Methods and compositions for expanding gamma delta T cell populations with multivalent agents

Abstract

The present invention relates to methods of using soluble multivalent activating agents for the selective in vitro and ex vivo activation and expansion of γδ T cell population(s), including specific γδ T cell subpopulation(s) of interest and mixtures thereof, and methods for their therapeutic use. The disclosed methods and compositions are useful for the treatment of various cancers, infectious diseases, and immune disorders.

Description

[Background technology]

[0001] Antigen recognition by T lymphocytes can be achieved through the diverse heterodimeric T cell receptor (TCR). Approximately 95% of human T cells in the blood and lymphoid organs express the heterodimeric αβTCR receptor (αβ T cell lineage). Approximately 5% of human T cells in the blood and lymphoid organs express the heterodimeric γδTCR receptor (γδ T cell lineage). These T cell subsets are sometimes referred to as "αβ" and "γδ" T cells, respectively. αβ and γδ T cells have different functions. Furthermore, activation of αβ T cells occurs when antigen-presenting cells (APCs) present the antigen in association with class I / II MHC. In contrast to αβ T cells, γδ T cells can recognize antigens independently of MHC constraints. Moreover, γδ T cells possess both innate and adoptive immune recognition and response capabilities.

[0002] γδT cells utilize a set of distinct variable (V), diversity (D), junction (J), and constant (C) genes reconstituted from somatic cells. γδT cells contain fewer V, D, and J segments than αβT cells. While the number of germline Vγ and Vδ genes is more limited than the repertoire of Vα and VβTCR genes, the potential γδTCR repertoire is broader than that of αβTCRs due to a more extensive junctional diversification process during the reconstitution of the γ and δ chains of the TCR (Carding and Egan, Nat Rev Immunol (2002) 2:336).

[0003] Human γδ T cells construct their TCRs using three major Vδ (Vδ1, Vδ2, Vδ3) region genes and up to six Vγ region genes (Hayday AC., Annu Rev Immunol. 2000;18, 975-1026). The two major Vδ subsets are γδ T cells of Vδ1 and Vδ2. Vδ1 T cells, which have different Vγs, constitute the majority of the intraepithelial subset of mucosal γδ T cells, and their TCRs appear to recognize stress molecules on epithelial cells (Beagley KW, Husband AJ. Crit Rev Immunol. 1998;18(3):237-254). Vδ2 T cells, which commonly co-express Vγ9, are abundant in peripheral blood and the lymphatic system.

[0004] The ability of γδT cells to directly recognize antigens on diseased cells and to kill tumor cells, demonstrating their inherent capabilities, makes them an attractive therapeutic tool. The abundant Vγ9Vδ2 subtype of γδT cells recognizes pyrophosphate compounds such as the microbial compound (E)-4-hydroxy-3-methyl-buta-2-enylpyrophosphate. However, the ligands recognized by other γδT cell subtypes remain unknown.

[0005] Adoptive transfer of Vγ9Vδ2 T cells yielded limited target clinical responses in cancer treatment at the research stage (Kondo et al, Cytotherapy, 10:842-856, 2008; Lang et al, Cancer Immunology, Immunotherapy:CII, 60:1447-1460, 2011; Nagamine et al, 2009; Nicol et al, British Journal of Cancer, 105:778-786, 2011; Wilhelm et al, Blood. 2003 Jul 1;102(1):200-6), suggesting the need to isolate a new γδ T cell population and test it clinically.

[0006] The ability to selectively proliferate a subset of γδT cells with potent antitumor activity at improved purity and clinically acceptable levels is highly desirable. While antibody-cytokine cocktails have been used to proliferate a wider variety of γδT cell sets, they have not been able to activate specific γδT cell subsets to sufficient purity and clinically acceptable levels (Dokouhaki et al, 2010; Kang et al, 2009; Lopez et al, 2000; Kress, 2006).

[0007] Selective proliferation of γδ T cell subtypes has been demonstrated in vitro and in vivo using known ligands for Vγ9Vδ2. For example, Pressey et al., Medicine (Baltimore). 2016 Sep;95(39):e4909 reports in vivo proliferation of Vγ9Vδ2 using intravenous zoledronate, synthetic pyrophosphate mimetic, and subcutaneous IL-2. Selective proliferation of other γδ T cell subtypes has been demonstrated in vitro using immobilized antibodies that selectively bind and crosslink, e.g., δ1, δ2, and δ3 subtypes. See WO2016 / 081518, WO2017 / 197347, and WO2019 / 099744, which incorporate the entire content of these references.

[0008] Unfortunately, antibody immobilization presents specific processing and reproducibility challenges as well as cost constraints, particularly in the context of scaled-up in vitro clinical cell therapy in accordance with Good Manufacturing Practices. Specifically, the plastic surfaces required for antibody immobilization also promote cell adhesion, and restimulation during proliferation with immobilized mAbs induces strong cell adhesion. Therefore, harvesting proliferated cells from plates requires consistent and appropriate physical disruption and scraping for suitable cells, which is highly variable between operators and can lead to consistency and reproducibility issues between and within different samples. Furthermore, activation of immobilized antibody-based cells can result in cell proliferation or cell death, depending on the antibody concentration, composition, and presentation. Finally, the plastic surfaces required for conventional antibody immobilization are rigid and not ideal for scale-up. Therefore, there remains a great need for a practical, consistent, reproducible, and clinically scalable method for proliferating γδT cells. [Overview of the project]

[0009] The inventors have surprisingly determined that robust γδT cell activation, proliferation, and / or maintenance can be achieved by using soluble polyvalent antibodies, such as trivalent, tetravalent, or pentavalent antibodies, as activators. As demonstrated herein for the first time, the soluble polyvalent antibodies of the present invention can effectively activate and proliferate chimeric antigen receptor (CAR) γδT cells and / or endogenous γδT cells in vitro at levels close to, but without, the limitations associated with, those obtained with immobilized antibodies, thereby facilitating the scale-up and reproducibility of these much-needed clinical therapies.

[0010] Methods and compositions for using these soluble polyvalent activators individually or in combination for the in vitro proliferation of T cells in general, and particularly for γδT cells, are described herein. In some embodiments, the soluble polyvalent agent activates and proliferates γδT cells by binding to at least one epitope of the γδTCR. In some embodiments, the soluble polyvalent agent binds to different epitopes on the constant or variable region of the γTCR and / or δTCR. In some embodiments, the soluble polyvalent agent is a γδTCR panagent described and illustrated herein. The methods and compositions are also suitable for the selective activation and proliferation of one or more γδT cell subtypes. In some embodiments, the soluble polyvalent agent selectively activates and proliferates δ1T cells by i) binding to an activation epitope specific to the δ1TCR, ii) selectively activating and proliferating δ2T cells by binding to an activation epitope specific to the δ2TCR, and / or iii) selectively activating and proliferating δ3T cells by binding to an activation epitope specific to the δ3TCR.

[0011] In some embodiments, a soluble polyvalent drug contains at least two or more antigen-binding sites that specifically bind to the same antigen, or a polyvalent drug contains at least two or more antigen-binding sites that specifically bind to the same epitope of the same antigen. In some embodiments, a soluble polyvalent drug contains at least three antigen-binding sites that specifically bind to the same antigen, or a polyvalent drug contains at least three antigen-binding sites that specifically bind to the same epitope of the same antigen. In some cases, a soluble polyvalent drug is divalent, trivalent, tetravalent, or pentavalent, or at least divalent, trivalent, tetravalent, or pentavalent. In some cases, a soluble polyvalent drug is trivalent, tetravalent, or pentavalent, or at least trivalent, tetravalent, or pentavalent and optionally monospecific. In some cases, a polyvalent drug is tetravalent, or at least tetravalent and optionally monospecific. In some cases, polyvalent drugs are trivalent, tetravalent, or pentavalent, or at least trivalent, tetravalent, or pentavalent, and optionally monospecific.

