical composition comprises a cell population or cell culture wherein at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 100% of the cells in the cell population or cell culture are the modified T cells described herein. In embodiments, the modified T cells are γδ T cells. In embodiments, the γδ T cells are δ1, δ2, δ3, or δ4 γδ T cells, preferably δ2. - γδ T cells, more preferably δ1 γδ T cells. In this embodiment, the cell population or cell culture does not contain other cells.

[0348] Modified T cells can be administered via any route appropriate to the condition to be treated. Administration is typically parenteral, such as via infusion, subcutaneous, intramuscular, intravenous, intradermal, intrathecal, and epidural.

[0349] Pharmaceutical compositions and formulations typically comprise one or more optional pharmaceutically acceptable carriers or excipients. In some embodiments, the composition comprises at least one additional therapeutic agent.

[0350] The choice of carrier can depend in part on the specific cells, binding molecules, and / or antibodies, and / or the method of administration. Therefore, a variety of suitable formulations exist. For example, carriers are described in Remington's Pharmaceutical Sciences, 16th edition, edited by Osol, A. (1980). Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the doses and concentrations used.

[0351] Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell population is administered parenterally. As used herein, the term "parenterally" includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the cell population is delivered to the subject using a peripheral system via intravenous, intraperitoneal, or subcutaneous injection.

[0352] Treatment

[0353] Pharmaceutical compositions containing the modified T cells described herein can be administered for prophylactic and / or therapeutic purposes. For example, the composition may be administered to a subject already suffering from the disease or condition in an amount sufficient to cure or at least partially prevent symptoms of the disease or condition. The composition may also be administered to reduce the likelihood of the development of symptoms, infection, or exacerbation of symptoms. The effective amount of modified T cells used for therapeutic purposes may vary depending on factors such as the severity and progression of the disease or condition, prior therapy, the subject's health status, weight, and / or response to the drug, and / or the judgment of the treating physician.

[0354] Modified T cells can be used to treat subjects with conditions requiring treatment. Examples of conditions include cancer, infectious diseases, autoimmune diseases, and sepsis, such as cancers, autoimmune diseases, and infections caused by the bacteria and viruses disclosed herein. Subjects can be humans, non-human primates such as chimpanzees and other ape and monkey species; farm animals such as cattle, horses, sheep, goats, and pigs; domestic animals such as rabbits, dogs, and cats; and laboratory animals, including rodents such as rats, mice, and guinea pigs. Subjects can be of any age. Subjects can be, for example, elderly people, adults, adolescents, pre-adolescent children, children, toddlers, and infants. In one embodiment, modified T cells can be used to treat cancer. In another embodiment, modified T cells can be used to treat autoimmune diseases. For example, methods of using modified T cells to treat autoimmune diseases are described in U.S. Provisional Application No. 63 / 738730, which is incorporated herein by reference in its entirety.

[0355] Modified T cells may also be administered to reduce the likelihood of developing symptoms, contracting symptoms, or worsening symptoms. The effective amount of modified T cells used for therapeutic purposes may vary depending on factors such as the severity and progression of the disease or symptoms, prior therapy, the subject's health status, weight and / or response to various medications, and / or the judgment of the treating physician.

[0356] In an implementation, a pharmaceutical composition comprising modified T cells may be administered in a first regimen. Subjects may be monitored, for example by a healthcare provider (e.g., a treating physician or nurse). In some instances, subjects are monitored to determine or measure the efficacy of the modified T cells in treating the subject's condition. In some cases, subjects may also be monitored to determine the in vivo expansion of the modified T cells in the subject's body. Another pharmaceutical composition comprising modified T cells may be administered to the subject in a second regimen. The pharmaceutical composition administered in the second regimen may contain the same type of modified T cells as those administered to the subject in the first regimen. However, within the scope of this disclosure, the pharmaceutical composition administered in the second regimen may contain different types of modified T cells, for example, expressing different CARs. In some instances, the second regimen is not performed, for example, if the first regimen is found to be effective (e.g., a single round of administration may be sufficient to treat the condition). In an implementation, the modified T cells may be administered to a variety of subjects (e.g., where the T cells have universal donor characteristics).

[0357] Modified T cells can be administered to subjects in any order or simultaneously. If administered simultaneously, modified T cells can be provided in a single, uniform form (such as intravenous injection) or in multiple forms (e.g., as multiple intravenous infusions, subcutaneous injections, or pills). Modified T cells can be packaged together or separately, in a single package or in multiple packages. Modified T cells can be administered in multiple doses. If not administered simultaneously, the time arrangement between multiple doses can vary up to about one week, one month, two months, three months, four months, five months, six months, or about one year. In some cases, modified T cells can proliferate in vivo in the subject's body after administration. Modified T cells can be frozen to provide cells for multiple treatments using the same cell preparation. Modified T cells and pharmaceutical compositions containing said cells can be packaged into kits. Kits may include instructions for use (e.g., written instructions) for one or more modified T cells and compositions containing said cells.

[0358] Modified T cells can be administered before, during, or after the onset of a disease or symptom, and the timing of administration of modified T cells can vary. For example, modified T cells can be used as a prophylactic agent and can be administered continuously to subjects with symptoms or a predisposition to disease in order to reduce the likelihood of the onset of the disease or symptom. Initial administration can be performed via any practical route, such as using any of the formulations described herein. In some instances, the administration of modified T cells is intravenous. One or more doses of modified T cells can be administered as soon as feasible after the onset of a specific symptom (e.g., cancer) and for the length of time required to continue treatment of the disease / symptom, such as about 24 hours to about 48 hours, about 48 hours to about 1 week, about 1 week to about 2 weeks, about 2 weeks to about 1 month, or about 1 month to about 3 months. In embodiments, one or more doses of modified T cells can be administered years after the onset of a disease / symptom (e.g., cancer) and before or after other treatments. In some instances, one or more of the compositions described herein may be applied for at least about 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years. The duration of treatment may vary for each subject.

[0359] Modified T cells can also be advantageously administered to patients in combination with any number of relevant forms of treatment (including, for example, chemotherapy, radiation therapy, or immunotherapy) (e.g., for cancer treatment) (e.g., before, during, or after). Patients may also be pre-conditioned with therapeutically effective amounts of modified T cells prior to chemotherapy, radiation therapy, or immunotherapy (e.g., cell therapy). Immunotherapy suitable for use in combination with modified T cells includes autologous and allogeneic cell therapies, engineered T and NK cells, immune conjugates, fusion proteins, or other immuno-oncology agents.