[0012] In one embodiment, the present invention provides a method for activating and / or proliferating γδT cells in an isolated composite sample or mixed cell population cultured in vitro by contacting the mixed cell population with one or more soluble polyvalent agents that proliferate γδT cells by specifically binding to epitopes of γδTCRs in order to result in a concentrated γδT cell population. In another embodiment, the method comprises selectively activating and / or proliferating one or more γδT cell subtypes in an isolated composite or mixed cell population sample cultured in vitro by contacting a mixed cell population with one or more soluble multimeric agents that selectively proliferate δ1T cells, δ2T cells, or δ3T cells, or a combination thereof, wherein one or more agents that selectively proliferate δ1T cells bind to activation epitopes specific to δ1TCRs, one or more agents that selectively proliferate δ2T cells bind to activation epitopes specific to δ2TCRs, and one or more agents that selectively proliferate δ3T cells bind to activation epitopes specific to δ3TCRs, thereby activating and proliferating the desired γδT cell subtype(s).

[0013] In one embodiment, the present invention provides in vitro and in vitro methods for producing enriched γδT cell populations, the method comprising directly contacting an isolated mixed cell population containing γδT cells or a purified fraction thereof with one or more soluble polyvalent agents, preferably the soluble polyvalent agents activating and proliferating γδT cells by binding to at least one epitope of γδTCR. In another embodiment, the method comprises producing enriched γδT cell subpopulations from an isolated mixed cell population, comprising directly contacting the mixed cell population with one or more soluble polyvalent agents that i) selectively proliferate δ1T cells by binding to an epitope specific to δ1TCR, ii) selectively proliferate δ2T cells by binding to an epitope specific to δ2TCR, and iii) selectively proliferate δ3T cells by binding to an epitope specific to δ3TCR, thereby resulting in enriched γδT cell subpopulations.

[0014] In one embodiment, the present invention provides an in vitro method for activating and proliferating γδT cells in an isolated mixed cell population, the method comprising contacting the isolated mixed cell population with one or more soluble polyvalent agents that activate and proliferate γδT cells by binding to at least one epitope of γδTCR. In another embodiment, the present invention provides an in vitro method for activating and proliferating one or more γδT cell subtypes in an isolated mixed cell population, the method comprising contacting the isolated mixed cell population with one or more soluble polyvalent agents that selectively activate and proliferate δ1T cells, δ2T cells, or δ3T cells, or a combination thereof, wherein one or more agents that selectively activate and proliferate δ1T cells bind to activation epitopes specific to δ1TCRs, one or more agents that selectively activate and proliferate δ2T cells bind to activation epitopes specific to δ2TCRs, and one or more agents that selectively activate and proliferate δ3T cells bind to activation epitopes specific to δ3TCRs, thereby activating and proliferating a desired γδT cell subtype in the mixed cell population.

[0015] In some embodiments, the method optionally further comprises manipulating one or more isolated γδT cells either before or after in vitro activation and proliferation, and then administering the isolated, manipulated and / or unmanipulated, and in vitro-proliferated populations of γδT cells to a subject requiring them. In some embodiments, the γδT cells are manipulated to stably express one or more tumor-recognizing moieties, and / or the γδT cells are manipulated to contain a transgene encoding a secreted cytokine. In some embodiments, the manipulated and / or unmanipulated γδT cells are a population of autologous cells to the subject. In some embodiments, the manipulated and / or unmanipulated γδT cells are a population of allogeneic cells to the subject.

[0016] In some embodiments, a soluble polyvalent agent selectively activates and proliferates δ1 T cells by i) binding to an activating epitope specific to δ1 TCR, ii) binding to an activating epitope specific to δ2 TCR, and / or iii) binding to an activating epitope specific to δ3 TCR.

[0017] In some embodiments, a soluble polyvalent drug that selectively proliferates δ1 T cells, δ2 T cells, or δ3 T cells, or a combination thereof, includes at least two antigen-binding sites that specifically bind to the same antigen, or the polyvalent drug includes at least two antigen-binding sites that specifically bind to the same epitope of the same antigen. In some embodiments, a polyvalent drug that selectively proliferates δ1 T cells, δ2 T cells, or δ3 T cells, or a combination thereof, includes at least three antigen-binding sites that specifically bind to the same antigen, or the polyvalent drug includes at least three antigen-binding sites that specifically bind to the same epitope of the same antigen. In some cases, a polyvalent drug that selectively proliferates δ1 T cells, δ2 T cells, or δ3 T cells, or a combination thereof, is divalent, trivalent, tetravalent, or pentavalent, or at least divalent, trivalent, tetravalent, or pentavalent. In some cases, polyvalent drugs that selectively proliferate δ1T cells, δ2T cells, or δ3T cells, or combinations thereof, are trivalent, tetravalent, or pentavalent, or at least trivalent, tetravalent, or pentavalent, and are optionally monospecific. In some cases, polyvalent drugs that selectively proliferate δ1T cells, δ2T cells, or δ3T cells, or combinations thereof, are tetravalent, or at least tetravalent, and are optionally monospecific. In some cases, polyvalent drugs that selectively proliferate δ1T cells, δ2T cells, or δ3T cells, or combinations thereof, are trivalent, tetravalent, or pentavalent, or at least trivalent, tetravalent, or pentavalent, and are monospecific.

[0018] In some embodiments, a soluble polyvalent agent that selectively proliferates δ1T cells includes at least two or more antigen-binding sites that specifically bind to the δ1TCR bin1δ1 epitope, bin1bδ1 epitope, bin2δ1 epitope, bin2bδ1 epitope, bin2cδ1 epitope, bin3δ1 epitope, bin4δ1 epitope, bin5δ1 epitope, bin6δ1 epitope, bin7δ1 epitope, bin8δ1 epitope, or bin9δ1 epitope of the human δ1TCR. In some embodiments, a soluble polyvalent agent that selectively proliferates δ1 T cells includes at least two or more antigen-binding sites that specifically bind to, or compete with, the same or essentially the same epitope as, or to, an antibody selected from the group consisting of δ1-05, δ1-08, δ1-18, δ1-22, δ1-23, δ1-26, δ1-35, δ1-37, δ1-39, δ1-113, δ1-143, δ1-149, δ1-155, δ1-182, δ1-183, δ1-191, δ1-192, δ1-195, δ1-197, δ1-199, δ1-201, δ1-203, δ1-239, δ1-253, δ1-257, δ1-278, δ1-282, and δ1-285. In some embodiments, the soluble polyvalent agent includes a CDR of an antibody selected from the group consisting of δ1-05, δ1-08, δ1-18, δ1-22, δ1-23, δ1-26, δ1-35, δ1-37, δ1-39, δ1-113, δ1-143, δ1-149, δ1-155, δ1-182, δ1-183, δ1-191, δ1-192, δ1-195, δ1-197, δ1-199, δ1-201, δ1-203, δ1-239, δ1-253, δ1-257, δ1-278, δ1-282, and δ1-285. In some embodiments, the soluble polyvalent agent selectively proliferates δ1 T cells and δ3 T cells. In some embodiments, a soluble polyvalent agent that selectively proliferates δ1 T cells selectively proliferates δ1, δ3, δ4, and δ5γδ T cells.

[0019] In some embodiments, a soluble polyvalent agent that selectively proliferates δ1 T cells includes at least two or more antigen-binding sites that specifically bind to the same epitopes as antibodies selected from TS-1 and TS8.2. In some embodiments, the soluble polyvalent agent includes a CDR of TS-1 or TS8.2 and / or is humanized TS-1 or TS8.2. In some embodiments, a soluble polyvalent agent that selectively proliferates δ1 T cells includes at least two or more antigen-binding sites that do not compete with TS-1, TS8.2, or R9.12. In some embodiments, a soluble polyvalent agent that selectively proliferates δ1 T cells includes at least two or more antigen-binding sites that specifically bind to epitopes containing the δ1 variable region. In some embodiments, a soluble polyvalent agent that selectively proliferates δ1 T cells includes at least two or more antigen-binding sites that specifically bind to epitopes containing the residues Arg71, Asp72, and Lys120 of the δ1 variable region. In some embodiments, a soluble polyvalent agent that selectively proliferates δ1 T cells comprises at least two or more antigen-binding sites having reduced binding to mutant δ1 TCR polypeptides, including mutations at K120 of delta J1 and delta J2.

[0020] In some embodiments, a drug that selectively proliferates δ2 T cells includes at least two or more antigen-binding sites that specifically bind to the δ2 ​​TCR bin1 δ2 epitope, bin2 δ2 epitope, bin3 δ2 epitope, or bin4 δ2 epitope of the human δ2 TCR. In some embodiments, a soluble polyvalent drug that selectively proliferates δ2 T cells includes at least two or more antigen-binding sites that specifically bind to, or compete with, the same or essentially the same epitopes as, or compete with, those of an antibody selected from the group consisting of δ2-14, δ2-17, δ2-22, δ2-30, δ2-31, δ2-32, δ2-33, δ2-35, δ2-36, and δ2-37. In some embodiments, the soluble polyvalent agent for selectively proliferating δ2 T cells comprises a CDR of an antibody selected from the group consisting of δ2-14, δ2-17, δ2-22, δ2-30, δ2-31, δ2-32, δ2-33, δ2-35, δ2-36, and δ2-37. In some embodiments, the soluble polyvalent agent for selectively proliferating δ2 T cells comprises at least two or more antigen-binding sites that specifically bind to the same epitopes as antibodies selected from 15D and B6. In some embodiments, the soluble polyvalent agent comprises a CDR of 15D or B6, and / or humanized 15D and B6. In some embodiments, the soluble polyvalent agent for selectively proliferating δ2 T cells comprises at least two or more antigen-binding sites that specifically bind to epitopes different from those of antibodies 15D and / or B6. In some embodiments, a soluble polyvalent agent that selectively proliferates δ2 T cells comprises at least two or more antigen-binding sites that specifically bind to an epitope containing the δ2 ​​variable region. In some embodiments, a soluble polyvalent agent that selectively proliferates δ2 T cells comprises at least two or more antigen-binding sites having reduced binding to mutant δ2 TCR polypeptides containing a mutation at G35 in the δ2 ​​variable region.

[0021] In some embodiments, the agent for selectively proliferating δ3 T cells includes at least two or more antigen-binding sites that specifically bind to, or compete with, the same or essentially the same epitopes as, or are equivalent to, those of, an antibody selected from the group consisting of δ3-08, δ3-20, δ3-23, δ3-31, δ3-42, δ3-47, and δ3-58. In some embodiments, the soluble polyvalent agent for selectively proliferating δ3 T cells includes a CDR of an antibody selected from the group consisting of δ3-08, δ3-23, δ3-31, δ3-42, δ3-47, and δ3-58. In some embodiments, the soluble polyvalent agent for selectively proliferating δ3 T cells includes at least two or more antigen-binding sites that specifically bind to, or are equivalent to, or are equivalent to, those of, an antibody selected from the group consisting of δ3-23, δ3-31, δ3-42, δ3-47, and δ3-58.

[0022] In some embodiments, the agent that selectively proliferates δ3T cells is an antibody or fragment selected from the group consisting of δ3-08, δ3-20, δ3-23, δ3-31, δ3-42, δ3-47, and δ3-58. In some embodiments, the agent that selectively proliferates δ3T cells is an antibody or fragment selected from the group consisting of δ3-08, δ3-23, δ3-31, δ3-42, δ3-47, and δ3-58. In some embodiments, the agent that selectively proliferates δ3T cells is an antibody or fragment selected from the group consisting of δ3-23, δ3-31, δ3-42, δ3-47, and δ3-58.

[0023] In some embodiments, the method further comprises culturing a γδ T cell population simultaneously or sequentially with cytokines, preferably common gamma chain cytokines. In some embodiments, the cytokines are selected from the group consisting of IL-2, IL-7, IL-9, IL-12, IL-15, IL-18, IL-21, and IL-33, preferably selected from the group consisting of IL-2, IL-7, IL-15, or IL-21, and even more preferably selected from the group consisting of IL-2, IL-7, and IL-15. In some embodiments, the method further comprises performing at least one depletion step for αβ T cells after activation and proliferation of the γδ T cell population and before administration to a subject.

[0024] In some embodiments, the proliferated manipulated or unmanipulated γδT cell population comprises at least 60% (e.g., at least 70%, 80%, or 90%, about 60% to about 80%, or about 60% to about 90%) of δ1γδT cells, and the method further comprises administering the γδT cells to a subject in need. In some embodiments, the proliferated manipulated or unmanipulated γδT cell population comprises at least 60% (e.g., at least 70%, 80%, or 90%, about 60% to about 80%, or about 60% to about 90%) of δ2γδT cells, and the method further comprises administering the γδT cells to a subject in need. In some embodiments, the proliferated manipulated or unmanipulated γδT cell population comprises at least 10% (e.g., at least 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, about 10% to about 80%, about 20% to about 40%, about 20% to about 50%, or about 20% to about 60%) δ3γδT cells, and the method further comprises administering the γδT cells to a subject in need thereof.

[0025] In another embodiment, the present invention comprises a soluble composition comprising one or more polyvalent agents for use in the present method, preferably the polyvalent agent(s) activating and proliferating γδT cells by binding to at least one epitope of the γδTCR. In some embodiments, the polyvalent agent activates and proliferates γδT cells by binding to at least one epitope of the γδTCR. In some embodiments, the polyvalent agent binds to different epitopes on the constant or variable region of the γTCR and / or δTCR. In some embodiments, examples of polyvalent agents include γδTCR panagents described and illustrated herein.

[0026] In some embodiments, a polyvalent drug selectively activates and proliferates δ1 T cells by i) binding to an activating epitope specific to the δ1 TCR, ii) binding to an activating epitope specific to the δ2 ​​TCR, and / or iii) binding to an activating epitope specific to the δ3 TCR. In some embodiments, a polyvalent drug that selectively proliferates δ1 T cells, δ2 T cells, or δ3 T cells, or a combination thereof, includes at least two antigen-binding sites that specifically bind to the same antigen, or the polyvalent drug includes at least two antigen-binding sites that specifically bind to the same epitope of the same antigen. In some embodiments, a polyvalent drug that selectively proliferates δ1 T cells, δ2 T cells, or δ3 T cells, or a combination thereof, includes at least three antigen-binding sites that specifically bind to the same antigen, or the polyvalent drug includes at least three antigen-binding sites that specifically bind to the same epitope of the same antigen. In some cases, polyvalent drugs that selectively proliferate δ1T cells, δ2T cells, or δ3T cells, or combinations thereof, are divalent, trivalent, tetravalent, or pentavalent, or at least divalent, trivalent, tetravalent, or pentavalent. In some cases, polyvalent drugs that selectively proliferate δ1T cells, δ2T cells, or δ3T cells, or combinations thereof, are trivalent, tetravalent, or pentavalent, or at least trivalent, tetravalent, or pentavalent, and are optionally monospecific. In some cases, polyvalent drugs that selectively proliferate δ1T cells, δ2T cells, or δ3T cells, or combinations thereof, are tetravalent, or at least tetravalent, and are optionally monospecific. In some cases, polyvalent drugs that selectively proliferate δ1T cells, δ2T cells, or δ3T cells, or combinations thereof, are trivalent, tetravalent, or pentavalent, or at least trivalent, tetravalent, or pentavalent, and are monospecific. In some cases, γδT cells are engineered to stably express one or more tumor-recognition regions, and / or to contain transgenes encoding secreted cytokines.