[0360] In implementations, the modified T cells may be administered in combination with another cell immunotherapy (e.g., CAR T or CAR NK cell or Treg therapy) for treating diseases such as cancer. For example, a treatment method (e.g., for treating cancer) may include a conditioning step, such as a preconditioning step, of administering a therapeutically effective amount of modified T cells to a subject concurrently with or sequentially with another cell immunotherapy targeting a disease (e.g., cancer). In implementations, the cell immunotherapy may include further administration of engineered T cell or NK cell therapy comprising a CAR that binds to any tumor-associated antigen of interest.

[0361] In the implementation scheme, the modified T cells may be advantageously administered in combination with another adoptive cell therapy (ACT) (for reviews of HSCT and adoptive cell therapy, see Roger and Porter, Ther Adv Hematol (2011) 2(6) 409-428; Roddie and Peggs, Expert Opin. Biol. Ther. (2011) 11(4):473-487; Wang et al. Int.J. Cancer: (2015)136, 1751-1768; and Chang, YJ and XJ Huang, Blood Rev,2013. 27(1): 55-62), each of which is incorporated herein by reference in its entirety. Such adoptive cell therapies include, but are not limited to, allogeneic and autologous hematopoietic stem cell transplantation, donor leukocyte (or lymphocyte) infusion (DLI), adoptive transfer of tumor-infiltrating lymphocytes, or adoptive transfer of T cells or NK cells (including recombinant cells, such as CAR T, CAR NK). In addition to the necessity for donor-derived cells to reconstitute hematopoietic function after radiotherapy and chemotherapy, immune reconstitution of the transferred cells is crucial for eliminating residual tumor cells. The efficacy of ACT as a therapeutic option for malignant tumors is influenced by a variety of factors, including the source, composition, and phenotype of the donor cells (lymphocyte subsets, activation status), underlying disease, pre-transplant conditioning protocols and post-transplant immune support (e.g., IL-2 therapy), and the donor-cell-mediated graft-versus-tumor (GVT) effect within the graft. Furthermore, these factors can be balanced with transplant-related mortality, often caused by conditioning protocols and / or excessive immune activity of donor cells within the host (i.e., graft-versus-host disease, cytokine release syndrome, etc.).

[0362] In some aspects, this disclosure provides methods for enhancing the activity and / or function of T cells by incorporating one or more modifications herein. In embodiments, the method uses modified T cells to enhance the potency of T cell therapy. In embodiments, the method enhances the cytotoxicity of the modified T cells. In some instances, the method includes disrupting the MED12 gene in the modified T cells and expressing membrane-bound IL-12 in the modified T cells. In some instances, the method includes disrupting the MED12 gene in the modified T cells, expressing membrane-bound IL-12 in the modified T cells, and disrupting the Fas gene in the modified T cells or expressing dnFas in the modified T cells.

[0363] Example

[0364] The following embodiments are provided to provide a complete disclosure and description to those skilled in the art regarding how to prepare and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors consider to be their invention. Efforts have been made to ensure the accuracy of the figures used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise stated, parts are parts by weight, molecular weights are average molecular weights, temperatures are degrees Celsius, and pressures are atmospheric pressure or close to atmospheric pressure.

[0365] Example 1. In vitro cytotoxicity of "bolt-fastened" anti-PSMA CAR engineered γδ T cells in the presence of TGFβ1.

[0366] The cytotoxic potential of anti-PSMA CAR-transduced γδ T cells and one of the same CAR constructs modified to express “bolt-fastening,” i.e., dominant-negative TGFβ receptor II (dnTGFβRII), was evaluated against Nuc Near Infrared (NIR) modified PC3-PSMA cells at an E:T ratio of 3:1. TGFβ1 was added under separate co-culture conditions to a final concentration of 20 ng / mL. The first stimulation was monitored for approximately 3 days, after which fresh target cells were seeded in another 96-well plate. Effector cells resulting from the first stimulation were then transferred to a plate containing fresh target cells. A second stimulation was monitored for an additional 3 days. Co-cultures were monitored every 4 hours using an Incucyte® SX5. The cytotoxicity index was calculated based on the remaining viable target tumor cells by dividing the total NIR target area at each time point by the value at time 0 for each stimulation. Figure 1 As shown, in the presence of TGFβ1, after a second stimulation with tumor cells, leader 1 cells (γδ T cells expressing a CAR containing SEQ ID NO: 401) with dnTFβRII “bolted-in” maintained higher cytotoxic activity against PC3-PSMA compared to the TGFβ1-free condition. In the presence of TGFβ1, after a second stimulation, naked leader 1 CAR Vδ1 γδ T cells were associated with minimal tumor control due to the lack of armoring to alleviate the inhibitory effect of TGFβ1.

[0367] Example 2. Expression of CAR, dnTGFβR2, CD103, and pSMAD2 / 3 in “bolt-tightening” modified PSMA-resistant CAR-engineered γδ T cells

[0368] Vδ1 T cells modified with the anti-PSMA CAR construct were counted on Novocyte (Agilent) using Annexin-DAPI. Cell viability was assessed using the cell viability dye Zombie Aqua (Biolegend) at approximately 1 x 10⁻⁶ cells per cell.5 Cells were stained. Further staining of the cell surface with Vδ1 (Biolegend), dnTGFβRII (APC anti-human TGF-β receptor II antibody, Biolegend), and CD103 (Biolegend) was performed, and CAR expression was detected using biotinylated protein L (AcroBiosystems) and PE-streptavidin (Biolegend). Cells were fixed before collection onto Novocytes (Agilent). Figures 2A to 2B As shown, compared to the naked CAR construct, CAR expression remained largely unchanged in γδ T cells engineered with the "bolt-tightened" anti-PSMA CAR, and dnTGFβRII expression was detected only in the "bolt-tightened" CAR construct. Figure 2C As shown, anti-PSMA CAR-transduced γδ T cells containing dnTGFβRII exhibited reduced CD103 expression compared to the control without the dnTGFβRII construct. Anti-PSMA CAR-transduced γδ T cells, modified with or without the dnTGFβRII construct, were subjected to serum starvation at 37°C and 5% CO2 for 2 hours to reduce pSMAD2 / 3 background levels. Cells were stained with Zombie Aqua viability dye (Biolegend) and seeded into the round-bottom wells of ULA 96-well plates. 10 ng / mL of recombinant human TGFβ1 (R&D Systems) was added at 37°C and 2% CO2 for 15 minutes. Cells were immediately fixed by adding an equal volume of pre-warmed BD Cytofix buffer (BD Biosciences) to the cell suspension. Cells were permeabilized with BD™ Phosflow Perm Buffer III (BD Biosciences) and incubated on ice in the dark for 30 minutes. After washing with staining buffer (2% FBS 1XPBS), anti-pSMAD2 / 3 antibody conjugated with PE (BD Biosciences) was added to the cells and incubated at room temperature in the dark for 1 hour. Cells were washed twice with staining buffer and collected on Novocytes. Figure 2D As shown, compared with the control without dnTGFβRII, anti-PSMA CAR transduced γδ T cells containing dnTGFβRII had reduced pSMAD2 / 3 levels.