[0027] In another embodiment, the present invention provides a method for treating cancer, infectious diseases, inflammatory diseases, or autoimmune diseases in a subject in need thereof, the method comprising performing one of the aforementioned in vitro and / or in vitro growth methods described herein, and administering the resulting proliferated γδT cell population to a subject in need thereof.

[0028] In another aspect, the present invention provides the use in the manufacture of a pharmaceutical product for the treatment of cancer, infectious diseases, inflammatory diseases, or autoimmune diseases in a subject requiring such treatment, comprising the resulting proliferated γδT cell population.

[0029] Embedding by reference All publications, patents, and patent applications referenced herein are incorporated herein by reference to the same extent as any individual publications, patents, or patent applications are specifically and individually indicated as being incorporated by reference.

[0030] The novel features of the present invention are expressed in detail in the claims set forth separately. A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description illustrating exemplary embodiments in which the principles of the present invention are utilized, and to the accompanying drawings (furthermore, "figure" and "Fig." as used herein). [Brief explanation of the drawing]

[0031] [Figure 1] The heavy chain framework and complementarity-determining region amino acid sequences of δ1-specific MAb are depicted (sequence numbers 1-27, in order of appearance). [Figure 2] Figure 1 depicts the light chain framework and complementarity-determining region amino acid sequences of δ1-specific MAb (sequence numbers 28-54, in order of appearance). [Figure 3]The heavy chain framework and complementarity-determining region amino acid sequences of δ2-specific MAb are depicted (sequence numbers 55-63, in order of appearance). [Figure 4] Figure 3 depicts the light chain framework and complementarity-determining region amino acid sequences of δ2-specific MAb (sequence numbers 64-73, in order of appearance). [Figure 5] The variable region sequences of δ3-specific anti-γδTCR antibodies are shown. Sequences above the heavy chain variable region (in order of appearance, sequence numbers 74-80, respectively). Sequences below the light chain variable region (in order of appearance, sequence numbers 81-87, respectively). [Figure 6A] This paper describes the effect of soluble mAb concentration on receptor crosslinking. High concentrations of mAbs result in monovalent single-arm binding that does not promote TCR complex crosslinking or clustering. Lower concentrations of mAbs promote TCR complex crosslinking and clustering, depending on the epitope and stoichiometry of the specific TCR subunit of the complex to which the mAb binds. [Figure 6B] This paper describes the effect of soluble mAb concentration on receptor crosslinking. High concentrations of mAbs result in monovalent single-arm binding that does not promote TCR complex crosslinking or clustering. Lower concentrations of mAbs promote TCR complex crosslinking and clustering, depending on the epitope and stoichiometry of the specific TCR subunit of the complex to which the mAb binds. [Figure 7] This describes how the geometry of mAb epitopes directs receptor and cellular involvement. Vertical, outward-facing epitopes promote synapse formation between adjacent cells (conventional bispecificity such as aCD3 × aTAA bsAb). [Figure 8] Describe the geometry of mAb epitopes that indicate receptor and cellular involvement. [Figure 9] This describes a specific embodiment of the soluble polyvalent agent of the present invention. [Figure 10A]This describes representative gating for PBMC activation on plate-bound D1-35_mlgG2 at 5 mg / mL for PBMC donor B88. FITC(CFSE) histograms for each T cell subset show cell division. The area under the histogram curve (AUC) represents the number of viable cells in each T cell subset. The TCRαβ- / Vδ1- / CD2+ population consists of non-Vδ1, γδ T cells (the pan-activated sample is likely mostly Vδ2 cells). [Figure 10B] This describes representative gating for PBMC activation on plate-bound D1-35 mlgG2 at 5 mg / mL for PBMC donor B91. FITC(CFSE) histograms for each T cell subset show cell division. The area under the histogram curve (AUC) represents the number of viable cells in each T cell subset. The TCRαβ- / Vδ1- / CD2+ population consists of non-Vδ1, γδ T cells (the pan-activated sample is likely mostly Vδ2 cells). [Figure 11] This describes plate-bound 5 mg / mL (donor B88) PBMC activation. Vδ1: D1-35_mIgG2 > Pan-05_hIgG1-Sc > D1-08_hIgG1-mSc (top 3). TCRαβ- / Vδ1- / CD2+: Pan-05_hIgG1-Sc > D1-08_hIgG1-ScAgg ~ Pan-07_hIgG1-Sc (top 3). [Figure 12] This describes plate-bound 5 mg / mL (donor B91) PBMC activation. Vδ1: D1-35_mIgG2 > D1-08_hIgG1-mSc > Pan-07_hIgG1-Sc (top 3). TCRαβ- / Vδ1- / CD2+: D1-08_hIgG1-ScAgg > Pan-07_hIgG1-Sc (top 2). [Figure 13] The 5 mg / ml (donor B88) soluble PBMC activation is described. Vδ1: Top 3 proliferation groups: Pan-05_hIgG1-Sc >> Pan-07_hIgG1-Sc > D1-08_hIgG1-Sc. TCRαβ- / Vδ1- / CD2+: Top 2 proliferation groups: Pan-05_hIgG1-Sc >> Pan-07_hIgG1-Sc. [Figure 14] This describes soluble 5ug / ml (donor B91) PBMC activation. Vδ1: Top 3 are D1-08_hIgG1-Sc~Pan-05_hIgG1-Sc~D1-08_hIgG1-mSc. TCRαβ- / Vδ1- / CD2+: Top 2 are D1-08_hIgG1-ScAgg>Pan-07_hIgG1-Sc. [Figure 15] This describes PBMC activation with soluble 50 ng / mL (donor B88). Vδ1: The superstructure is Pan-07_hIgG1-Sc. TCRαβ- / Vδ1- / CD2+: The superstructure is Pan-07_hIgG1-Sc. [Figure 16] This describes PBMC activation with soluble 50 ng / mL (donor B91). Vδ1: The top 3 activators are D1-35_mIgG2 > D1-08_hIgG1-mSc > Pan-07_hIgG1-Sc. TCRαβ- / Vδ1- / CD2+: No clearly dominant activators. [Figure 17] This provides the nucleic acid sequence and amino acid sequence (sequence numbers 88 and 89, respectively) of PL426 (pCI-D1-08-chimeric-Scorpion), as well as a table of regions of the polynucleotide construct. [Figure 18] This provides the nucleic acid sequence and amino acid sequence (sequence numbers 90 and 91, respectively) of PL42 (pCI-D1-08-MiniScorpion), as well as a table of regions of the polynucleotide construct. [Figure 19] This provides the nucleic acid sequence and amino acid sequence (sequence numbers 92 and 93, respectively) of PL478 (pCI-D1-08-chimeric-Scorpion-hIgG4), as well as a table of regions of the polynucleotide construct. [Figure 20] This provides the nucleic acid sequence and amino acid sequence (SEQ ID NOs. 94 and 95, respectively) of PL502 (pCI-Pan05-chimeric-Scorpion-hIgG1), as well as a table of regions of the polynucleotide construct. [Figure 21] This provides the nucleic acid sequence and amino acid sequence (SEQ ID NOs. 96 and 97, respectively) of PL503(pCI-Pan05-LC), as well as a table of regions of the polynucleotide construct. [Figure 22] This document provides the nucleic acid sequence and amino acid sequence (SEQ ID NOs. 98 and 99, respectively) of PL504 (pCI-Pan05-MiniScorpion-hIgG1), as well as a table of regions of the polynucleotide construct. [Figure 23] This provides the nucleic acid sequence and amino acid sequence (SEQ ID NOs. 100 and 101, respectively) of PL505 (pCI-Pan07-chimeric-Scorpion-hIgG1), as well as a table of regions of the polynucleotide construct. [Figure 24] This provides the nucleic acid sequence and amino acid sequence (SEQ ID NOs. 102 and 103, respectively) of PL506(pCI-Pan07-LC), as well as a table of regions of the polynucleotide construct. [Figure 25] This document provides the nucleic acid sequence and amino acid sequence (SEQ ID NOs. 104 and 105, respectively) of PL507 (pCI-Pan07-MiniScorpion-hIgG1), as well as a table of regions of the polynucleotide construct. [Modes for carrying out the invention]

[0032] While various embodiments of the present invention are shown and described herein, it will be clear to those skilled in the art that such embodiments are provided merely as examples. Those skilled in the art can conceive of many variations, modifications, and substitutions without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention may be adopted.