[0369] Example 3. Gene editing of γδ T cells

[0370] To knock out the CISH gene in Vδ1 T cells, a ribonucleoprotein (RNP) complex consisting of CRISPR-Cas9 (Integrated DNA Technologies) and CISH sgRNA (Synthego Corporation) was combined with activated Vδ1 T cells from PBMCs (P3 primary cells 4D-Nucleofector X kit L, Lonza). The RNP complex was delivered into Vδ1 T cells via electroporation (Lonza 4D-Nucleofector). After electroporation, cells were transferred to T-flasks (Corning) containing X-Vivo15 (Lonza) for cell recovery and expansion in a 37°C CO2 incubator (ThermoFisher). To assess CISH KO efficiency, cell pellets were collected on day 7 post-gene editing after αβ T cell exhaustion (StemCell Technologies), and genomic DNA was extracted using the NucleoSpin tissue kit (MACHEREY-NAGEL). The edited region of the CISH gene was amplified by PCR and then sequenced using Sequetech. Gene knockout efficiency was determined using the ICE analysis tool (Synthego). Figure 3 As shown, efficient knockout of the CISH gene was observed in Vδ1 T cells.

[0371] Following CISH gene knockout, Vδ1 T cell enrichment in PBMCs was monitored over time using flow cytometry (anti-Vδ1, BioLegend). Figure 4A As shown, CISH gene knockout did not affect Vδ1 T cell enrichment compared to unedited simulated control cells. CISH gene-edited Vδ1 T cells achieved similar purity after TCRαβ depletion (StemCell Technologies). Cell viability of PBMCs enriched with Vδ1 T cells was monitored after CISH gene knockout (Countess II, ThermoFisher). Figure 4B As shown, CISH KO cells maintained similar high cell viability to unedited simulated control cells.

[0372] Example 4: CISH KO enhances the function of Vδ1 T cells

[0373] Healthy donor peripheral blood mononuclear cells (PBMCs) were used for activation, expansion, CRISPR gene editing, and engineered Vδ1γδ T cells to express CAR. To knock out the CISH gene in Vδ1 T cells, a ribonucleoprotein (RNP) complex containing CRISPR-Cas9 (Integrated DNA Technologies) or -MAD7 (Aldevron) and CISH sgRNA was conjugated with activated Vδ1 T cells from PBMCs (P3 Primary Cells 4D-Nucleofector X Kit L, Lonza). The RNP complex was delivered into Vδ1 T cells via electroporation (Lonza 4D-Nucleofector). After electroporation, cells were transferred to T-flasks (Corning) containing X-Vivo15 (Lonza) for cell recovery, transduced with a CAR-encoding gamma retroviral vector, and then expanded. To assess CISH KO efficiency, cell pellets were collected, and genomic DNA was extracted using the NucleoSpin Tissue Kit (MACHEREY-NAGEL). The edited region of the CISH gene was amplified by PCR and sequenced using Sanger sequencing (Sequetech). Gene knockout efficiency was determined using the ICE analysis tool (Synthego). To assess the knockout of CISH protein in CAR Vδ1 T cells, WT (unedited) and CISH KO CAR Vδ1 T cells were incubated with and without IL-2 (Peprotech) for 5 hours. Cell lysates were generated for Western blot detection of CISH protein using anti-CISH rabbit mAb (CellSignaling). The primary antibody was detected using IRDye 800CW goat anti-rabbit IgG secondary antibody (LI-COR), and the presence of IRDye was imaged using the Odyssey imaging system (LI-COR). Results were presented in […]. Figures 5A to 5B As shown in the image. Figure 5A When exposed to IL-2, WT CAR Vδ1 T cells showed increased CISH protein levels. Conversely, in the presence of IL-2, which supports CISH KO, CISH protein levels in CISH KO CAR Vδ1 T cells were significantly reduced. Figure 5BTo demonstrate the enhanced function of CISH KO CAR-transduced and untransduced Vδ1 T cells, cytolytic activity against the B7-H6+ HCT-15 cell line (ATCC) was assessed using an in vitro repeated stimulation assay. HCT-15 NIR-labeled cell lines and effector cells were added to 96-well plates with or without different concentrations of IL-2 at a 5:1 E:T ratio. Each stimulation was monitored for 3 days, after which fresh target cells were seeded into another 96-well plate. Effector cells from each stimulation were then transferred to a plate containing fresh target cells. Co-cultures were monitored every 4 hours using an Incucyte® SX5 (Satorius). The cytotoxicity index was calculated based on the remaining viable target tumor cells by dividing the total NIR target area at each time point by the value at time 0 of each stimulation. A lower cytotoxicity index indicated better cytotoxic potential. After the second stimulation, CISH KO untransduced Vδ1 T cells were associated with higher tumor cell killing compared to WT untransduced Vδ1 T cells only in the presence of IL-2. In the absence of IL-2, CISH KO CAR Vδ1 T cells demonstrated superior tumor control compared to WT CAR Vδ1 T cells. These results indicate that CISH KO enhances the in vitro cytotoxicity of Vδ1 cells.

[0374] CAS9 CISH sgRNA: TGTACAGCAGTGGCTGGTGG (AGG) (SEQ ID NO: 339)

[0375] MAD7 CISH sgRNA: (TTTA) GGTGTACAGCAGTGGCTGGTG (SEQ ID NO: 340).