[0033] definition Unless otherwise specified, all scientific and technical terms used herein have the same meaning as that commonly understood by a person of ordinary skill in the art to which the inventions described herein pertain. For explanatory purposes of this specification, the following definitions shall apply, including the plural form of a singular term where appropriate, and vice versa. In the event of any conflict between any explicitly stated definition and any document referenced herein by reference, the definitions expressed below shall prevail.

[0034] As used herein, the term “γδT cells (gamma delta T cells)” refers to a subset of T cells that express a unique T cell receptor (TCR) consisting of one gamma chain and one delta chain, the γδTCR, on their surface. Specifically, the term “γδT cells” includes, without limitation, all subsets of γδT cells and their combinations, including Vδ1, Vδ2, and Vδ3γδT cells, as well as naive, effector memory, central memory, and terminally differentiated γδT cells. For further example, the term “γδT cells” includes Vδ4, Vδ5, Vδ7, and Vδ8γδT cells, as well as Vγ2, Vγ3, Vγ5, Vγ8, Vγ9, Vγ10, and Vγ11γδT cells.

[0035] As used herein, the terms “T lymphocyte” or “T cell” refer to immune cells that express CD3 (CD3+) and the T cell receptor (TCR+). T cells play a central role in cellular immunity.

[0036] As used herein, the terms “TCR” or “T cell receptor” refer to heterodimeric cell surface signaling proteins that form alpha-beta or gamma-delta receptors. αβTCRs recognize antigens presented by MHC molecules, while γδTCRs recognize antigens independently of MHC presentation.

[0037] The term "MHC" (Major Histocompatibility Complex) refers to a subset of genes that encode cell surface antigen-presenting proteins. In humans, these genes are called human leukocyte antigen (HLA) genes. In this specification, the abbreviations MHC and HLA are used interchangeably.

[0038] As used herein, the terms “peripheral blood lymphocytes” or “PBL” are used in their broadest sense to refer to leukocytes, including T and B cells, plasma cells, monocytes, macrophages, natural killer cells, basophils, eosinophils, and other leukocytes at a wide range of differentiation and functional stages. T lymphocytes make up approximately 20–80% of peripheral blood.

[0039] As used herein, the term “cell population” refers to a large number of cells obtained by direct isolation from a suitable source (usually from mammals). The isolated cell population can then be cultured in vitro. Those skilled in the art will be familiar with the various methods for isolating and culturing cell populations used in conjunction with the present invention, as well as the various numbers of cells in a cell population suitable for use in the present invention. Cell populations can be purified to homogeneity, substantial homogeneity, or depletion of one or more cell types (e.g., αβT cells) by various culture techniques and / or negative or positive selection of specific cell types. Cell populations may be mixed heterogeneous cell populations derived from, for example, peripheral blood samples, umbilical cord blood samples, tumors, stem cell precursors, tumor biopsies, tissues, lymph, skin, tumor-infiltrating lymphocytes, or samples containing these, or from epithelial sites of an object in direct contact with the external environment, or derived from stem progenitor cells. Alternatively, the mixed cell population may originate from in vitro cultures of mammalian cells established from peripheral blood samples, umbilical cord blood samples, tumors, stem cell precursors, tumor biopsies, tissues, lymph, skin, tumor-infiltrating lymphocytes, or samples containing these, or from epithelial sites of the subject in direct contact with the external environment, or from stem progenitor cells.

[0040] A “concentrated” cell population or formulation refers to a cell population derived from a starting mixed cell population in which the percentage of a particular cell type it contains is higher than the percentage of that cell type in the starting population. For example, a starting mixed cell population can be concentrated with respect to a particular γδT cell population. In one embodiment, the concentrated γδT cell population contains a higher percentage of δ1 cells than the percentage of that cell type in the starting population. In another embodiment, the concentrated γδT cell population contains a higher percentage of δ2 cells than the percentage of that cell type in the starting population. In yet another embodiment, the concentrated γδT cell population contains a higher percentage of δ3 cells than the percentage of that cell type in the starting population. To give yet another example, the concentrated γδT cell population may contain a higher percentage of δ1 cells and a higher percentage of δ3 cells than the percentages of each cell type in the starting population. To give yet another example, the concentrated γδT cell population may contain a higher percentage of δ1 cells and a higher percentage of δ4 cells than the percentages of each cell type in the starting population. To give another example, the enriched γδT cell population may have higher percentages of δ1 cells and δ5 cells than the percentages of each cell type in the starting population. To give yet another example, the enriched γδT cell population may have higher percentages of δ1T cells, δ3T cells, δ4T cells, and δ5T cells than the respective percentages of each cell type in the starting population. In yet another embodiment, the enriched γδT cell population has higher percentages of both δ1 cells and δ2 cells than the percentages of each cell type in the starting population. In yet another embodiment, the enriched γδT cell population has higher percentages of δ1 cells, δ2 cells, and δ3 cells than the percentages of each cell type in the starting population. In all embodiments, the enriched γδT cell population contains a lower percentage of αβT cells.

[0041] As used herein, "proliferated" means that the number of desired cell types, i.e., target cell types (e.g., δ1 and / or δ2 T cells and / or δ3 T cells) in the concentrated formulation is greater than the number in the initial cell population, i.e., the starting cell population.

[0042] "Selective proliferation" means preferentially proliferating a target cell type (e.g., δ1, δ2, or δ3 T cells) over other non-target cell types, such as αβ T cells or NK cells, or γδ T cells in untargeted subpopulations. In certain embodiments, the activator of the present invention selectively proliferates, for example, manipulated or unmanipulated δ1, δ2, and / or δ3 T cells without the proliferation of αβ T cells, or without significant proliferation of αβ T cells. In certain embodiments, the activator of the present invention selectively proliferates, for example, manipulated or unmanipulated δ1 T cells without the proliferation of δ2 T cells, or without significant proliferation of δ2 T cells. In other embodiments, the activator of the present invention selectively proliferates, for example, manipulated or unmanipulated δ2 T cells without the proliferation of δ1 T cells, or without significant proliferation of δ1 T cells. In certain embodiments, the activator of the present invention selectively proliferates, for example, manipulated or unmanipulated δ3 T cells without proliferation of δ2 T cells or without significant proliferation of δ2 T cells, without proliferation of δ1 T cells or without significant proliferation of δ1 T cells. In certain embodiments, the activator of the present invention selectively proliferates, for example, manipulated or unmanipulated δ1 and δ3 T cells without proliferation of δ2 T cells or without significant proliferation of δ2 T cells. In certain embodiments, the activator of the present invention selectively proliferates, for example, manipulated or unmanipulated δ1 and δ4 T cells without proliferation of δ2 T cells or without significant proliferation of δ2 T cells. In certain embodiments, the activator of the present invention selectively proliferates, for example, manipulated or unmanipulated δ1 and δ5 T cells without proliferation of δ2 T cells or without significant proliferation of δ2 T cells. In certain embodiments, the activator of the present invention selectively proliferates, for example, manipulated or unmanipulated, δ1, δ3, δ4, and δ5 T cells without or without significant proliferation of δ2 T cells. In this context, the term "without significant proliferation" means that the preferentially proliferated cell population is at least 10 times, preferably 100 times, and more preferably 1,000 times greater than the reference cell population.

[0043] As used herein, the term “mixture” refers to a combination of two or more isolated and enriched cell populations derived from a mixed heterogeneous cell population. According to certain embodiments, the cell population of the present invention is an isolated γδT cell population. According to certain embodiments, the cell population of the present invention is grown in vitro and / or provided in vitro and administered to a subject to become an in vivo γδT cell population. According to certain embodiments, the cell population of the present invention may also be further grown and / or maintained in vivo by administering one or more agents that selectively promote the growth of the γδT cell population.

[0044] The term "soluble" is used in its conventional sense to specify a composition that, for example, can be dissolved or liquefied in an aqueous solution, and which certainly excludes agents that are covalently bonded to a plate or beads.

[0045] The term “isolated” as applied to a cell population refers to a cell population isolated from the body of a human or animal that substantially does not contain one or more cell populations associated with the aforementioned cell population in vivo or in vitro.

[0046] In relation to cell populations, the term “contact,” as used herein, refers to incubating an isolated cell population with a reagent, such as an antibody, cytokine, ligand, mitogen, or co-stimulatory molecule that can be linked to either beads or cells. The antibody or cytokine may be in a soluble form or immobilized. In one embodiment, the immobilized antibody or cytokine is tightly or covalently bound to the beads or plate. In one embodiment, the antibody is immobilized in an Fc-coated well. In a preferred embodiment, contact occurs in vivo.

[0047] As used herein, the term “antibody” refers to immunoglobulin molecules and molecules containing the immunoactive portion of an immunoglobulin (Ig) molecule, i.e., an antigen-binding site that specifically binds to (immunely reacts with) an antigen. “Specifically binds,” “immunely reacts with,” or “against” means that the antibody reacts with one or more antigenic determinants of a desired antigen, but does not react with other polypeptides, or has a much lower affinity (K). D >10 -6 This means binding in moles. Antibodies are not limited to these, but include polyclonal, monoclonal, chimeric, sdAb (heavy or light chain single-domain antibody), single-chain, and F ab F ab’ , and F (ab’)2 Fragment, scFv, diabody, minibody, nanobody, and F ab Expression libraries are one example.

[0048] As used herein, the term “chimeric antigen receptor (CAR)” may refer to an artificial T cell receptor, T body, single-chain immune receptor, chimeric T cell receptor, or chimeric immune receptor, and may include, for example, manipulated receptors that fuse artificial specificity to specific immune effector cells. CARs may be employed to confer the specificity of a monoclonal antibody to T cells, thereby generating a large number of specific T cells, for example, for adoptive cell therapy. In specific embodiments, CARs direct cell specificity to, for example, tumor-associated antigens. In some embodiments, a CAR comprises an intracellular activation domain (which activates T cells upon engagement of the target-directing portion with target cells such as target tumor cells), a transmembrane domain, and an extracellular domain, which may vary in length, containing a disease or disorder-associated, e.g., tumor antigen-binding region. In more detailed embodiments, a CAR comprises a fusion of a single-chain variable fragment (scFv) derived from a monoclonal antibody, fused with a CD3-zeta transmembrane domain and an intracellular domain. Specificity of other CAR designs may derive from a ligand of a receptor (e.g., a peptide) or a pattern recognition receptor such as dectin. In certain cases, the spacing of antigen recognition domains can be adjusted to reduce activation-induced cell death. In certain cases, CARs may include domains for additional co-stimulatory signaling, such as CD3-zeta, FcR, CD27, CD28, CD137, DAP10 / 12, and / or OX40, ICOS, TLR, etc. In some cases, molecules including co-stimulatory molecules, reporter genes for contrast (e.g., for positron emission tomography), gene products that conditionally remove T cells by the addition of prodrugs, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors can be co-expressed with CARs.

[0049] The basic antibody structural unit is known to comprise a tetramer. Each tetramer consists of two identical pairs of polypeptide chains, with each pair having one "light chain" (about 25 kDa) and one "heavy chain" (about 50 - 70 kDa). The amino-terminal portion of each chain contains a variable region of about 100 - 110 or more amino acids that is mainly responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region that is mainly responsible for effector functions. Generally, antibody molecules obtained from humans are related to one of the classes IgG, IgM, IgA, IgE, and IgD, which differ from each other in the nature of the heavy chains present in the molecule. Specific classes also have subclasses such as IgG1, IgG2, etc. Furthermore, in humans, the light chain can be a kappa chain or a lambda chain.

[0050] The term "Fab" refers to an antibody fragment composed of the entire L chain (V L and C L ), in addition to the variable region domain of the H chain (V H ) and the first constant domain of one heavy chain (CH1). Two Fab fragments can be generated using papain digestion of intact antibodies, each of which contains one antigen-binding site. Typically, the L chain and H chain fragments of Fab generated by papain digestion are linked by interchain disulfide bonds.

[0051] The term "Fc" refers to an antibody fragment that includes the carboxy-terminal portions (CH2 and CH3) of both H chains and part of the hinge region, joined together by sulfide bonds. The effector function of an antibody is determined by the sequence of the Fc region, which is also the part recognized by Fc receptors (FcR) found on certain types of cells. One Fc fragment can be obtained by papain digestion of intact antibodies.

[0052] The term "F(ab’)2" refers to an antibody fragment produced by pepsin digestion of intact antibodies. The F(ab’)2 fragment contains two Fab fragments and part of the hinge region joined together by disulfide bonds. The F(ab’)2 fragment has bivalent antigen-binding activity and can cross-link antigens.

[0053] The term Fab' refers to an antibody fragment that is the product of the reduction of the F(ab')2 fragment. The Fab' fragment differs from the Fab fragment in that it has a few additional residues at the carboxyl terminus of the CH1 domain, which contains one or more cysteines from the antibody hinge region. Fab'-SH is the herein designation for Fab' in which the cysteine ​​residue(s) of the constant domain have a free thiol group.

[0054] The term "Fv" refers to an antibody fragment composed of a dimer of one heavy chain variable domain and one light chain variable domain that are tightly and non-covalently associated. The folding of these two domains creates six hypervariable loops (three from the H chain and three from the L chain) that contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) can recognize and bind to an antigen, although its affinity is usually lower than that of a complete binding site.

[0055] The term "single-stranded Fv," also abbreviated as "sFv" or "scFv," refers to an antibody fragment containing VH and VL antibody domains linked together as a single polypeptide chain. Typically, an scFv polypeptide further includes a polypeptide linker between the VH and VL domains, which allows the scFv to form a desired structure for antigen binding. For a review of scFv, see, for example, Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994), and Malmborg et al., J. Immunol. Methods 183:7-13, 1995.

[0056] The term "linear antibody" refers to a pair of tandem V antibodies that form a pair of antigen-binding regions. H -C H 1 segment (V H -C H 1-V H -C H 1) is used to refer to polypeptides containing the above. Linear antibodies can be bispecific or monospecific, as described, for example, in Zapata et al., Protein Eng. 8(10):1057-1062 (1995).

[0057] The term "variable" refers to the fact that certain portions of the variable domain exhibit extensive sequence differences between antibodies, which are used in the binding affinity and specificity of each particular antibody to its specific antigen. However, variability is not uniformly distributed throughout the entire variable domain of an antibody. It is concentrated in three segments called hypervariable regions in both the light and heavy chain variable domains. The more highly conserved portions of the variable domain are called framework regions (FRs). Each of the natural heavy and light chain variable domains contains four FRs, generally in a beta-sheet configuration, which are linked by three hypervariable regions that form loops that connect the beta-sheet structures and, in some cases, form part of the beta-sheet structures. The hypervariable regions within each chain are grouped together in close proximity by the FRs and, together with the hypervariable region from the other chain, contribute to the formation of the antibody's antigen-binding site (see Kabat et al (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.). The constant domain does not directly participate in the binding of antibodies to antigens, but it exhibits various effector functions, such as the involvement of antibodies in antibody-dependent cytotoxicity (ADCC).