[0376] Example 5: Enhancing CAR Vδ1 T cell effector function using membrane-bound IL-12 (mbIL-12)

[0377] A CAR vector, consisting of an anti-CD19 (clone FMC63) scFv (as described in Kochendefer JN et al., Adoptive transfer of syngeneic T cells transduced with a chimeric antigen receptor that recognizes murine CD19 can eradicate lymphoma and normal B cells, Blood. 2010 Nov 11; 116(19):3875-86), a CD8α hinge and transmembrane domain, and 4-1BB and CD3ζ signaling domains, followed by a self-cleaving P2A sequence for separating mbIL-12 molecules (as described in Lee et al., Antigen-dependent IL-12 signaling in CAR T cells promotes regional to systemic disease targeting, bioRxiv. 2023 Jan 7; 2023.01.06.522784), is created by cloning a DNA fragment containing all CAR domains (Integrated DNA). The construct was constructed by incorporating a self-inactivated (SIN) Moloney Murine Leukemia Virus (MMLV) gamma retrovirus plasmid. The construct design is as follows: Figure 6 As shown: (Constructor A) CAR (FMC63) construct, (Constructor B) CAR-mbIL-12 construct, and (Constructor C) mbIL-12 construct.

[0378] Healthy donor peripheral blood mononuclear cells (PBMCs) were used to activate, expand, and engineer Vδ1γδ T cells to express the CD19 CAR-mbIL-12 transgene. Results were... Figures 7A to 7D As shown in the image. Figure 7AVδ1 γδ T cells (post-transduced) were sampled at different time points during the expansion process to determine cell counts. There was no difference in total Vδ1 γδ T cell counts between CAR and CAR-mbIL-12, indicating that mbIL-12 had no negative impact on Vδ1 γδ T cell expansion. Cells were harvested and stained against CAR and mbIL-12 using PE-conjugated anti-FMC63 antibody (Acrobio Systems, Newark, DE) and APC-conjugated anti-IL12 antibody (Miltenyi Biotec), respectively, and collected on a Novocyte flow cytometer (Agilent Technologies) to determine the percentage of CAR and mbIL-12 positive Vδ1 γδ T cells. Figure 7B At the end of the process, no % CAR difference was observed between Vδ1 γδ T cells expressing CAR and CAR-mbIL-12, further supporting that mbIL-12 does not affect CAR surface expression. Figure 7C During the expansion process, the percentage of mbIL-12 expressed on the cell surface of Vδ1 γδ T cells decreased over time. Figure 7D CAR-mbIL-12 Vδ1 γδ T cells were co-cultured with and without CD19+ Raji cells for 18 hours. After stimulation with target cells, mbIL-12 was increased on the cell surface of Vδ1 γδ T cells compared with the unstimulated condition, indicating that the surface expression of mbIL-12 depends on CAR activation.

[0379] To demonstrate the function of CAR-mbIL-12, the cytolytic activity against CD19-positive cell lines using Vδ1 γδ T cells transduced with CAR, CAR-mbIL-12, and mbIL-12 was evaluated in an in vitro repeated stimulation assay. Raji-Nuc NIR-labeled cell lines and effector cells were added to 96-well plates at E:T ratios of 5:1, 2.5:1, and 1.25:1. The first stimulation was monitored for 3 days, after which fresh target cells were seeded into another 96-well plate. The effector cells produced by the first stimulation were then transferred to a plate containing fresh target cells. The second stimulation was monitored for an additional 3 days, and the same procedure was performed for the third stimulation. Co-cultures were monitored every 4 hours using an Incucyte® SX5 (Satorius). The cytotoxicity index was calculated based on the remaining viable target tumor cells by dividing the total NIR target area at each time point by the value at time 0 for each stimulation. A lower cytotoxicity index indicated better cytotoxic potential. Following the second and third stimulations, in the tested E:T ratio, CAR-mbIL-12 Vδ1 γδ T cells were associated with higher tumor cell killing compared to CAR and mbIL12 Vδ1 γδ T cells. Co-culture conditions with a cytotoxicity index >1 (indicating incomplete tumor control) were not analyzed in subsequent stimulations. Results are as follows. Figure 8 The results showed that CAR-mbIL-12 expressed by Vδ1 γδT cells maintained the cell lysis and killing function of tumor cells.

[0380] In a Raji cell subcutaneous xenograft tumor model, a suboptimal dose of CAR-mbIL-12 Vδ1 γδ T cells (2.5e6 CAR+ live cells) was evaluated. Raji cell subcutaneous xenografts were established by inoculating the right posterior flank of female NSG mice with 1E6 cells / mouse. The tumor volume was approximately 200 mm². 3 Mice were randomly assigned to study cohorts (N=5 mice per group). Vδ1 γδ T cells expressing CAR and CAR-mbIL-12 were administered as a single bolus dose on day 0. Human IL-2 was supplemented three times weekly during the study period. Tumor volume and mouse weight were monitored throughout the experiment. Results were presented in... Figure 9 As shown in the figure, CAR-mbIL-12 Vδ1 γδ T cells were associated with significantly higher tumor cell growth inhibition compared to the suboptimal dose of CAR Vδ1 γδ T cells. The data support enhanced function of Vδ1 γδ T cells expressing CAR-mbIL-12.