[0058] The term "antigen-binding site" or "binding region" refers to the portion of an immunoglobulin molecule involved in antigen binding. The antigen-binding site is formed by amino acid residues in the variable ("V") regions at the N-terminus of the heavy ("H") and light ("L") chains. Three highly variable regions within the V region of the heavy and light chains are called "hypervariable regions" and are sandwiched between more conserved regions on either side known as "framework regions" or "FR." Thus, the term "FR" refers to the naturally occurring amino acid sequences adjacent to and between the hypervariable regions of immunoglobulins. In antibody molecules, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged in three-dimensional space relative to each other to form the antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each heavy and light chain are called "complementarity-determining regions" or "CDRs." The amino acid assignments to each domain are based on the definitions in Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia & Lesk J. Mol. Biol. 196:901-917 (1987) and Chothia et al. Nature 342:878-883 (1989).

[0059] The terms "hypervariable region," "HVR," or "HV" refer to the region of the antibody variable domain whose sequence is hypervariable and / or forms a structurally defined loop. Generally, antibodies contain six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). In natural antibodies, H3 and L3 exhibit the greatest diversity among the six HVRs, and H3 in particular is thought to play a unique role in conferring subtle specificity to the antibody. See, for example, Xu et al., Immunity 13:37-45 (2000) and Johnson and Wu, in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, NJ, 2003). In fact, naturally occurring camel antibodies, composed solely of heavy chains, are functional and stable in the absence of light chains. For example, see Hamers-Casterman et al., Nature 363:446-448 (1993) and Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0060] The "framework region" (FR) consists of variable domain residues other than the CDR residues. Each variable domain typically has four FRs, identified as FR1, FR2, FR3, and FR4. When the CDR is defined by Kabat, the light chain FR residues are located at approximately residues 1–23 (LCFR1), 35–49 (LCFR2), 57–88 (LCFR3), and 98–107 (LCFR4), while the heavy chain FR residues are located at approximately residues 1–30 (HCFR1), 36–49 (HCFR2), 66–94 (HCFR3), and 103–113 (HCFR4). If a CDR contains amino acid residues from a hypervariable loop, the light chain FR residues are located at approximately 1–25 (LCFR1), 33–49 (LCFR2), 53–90 (LCFR3), and 97–107 (LCFR4) of the light chain, and the heavy chain FR residues are located at approximately 1–25 (HCFR1), 33–52 (HCFR2), 56–95 (HCFR3), and 102–113 (HCFR4) of the heavy chain. In some cases, if a CDR contains amino acids from both a CDR defined by Kabat and one from a hypervariable loop, the FR residues will be adjusted accordingly. For example, if CDRH1 contains amino acids H26–H35, the heavy chain FR1 residues are at positions 1–25 and the FR2 residues are at positions 36–49.

[0061] The "Human Consensus Framework" is a framework that represents the most commonly occurring amino acid residues among the choices of VL or VH framework sequences for human immunoglobulins. Typically, the choices of VL or VH sequences for human immunoglobulins are derived from subgroups of variable domain sequences. Typically, these sequence subgroups are subgroups like those in Kabat. In some cases, for VL, the subgroup is subgroup Kappa I, as in Kabat. In other cases, for VH, the subgroup is subgroup III, as in Kabat.

[0062] The antibodies described herein can be humanized. The “humanized” form of a non-human (e.g., rodent) antibody is a chimeric antibody containing the minimal sequence derived from the non-human antibody. For the most part, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient’s hypervariable region are replaced by residues from the hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, having the desired antibody specificity, affinity, and capabilities. In some cases, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, a humanized antibody may contain residues not found in the recipient antibody or donor antibody. These modifications are made to further refine antibody performance. Generally, a humanized antibody will contain at least one, typically two, substantially all of the variable domains, where all or substantially all of the hypervariable loops correspond to the hypervariable loops of the non-human immunoglobulin, and all or substantially all of the FRs are FRs of the human immunoglobulin sequence. Humanized antibodies will also optionally contain the constant region (Fc) of immunoglobulins, typically at least a portion of human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986), Riechmann et al., Nature 332:323-329 (1988), and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the following review articles and the references cited therein: Vaswani and Hamilton, Ann. Allergy, Asthma and Immunol., 1:105-115 (1998), Harris, Biochem. Soc. Transactions, 23:1035-1038 (1995), Hurle and Gross, Curr. Op. Biotech., 5:428-433 (1994).

[0063] A “human antibody” is one having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human, and / or one prepared using any of the techniques for producing human antibodies disclosed herein. This definition of a human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries. (Hoogenboom and Winter, J.Mol.Biol., 227:381 (1991), Marks et al., J.Mol.Biol., 222:581 (1991)). Methods for preparing human monoclonal antibodies are also available, as described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985), and Boerner et al., J.Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5:368-74 (2001). Human antibodies can be prepared by administering an antigen to transgenic animals, such as immunized xenomous mice, which are modified to produce such antibodies in response to antigen challenge but whose endogenous gene locus is deactivated (see, for example, U.S. Patents 6,075,181 and 6,150,584 relating to XENOMOUSE® technology). For human antibodies produced via human B-cell hybridoma technology, see, for example, Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006).

[0064] For example, antigen-binding moieties described herein that are useful for activating γδT cells, such as antibodies or antigen-binding fragments described herein, are preferably polyvalent. For example, the F(ab')2 fragment has bivalent antigen-binding activity and can crosslink antigens. Similarly, antigen-binding moieties, such as IgG or other canonical antibody architectures, may have a bivalent structure. In some cases, the antigen-binding moiety is greater than bivalent. In some cases, the antigen-binding moiety may be a trivalent moiety, such as a trivalent antibody. In some cases, the antigen-binding moiety may be a tetravalent antibody, such as an IgA antibody. In some cases, the antigen-binding moiety may have a value of 10. For example, the antigen-binding moiety may be an IgM antibody. Preferred polyvalent antigen-binding moieties described herein, such as antibodies or fragments thereof, typically bind to the same antigen, and in some cases to the same epitope of the same antigen, at each antigen-binding site. In some cases, the polyvalent antigen-binding moiety includes at least one antigen-binding site that is different from one of the other antigen-binding sites of the polyvalent antigen-binding moiety.

[0065] As used herein, "Kd" or "Kd value" refers to the dissociation constant measured by surface plasmon resonance assay at 25°C using a CM5 chip immobilized with approximately 10 response units (RUs) of antigen or antibody, for example, using a BIAcore.TM.-2000 or BIAcore.TM.-3000 (BIAcore, Inc., Piscataway, NJ). In the case of bivalent or other polyvalent antibodies, it is common practice to immobilize the antibody to avoid interference with the measurement of the dissociation constant induced by avidity. For further details, see, for example, Chen et al., J.Mol.Biol.293:865-881 (1999).

[0066] As used herein to refer to binding affinity, "greater than or equal to" refers to a stronger bond between a molecule and its binding partner. As used herein, "greater than or equal to" refers to a smaller number K. DThis refers to a stronger binding, expressed by a specific value. For example, an antibody has an affinity for an antigen of "0.6 nM or greater," meaning the antibody's affinity for the antigen is ≤0.6 nM, i.e., 0.59 nM, 0.58 nM, 0.57 nM, or any value less than or equal to 0.6 nM.

[0067] The term "epitope" includes any protein, lipid, or hydrocarbon determinant that can specifically bind to immunoglobulins or T cell receptors. Epitope determinants are usually composed of active surfaces of molecules such as amino acids, lipids, or sugar side chains, and typically possess distinctive three-dimensional structural and charge characteristics. Antibodies have an equilibrium dissociation constant (K). D ) is 10 -6 ~10 -12 If the dissociation constant is within the range of M or higher, it is considered to bind specifically to the antigen. Specific binding is considered to be at least 10 times, preferably 100 times, or more preferably 1,000 times stricter than the dissociation constant for binding to other non-target epitopes (lower K). D This can refer to binding to a target epitope having γδ-TCR and / or αβ-TCR. In some cases, the target epitope is the epitope of the δ1, δ2, or δ3 chain of the delta-3 TCR. In some cases, the non-target epitope is the αβ TCR. In some cases, the non-target epitope is a different subtype of delta chain. The specificity of binding can be determined in the context of binding to the extracellular domain of the γδ-TCR and / or αβ-TCR (e.g., as an Fc fusion immobilized on an ELISA plate or expressed on cells).