[0381] Example 6: CBL-B KO enhances the function of Vδ1 T cells

[0382] Healthy donor peripheral blood mononuclear cells (PBMCs) were used for activation, expansion, CRISPR gene editing, and engineered Vδ1γδ T cells to express CAR. To knock out the CBL-B gene in Vδ1 T cells, a ribonucleoprotein (RNP) complex containing CRISPR-Cas9 (Integrated DNA Technologies) and CBL-B sgRNA was combined with activated Vδ1 T cells from PBMCs (P3 Primary Cell 4D-Nucleofector X Kit L, Lonza). The RNP complex was delivered into Vδ1 T cells via electroporation (Lonza 4D-Nucleofector). After electroporation, cells were transferred to T-flasks (Corning) containing X-Vivo15 (Lonza) for cell recovery, then transduced with a CAR-encoding gamma retroviral vector, and subsequently expanded. Results were... Figures 10A to 10B As shown in the image. Figure 10A To determine CBL-B KO efficiency, genomic DNA was extracted from cell pellet using the NucleoSpin Tissue Kit (MACHARY-NAGEL). Editing regions were amplified by PCR using previously internally optimized primers. PCR reactions were run on agarose gels, and prominent bands were excised. The gels were then extracted using the NucleoSpin Gel and PCR Cleanup Kit (MACHARY-NAGEL). The extracted DNA was sequenced using Sanger sequencing (Sequetech). %Indel was determined using the ICE analysis tool (Synthego). Figure 10B To demonstrate enhanced function of CBL-B KO CAR-transduced and untransduced Vδ1 T cells, cytolytic activity against PSMA+ PC3 cell lines was assessed using an in vitro repeated stimulation assay (utilizing the PC3-PSMA target in prostate adenocarcinoma). PC3-PSMA NIR-labeled cell lines and effector cells were added to 96-well plates at an E:T ratio of approximately 3:1, with or without IL-2. Each stimulation was monitored for 3 days, after which fresh target cells were seeded into another 96-well plate. Effector cells from each stimulation were then transferred to a plate containing fresh target cells. Co-cultures were monitored every 4 hours using an Incucyte® SX5 (Satorius). The cytotoxicity index was calculated based on the remaining viable target tumor cells by dividing the total NIR target area at each time point by the value at time 0 for each stimulation.

[0383] Cas9 multi-guided sgRNA sequence: AAGACUCUUUAAAGAAGGCA (SEQ ID NO: 341) AGUACUCAUUCUCACUGAGU (SEQ ID NO: 342) CGUAAAUGCUGAUAUGUAUC (SEQ ID NO: 343) Cas9 single guide: TAATCTGGTGGACCTCATGA (AGG) (SEQ ID NO: 344).

[0384] Example 7: Roquin-1 KO enhances Vδ1 T cell function

[0385] Healthy donor peripheral blood mononuclear cells (PBMCs) were used for activation, expansion, CRISPR gene editing, and engineered Vδ1γδ T cells to express CAR. To knock out the Roquin-1 gene in Vδ1 T cells, a ribonucleoprotein (RNP) complex containing CRISPR-Cas9 (Integrated DNA Technologies) and Roquin-1 sgRNA was combined with activated Vδ1 T cells from PBMCs (P3 Primary Cell 4D-Nucleofector X Kit L, Lonza). The RNP complex was delivered into Vδ1 T cells via electroporation (Lonza 4D-Nucleofector). After electroporation, cells were transferred to T-flasks (Corning) containing X-Vivo15 (Lonza) for cell recovery, then transduced with a CAR-encoding gamma retroviral vector, and subsequently expanded. Results were... Figures 11A to 11B As shown in the image. Figure 11A To determine Roquin KO efficiency, genomic DNA was extracted from cell pellet using the NucleoSpin Tissue Kit (MACHARY-NAGEL). Editing regions were amplified by PCR using previously internally optimized primers. PCR reactions were run on agarose gels, and prominent bands were excised. The gels were then extracted using the NucleoSpin Gel and PCR Cleanup Kit (MACHARY-NAGEL). The extracted DNA was sequenced using Sanger sequencing (Sequetech). % Indel was determined using the ICE analysis tool (Synthego). Edits were first generated using three gRNA sequences in a 1:1:1 ratio to demonstrate the ability to edit genes in Vd1 T cells, followed by single-guided optimization to identify the gRNA with the highest KO efficiency. Figure 11BTo demonstrate enhanced function of Roquin-1 KO CAR-transduced and untransduced Vδ1 T cells, cytotoxicity against PSMA+ PC3 cell lines was assessed using an in vitro repeated stimulation assay (utilizing the PC3-PSMA target in prostate adenocarcinoma). PC3-PSMA NIR-labeled cell lines and effector cells were added to 96-well plates at an E:T ratio of approximately 3:1, with or without IL-2. Each stimulation was monitored for 3 days, after which fresh target cells were seeded into another 96-well plate. Effector cells from each stimulation were then transferred to a plate containing fresh target cells. Co-cultures were monitored every 4 hours using an Incucyte® SX5 (Satorius). The cytotoxicity index was calculated based on the remaining viable target tumor cells by dividing the total NIR target area at each time point by the value at time 0 for each stimulation.

[0386] Multiple guidance sgRNA sequences: AAGCCCAUCAGUUUGGGUUG (SEQ ID NO: 345) UGUACAAGCUCCACAAUGGA (SEQ ID NO: 346) CAAAUGGGCAAGCCUUGCGG (SEQ ID NO: 347) Internally optimized single gRNA sequence: CCTGAATAAACTCCACCGCA (AGG) (SEQ ID NO: 348).

[0387] Example 8: ICAM-1 and CD58 KO enhance in vitro cell survival of CAR Vδ1 T cells in the presence of allogeneic PBMCs

[0388] Healthy donor peripheral blood mononuclear cells (PBMCs) were used for activation, expansion, CRISPR gene editing, and engineering of Vδ1γδ T cells to express CAR. To knock out the CISH gene in Vδ1 T cells, a ribonucleoprotein (RNP) complex consisting of CRISPR-Cas9 (Integrated DNA Technologies) or -MAD7 (Aldevron) and ICAM-1 or CD58 sgRNA was conjugated with activated Vδ1 T cells from PBMCs (P3 Primary Cell 4D-Nucleofector X Kit L, Lonza). The RNP complex was delivered into Vδ1 T cells via electroporation (Lonza 4D-Nucleofector). After electroporation, cells were transferred to T-flasks (Corning) containing X-Vivo15 (Lonza) for cell recovery, then transduced with a CAR-encoding gamma retroviral vector, and subsequently expanded. To determine the KO efficiency of ICAM-1 and CD58, WT (unedited), ICAM-1 KO, and CD58 KO CAR Vδ1 γδT cells were stained with anti-ICAM-1, anti-CD58, or allotype control antibodies conjugated to PE (BioLegend) and collected using a Novocyte flow cytometer (Agilent Technologies).

[0389] The result is Figures 12A to 12B As shown in the image. Figure 12A In CAR Vδ1 γδ T cells, the KO efficiency of both ICAM-1 and CD58 KO exceeded 80%. Figure 12BTo assess whether ICAM-1 and CD58 could enhance the survival of CAR Vδ1 T cells in the presence of allogeneic PBMCs, a 10-day mixed lymphocyte reaction (MLR) assay was performed. WT (unedited), CISH KO, ICAM-1 KO, and CD58 KO CAR Vδ1 γδ T cells (target cells) were co-cultured with allogeneic PBMCs (effector cells) at a 10:1 E:T ratio in the presence of IL-2. During co-culture, half of the medium was replaced with fresh medium containing IL-2 every 2–3 days. Cells were harvested from the co-culture at days 3, 6, and 10, and CAR+ and Vδ1+ γδ T cells were stained with B7-H6 conjugated to APC (generated by Adicet Bio) (for CAR assay) and anti-Vδ1 antibody conjugated to PE-CY7 (generated by Adicet Bio) (for Vδ1 T cell assay). Cells were collected using Novocyte flow cytometry to obtain absolute counts of double-positive CAR+ Vδ1+ T cells. To determine the % target cell change at each time point, the absolute counts of WT CAR+ Vδ1+ γδ T cells were compared with the KO CAR+ Vδ1+ γδ T cell count using the following formula: [(WT CAR+ Vδ1+ γδ T cell count - KO CAR+ Vδ1+ γδ T cell count) ÷ (WT CAR+ Vδ1+ γδ T cell count)] x 100. Based on the % target cell change relative to the WT CAR+ Vδ1+ γδ T cell count, only ICAM-1 KO and CD58 KO CAR+ Vδ1+ γδ T cells showed increased % target cell change, indicating that disruption of ICAM-1 and CD58 reduced in vitro killing of allogeneic PBMCs, thereby improving cell survival.

[0390] CAS9 ICAM-1 sgRNA: AAAGUCAUCCUGCCCCGGGG (SEQ ID NO: 349)

[0391] MAD7 ICAM-1 sgRNA: TTTG / AATAGCACATTGGTTGGCTAT (SEQ ID NO: 350)

[0392] CAS9 CD58 sgRNA:AAAUAUAUGGUGUGUGUAU (SEQ ID NO: 351)

[0393] MAD7 CD58 sgRNA: TTTA / GACACTGTGTCAGGTAGCCTC (SEQ ID NO: 352).

[0394] Example 9: TGFβRII KO alleviates the immunosuppressive effect of TGFβ1 on CAR Vδ1 T cells

[0395] Healthy donor peripheral blood mononuclear cells (PBMCs) were used to activate, expand, CRISPR-edit, and engineer Vδ1γδ T cells to express PSMA-targeting CARs. To knock out the TGFβRII gene in Vδ1 T cells, a ribonucleoprotein (RNP) complex consisting of CRISPR-Cas9 (Integrated DNA Technologies) or -MAD7 (Aldevron) and TGFβRII sgRNA (Cas9 target: CCCCTACCATGACTTTATTC / TGG or Eureca-V (MAD7) target: TTTC / TGGTTGTCACAGGTGGAAAAT) was combined with activated Vδ1 T cells from PBMCs (P3 Primary Cell 4D-Nucleofector X Kit L, Lonza). The RNP complex was then delivered into the Vδ1 T cells via electroporation (Lonza 4D-Nucleofector). Following electroporation, cells were transferred to T-flasks (Corning) containing X-VIVO 15 (Lonza) supplemented with 10% FBS (Cytiva) and 100 U / mL IL-2 (Miltenyi Biotech) for cell recovery. Cells were then transduced using a CAR-encoding gamma retroviral vector and amplified. To obtain a pure Vδ1 population at the end of amplification, TCRαβ-positive T cells were depleted from the culture using the EasySep™ Human TCR α / β Depletion Kit (StemCell Technologies).

[0396] To determine the knockout efficiency of TGFβRII in CAR Vδ1 T cells, genomic DNA was extracted from cell pellet using the NucleoSpin Tissue Kit (MACHARY-NAGEL). Edited regions were amplified using PCR primers, sequenced using Sanger sequencing, and %Indels were determined by degradation-based insertion / deletion tracking (TIDE) analysis (shinyapps.datacurators.nl / tide). Editing with CRISPR-Cas9 and CRISPR-MAD7 resulted in a TGFβRII knockout efficiency greater than 70%. Editing efficiency is relative to WT in donor-matched Vδ1 cells. Results were presented in... Figure 13A As shown in the image.

[0397] The inhibition of TGFβ signaling by TGFβRII KO CAR Vδ1 T cells was assessed by detecting SMAD2 / 3 phosphorylation (pSMAD2 / 3) using flow cytometry. WT or TGFβRII KO CAR Vδ1 T cells derived from two donors were cultured overnight in X-VIVO 15 medium containing 0.1% FBS, followed by incubation at 37°C for 30 min with 20 ng / mL human TGFβ1 (BioLegend). Cells were fixed and permeabilized using CytoFix buffer and Perm buffer III (BD Biosciences), and intracellular staining was performed using PE-conjugated anti-human pSMAD2 / 3 (BD ​​Biosciences). In the presence of TGFβ1, decreased pSMAD2 / 3 levels were detected in TGFβRII KO CAR Vδ1 T cells compared to WT cells. Results were obtained in… Figure 13B As shown in the figure. The data support the disruption of TGFβ-mediated signaling by the knockout of TGFβRII in CAR Vδ1 T cells.

[0398] In the presence of exogenous TGFβ1 (20 ng / mL), the cytotoxic potential of WT CAR Vδ1 T cells and TGFβR2 KO CAR Vδ1 T cells against PSMA+ tumor cell lines (22RV1, PC3 / PSMA, and DU145 / PSMA) expressing NIR nuclear marker fluorescent dyes was assessed using an in vitro repeated stimulation assay. CAR-positive Vδ1 T cells derived from two donors were co-cultured with tumor cells in 96-well plates at a 1:1 effector-to-target (E:T) cell ratio. After three days of co-culture, half of the cells were transferred to new containers with freshly seeded target cells and cultured for another three days. Co-cultures were monitored every 4 hours using an Incucyte® SX5 (Satorius). The cytotoxicity index was calculated by dividing the total NIR target area (µm² / well) at all time points by the value at tumor challenge. A lower cytotoxicity index indicated better cytotoxic potential. Following the second stimulation, TGFβRII KO CAR Vδ1 T cells were associated with higher tumor cell killing compared to WT CAR Vδ1 T cells. Results in Figure 13C The results show that TGFβRII KO alleviates the immunosuppressive effect of TGFβ1 in CAR Vδ1 T cells.

[0399] Example 10: In vitro activity of PSMA CAR-transduced γδ T cells expressing mbIL-12 with gene editing

[0400] PSMA-CAR or PSMA-CAR+mbIL12 transduced γδ T cells with or without gene editing (circle = unedited, square = MED12 knockout, triangle = MED12 knockout and TGFβRII knockout) were co-cultured with PC3-PSMA-expressing tumor cells at a 3:1 ratio in the presence of 20 ng / mL TGFβ1. PC3-PSMA survival was monitored on the Incucyte platform. Every 3 days, the effector γδ T cells were restimulated with PC3-PSMA-expressing tumor cells by transferring half of the effector γδ T cells from the existing co-culture to a new plate of tumor targets and replenishing the medium with TGFβ. At the final time point of each round of stimulation, %cytotoxicity was calculated based on the cytotoxicity index (CI). The cytotoxicity index was calculated by dividing the total integrated intensity of the NucNIR signal at each time point by the value at the start of each new round of stimulation. %cytotoxicity was then calculated as (CI) 单独肿瘤 -CI 共培养物 ) / CI 单独肿瘤 100. Once effector γδ T cells are no longer able to suppress tumor cells, they are removed from future analyses and reported as 0% cytotoxicity. Figure 14 The results support the conclusion that the combination of mbIL-12 armoring and MED12 KO significantly enhances the cytotoxicity of PSMA-CAR-transduced γδ T cells compared with the application of each modification alone.

[0401] Example 11: In vivo activity of PSMA CAR-transduced γδ T cells expressing mbIL-12 with gene editing

[0402] To evaluate the efficacy of the combination of gene editing (MED12 KO ± TGFβRII KO) and mbIL-12 on PSMA CAR-transduced γδ T cells, an in vivo efficacy study was conducted using a subcutaneous human prostate PC3 tumor cell (ATCC) model modified to express PSMA (PC3-PSMA). Female NSG mice (The Jackson Laboratory) were subcutaneously implanted with 1 x 10⁻⁶ cells mixed with Matrigel (1:1 ratio) (Corning). 6 PC3-PSMA cells. When the tumor volume is approximately 150 mm. 3 Mice were randomly assigned to study cohorts (n=5 mice per group). On day 0, each mouse was administered 1 x 10⁻⁶ mg of the drug as a single bolus dose via intravenous (iv) injection. 6Suboptimal treatment with CAR-positive γδ T cells. The cohort included PSMA-CAR, PSMA-CAR MED12 KO, PSMA-CAR MED12 KO + TGFβRII KO, PSMA-CAR + mbIL12, PSMA-CAR + mbIL-12 MED12 KO, and PSMA-CAR + mbIL-12 MED12 KO + TGFβRII KO γδ T cells. Tumor volume and mouse body weight were closely monitored throughout the experiment. Results were... Figure 15A and Figure 15B As shown in the image. Figure 15A The mean tumor volume (MTV) and standard error bar are shown for each group during the study period, up to day 36 when the endpoint was reached, at which point the MTV of the individual tumor cohorts reached approximately 1000 mm. 3 Statistical analysis of tumor volume data on day 36 (at the end of the study) was performed using one-way ANOVA and Dunnett's multiple comparison test. This indicates that the p-value is ≤ 0.0001. Indicates p-value ≤ 0.01. At suboptimal dose levels, PSMA-CAR+mbIL12 and PSMA-CAR+mbIL12 MED12 KO (± TGFβRII KO) γδ T cells exhibited statistically significant tumor control. Notably, PSMA-CAR+mbIL-12 MED12 KO (± TGFβRII KO) γδ T cells demonstrated potent tumor growth inhibition. This finding confirms the synergistic effect of combining MED12 KO (± TGFβRII KO) with mbIL-12 expression, enhancing the efficacy of PSMA-CAR Vδ1 T cells.

[0403] Figure 15BThe percentage tumor growth inhibition (%TGI) of each treatment group compared to the tumor-only control group is shown, which is performed using the formula %TGI = (1 - (MTV of the treatment group ÷ MTV of the tumor-only group)) x 100. Further statistical analyses were performed using Bliss-independent models (e.g., the model described in Duarte, Diana, and Nuno Vale. Evaluation of synergism in drug combinations and reference models for future orientations i...

Claims

1. A modified T cell comprising: a) An antigen recognition portion expressed on the surface of the cell, wherein the antigen recognition portion comprises an affinity-binding domain that is specific to disease-associated antigens; b) at least one modification for modulating the disease microenvironment and / or at least one modification for modulating cytokine activity; and c) At least one allogeneic escape modification and / or at least one modification for regulating T cell metabolism.

2. The modified T cell as described in claim 1, wherein it is a γδ T cell.

3. The modified T cells of claim 2, wherein the γδ T cells are δ1, δ2, δ3, or δ4 γδ T cells, preferably δ2. - γδ T cells, more preferably δ1 γδ T cells.

4. The modified T cell as claimed in any of the preceding claims, wherein the at least one modification for regulating the disease microenvironment comprises disruption of the CBL-B gene, disruption of the REGNASE-1 gene, disruption of the roquin gene, disruption of the TGF-β receptor type 2 (TGFβR2) gene, expression of dominant-negative TGF-β receptor type 2 (dnTGFβR2), or any combination thereof; optionally / preferredly wherein the at least one modification for regulating the disease microenvironment comprises either disruption of the TGFβR2 gene or expression of dnTGFβR2.

5. The modified T cell as claimed in any of the preceding claims, wherein the at least one modification for regulating cytokine activity includes the expression of membrane-bound IL-12, disruption of the CISH gene, expression of a cytokine switching receptor, or any combination thereof; optionally / preferably wherein the at least one modification for regulating cytokine activity includes the expression of membrane-bound IL-12.

6. The modified T cell as claimed in any of the preceding claims, wherein the at least one allogeneic escape modification comprises disruption of the ICAM-1 gene, disruption of the CD58 gene, disruption of the Fas gene, expression of a dominant-negative Fas receptor (dnFas), expression of a chimeric antigen receptor (CAR) that binds to CD70, or any combination thereof; optionally / preferredly wherein the at least one allogeneic escape modification comprises either disruption of the Fas gene or expression of dnFas.

7. The modified T cell as claimed in any of the preceding claims, wherein the at least one modification for regulating T cell metabolism includes disruption of the RASA2 gene, disruption of the MED12 gene, or any combination thereof; optionally / preferredly, wherein the at least one modification for regulating T cell metabolism includes disruption of the MED12 gene.

8. The modified T cell as claimed in any of the preceding claims, comprising at least one heterologous cytokine receptor expressed on the surface of the cell; preferably, the at least one heterologous cytokine receptor is dnTGFβR2.

9. The modified T cell as described in any of the preceding claims, which expresses exogenous stem cell factor (SCF).

10. A modified T cell as claimed in any of the preceding claims, wherein the antigen recognition portion is selected from the group consisting of: TCR, αβ TCR, γδ TCR, chimeric antigen receptor (CAR), whole antibody or antigen-binding fragment thereof, single-chain variable fragment (scFv), heavy chain or light chain single-domain antibody (sdAb), Fab, F(ab)2 or any combination thereof, wherein the antigen recognition portion binds to: (i) a cell surface tumor antigen, (ii) a peptide derived from a cell surface expressed as a tumor antigen in a complex with MHC (peptide-MHC complex), (iii) a cell surface antigen associated with an autoimmune disease or pathogen, or (iv) a peptide derived from a cell surface expressed as a complex with MHC (peptide-MHC complex) associated with an autoimmune disease or pathogen.

11. The modified T cell of claim 10, wherein the antigen recognition portion is a CAR.

12. The modified T cell of claim 11, wherein the CAR further comprises a transmembrane (TM) domain, a hinge domain, a co-stimulatory domain, and an intracellular signal transduction domain.

13. The modified T cell of claim 12, wherein the hinge domain is a CD8α hinge domain.

14. The modified T cell of claim 12, wherein the TM domain comprises the TM domain of CD8α.

15. The modified T cell of claim 12, wherein the intracellular signal transduction domain is the CD3ζ intracellular signal transduction domain.

16. The modified T cell as described in any of the preceding claims, wherein the disease-associated antigen is a tumor antigen, an autoimmune disease-associated antigen, or a pathogen antigen.

17. The modified T cell as described in any of the preceding claims, comprising disruption of the CISH gene, disruption of the CBL-B gene, disruption of the ICAM-1 gene, disruption of the CD58 gene, disruption of the Roquin-1 gene, disruption of the Regnase-1 gene, disruption of the TGFβR2 gene, disruption of the RASA2 gene, disruption of the MED12 gene, or a combination thereof.

18. The modified T cell as described in any of the preceding claims, comprising disruption of the MED12 gene and expression of membrane-bound IL-12.

19. The modified T cell of claim 18, further comprising disruption of the TGFβR2 gene or expression of dnTGFβR2, and / or disruption of the Fas gene or expression of dnFas.

20. The modified T cell according to any one of claims 1-17, comprising disruption of the TGFβR2 gene or expression of dnTGFβR2, and disruption of the MED12 gene.

21. The modified T cell of claim 20, further comprising disruption of the Fas gene or expression of dnFas, and / or expression of membrane-bound IL-12.

22. A pharmaceutical composition comprising a plurality of modified T cells as described in any one of the preceding claims.

23. The pharmaceutical composition of claim 22, wherein the plurality of modified T cells are a plurality of γδ T cells comprising at least about 60%, at least about 80%, or at least about 80% to about 90% δ1 γδ T cells, and a pharmaceutically acceptable carrier.

24. The pharmaceutical composition of claim 22 or 23, wherein the plurality of T cells comprises at least about 10 7 γδ T cells, optionally about 10 8 To about 10 10 One γδ T cell.

25. A method for treating or preventing a disease in a subject, comprising administering to a subject in need the pharmaceutical composition as described in any one of claims 22-24.

26. The method of claim 25, wherein the disease is an autoimmune disease.

27. The method of claim 25, wherein the disease is cancer or a precancerous condition.

28. A method for enhancing the efficacy of a T-cell therapy, comprising disrupting the MED12 gene in the T cells and expressing membrane-bound IL-12 in the T cells, thereby enhancing the cytotoxicity of the modified T cells.

29. The method of claim 28, wherein the modified T cell further comprises an antigen recognition portion expressed on the surface of the cell, wherein the antigen recognition portion comprises an affinity-binding domain specific to disease-associated antigens.

30. The method of claim 28 or 29, further comprising disrupting the Fas gene in the modified T cells or expressing dnFas in the modified T cells.

31. The method of any one of claims 28-30, wherein the T cell is a γδ T cell.

32. The method of claim 31, wherein the γδ T cells are δ1, δ2, δ3, or δ4 γδ T cells, preferably δ2. - γδ T cells, more preferably δ1 γδ T cells.

33. A method for enhancing the cytotoxicity of T cells, comprising disrupting the MED12 gene in the T cells and expressing membrane-bound IL-12 in the T cells, thereby enhancing the cytotoxicity of the modified T cells.

34. The method of claim 33, wherein the modified T cell further comprises an antigen recognition portion expressed on the surface of the cell, wherein the antigen recognition portion comprises an affinity-binding domain specific to disease-associated antigens.

35. The method of claim 33 or 34, further comprising disrupting the Fas gene in the modified T cells or expressing dnFas in the modified T cells.

36. The method of any one of claims 33-35, wherein the T cell is a γδ T cell.

37. The method of claim 36, wherein the γδ T cells are δ1, δ2, δ3, or δ4 γδ T cells, preferably δ2. - γδ T cells, more preferably δ1 γδ T cells.

38. A modified T cell comprising disruption of the MED12 gene in the T cell and expression of membrane-bound IL-12 in the T cell, wherein the modification enhances the cytotoxicity of the modified T cell.

39. The modified T cell of claim 38, wherein the modified T cell further comprises an antigen recognition portion expressed on the surface of the cell, wherein the antigen recognition portion comprises an affinity-binding domain specific to disease-associated antigens.

40. The modified T cell of claim 38 or 39, further comprising disruption of the Fas gene or expression of dnFas in the modified T cell.

41. The modified T cell according to any one of claims 38-40, wherein the T cell is a γδ T cell.

42. The method of claim 41, wherein the γδ T cells are δ1, δ2, δ3, or δ4 γδ T cells, preferably δ2. - γδ T cells, more preferably δ1 γδ T cells.

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