[0068] An "activating epitope" can activate a specific population of γδ T cells through binding. T cell proliferation indicates T cell activation and proliferation.

[0069] An antibody is said to bind to the "essentially identical epitope" as a reference antibody when the two antibodies recognize the same or sterically overlapping epitopes. The most widely used rapid method for determining whether two epitopes bind to the same or sterically overlapping epitopes is a competitive assay, which can consist of a wide variety of methods using either a labeled antigen or a labeled antibody. In some embodiments, the antigen is immobilized in a 96-well plate, and the ability of an unlabeled antibody to block the binding of the labeled antibody is measured using radiolabeling or enzymatic labeling. Alternatively, competitive tests using labeled and unlabeled antibodies are performed using flow cytometry on antigen-expressing cells.

[0070] Epitope localization is the process of identifying the antibody's binding site or epitope on the target antigen. Antibody epitopes can be linear epitopes or conformational epitopes. Linear epitopes are formed by a continuous sequence of amino acids in a protein. Conformational epitopes are formed from amino acids that are discontinuous in the protein sequence but come together as the protein folds into its three-dimensional structure.

[0071] As defined herein, “epitope binning” is a process of classifying antibodies based on the epitopes they recognize. More specifically, epitope binning includes methods and systems for distinguishing the epitope recognition properties of different antibodies, combined with computer processes for clustering antibodies based on their epitope recognition properties and for identifying antibodies with distinct binding specificities.

[0072] The “agents” or “compounds” according to the present invention include small molecules, polypeptides, proteins, antibodies, or antibody fragments. In connection with the present invention, small molecules, in one embodiment, mean chemical substances with a molecular weight of less than 1000 daltons, more specifically less than 800 daltons, and more specifically less than 500 daltons. The term “therapeutic agent” refers to a drug having biological activity. The term “anticancer agent” refers to a drug having biological activity against cancer cells.

[0073] As used herein, the term “cell culture” refers to any in vitro culture of cells. This term includes serial cell lines (e.g., those with an immortalized phenotype), primary cell cultures, finite cell lines (e.g., non-transformed cells), and other cell populations maintained in vitro, such as stem cells, blood cells, embryonic umbilical cord blood cells, tumor cells, transduced cells, and the like.

[0074] The term “to treat” or “treatment” refers to both therapeutic treatments and preventive or protective measures aimed at preventing or slowing (reducing) undesirable physiological changes or impairments. Beneficial or desired clinical outcomes include, but are not limited to, symptom reduction, attenuation of disease severity (e.g., reduction of tumor size, tumor burden, or tumor distribution), stabilized (i.e., non-worsening) disease status, delayed or slowed disease progression, improvement or mitigation of disease status, and remission (whether partial or complete), whether detectable or undetectable. “Treatment” may also mean extending survival beyond what would be expected without treatment. Those who require treatment include not only those who already have symptoms or impairments, but also those who are prone to developing symptoms or impairments or those for whom symptoms or impairments should be prevented.

[0075] Administration "in combination" with one or more additional therapeutic agents includes simultaneous (contemporaneous) administration and sequential administration in any order.

[0076] The term “identical,” as used herein, refers to two or more sequences or sub-sequences that are the same. Furthermore, the term “substantially identical,” as used herein, refers to two or more sequences that have a percentage of identical sequence units when compared and aligned to obtain the greatest match across a specified region, measured using a comparison window or comparison algorithm, or by manual alignment and visual inspection. For example, two or more sequences may be “substantially identical” if their sequence units are approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical across a particular region. Such percentages represent the “identity percentage” of the two or more sequences. Sequence identity may exist over regions of at least approximately 75–100 sequence units in length, over regions of approximately 50 sequence units in length, or, where not specified, over the entire sequence. This definition also refers to the complements of test sequences. Furthermore, to give a simple example, two or more polynucleotide sequences are identical if they have the same nucleic acid residues, but they are "substantially identical" if the nucleic acid residues are approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical across a specific region. Identity can exist over a region of at least approximately 75 to 100 nucleic acids, a region of approximately 50 nucleic acids, or, if not specified, over the entire sequence of the polynucleotide sequence.

[0077] Where used herein, the term "pharmaceutically acceptable" means a material, not limited to salts, carriers, or diluents, that does not negate the biological activity or properties of a compound and is relatively non-toxic, i.e., a material that can be administered to an individual without causing undesirable biological effects or harmful interactions with any component contained in the composition.

[0078] Where used herein, the terms “subject” or “patient” refer to a vertebrate. In certain embodiments, a vertebrate is a mammal. Mammals include, but are not limited to, humans, non-human primates, domestic animals (e.g., cattle), sports animals, and pets (e.g., cats, dogs, and horses). In certain embodiments, a mammal is a human.

[0079] The term antigen-presenting cells (APCs) refers to wild-type APCs or manipulated or artificial antigen-presenting cells (aAPCs). APCs can be supplied as an irradiated population of APCs. APCs can be supplied from immobilized cell lines (e.g., K562 or manipulated aAPCs derived from immobilized cell lines) or as a fraction of cells from a donor (e.g., PBMCs).

[0080] As used herein, the terms “structurally different” and “structurally distinct” with respect to a protein or its polypeptide fragment or epitope refer to covalent (i.e., structural) differences between at least two different proteins, their polypeptide fragments, or epitopes. For example, two structurally different proteins (e.g., antibodies) may refer to two proteins having different primary amino acid sequences. In some cases, structurally different activators bind to structurally different epitopes, for example, epitopes having different primary amino acid sequences.

[0081] As used herein, the term “antitumor cytotoxicity” “independent of” specific receptor activity (e.g., NKp30 activity, NKp44 activity, and / or NKp46 activity) refers to antitumor cytotoxicity exerted regardless of whether a specific receptor or a particular combination of receptors is expressed or functional in the cell. Thus, γδT cells exhibiting antitumor cytotoxicity independent of NKp30 activity, NKp44 activity, and / or NKp46 activity may also exhibit NKp30-dependent antitumor cytotoxicity, NKp44-dependent antitumor cytotoxicity, and / or NKp46-dependent antitumor cytotoxicity.

[0082] As used herein, the terms “NKp30 activity-dependent antitumor cytotoxicity,” “NKp44 activity-dependent antitumor cytotoxicity,” and “NKp46 activity-dependent antitumor cytotoxicity” refer to antitumor cytotoxicity that requires the functional expression of a specific receptor. The presence or absence of such receptor-dependent antitumor cytotoxicity can be determined by performing a standard in vitro cytotoxicity assay, such as that performed in Example 48 of PCT / US17 / 32530, in or without an antagonist to the specific receptor. For example, the presence or absence of NKp30 activity-dependent antitumor cytotoxicity can be determined by comparing the results of an in vitro cytotoxicity assay in the presence or absence of an anti-NKp30 antagonist.

[0083] Furthermore, it is understood that γδT cells or populations of γδT cells can be assayed for mRNA expression of one or more cytotoxic receptors NKp30, NKp44, and / or NKp46. In such cases, the expression assay can indicate the presence or absence of receptor-dependent antitumor cytotoxicity. For example, the measured mRNA expression of γδT cells or populations of γδT cells can be compared to a positive control using cells or cell lines that do not exhibit specific receptor-dependent cytotoxicity (e.g., verified by in vitro cytotoxicity assays in the presence and absence of an antagonist).

[0084] A population of γδT cells containing antitumor cytotoxicity in which at least a specific "%" of antitumor cytotoxicity is "unrelated" to specific receptor activity (e.g., NKp30 activity, NKp44 activity, and / or NKp46 activity), as used herein, refers to cells in which blocking a specific receptor does not reduce the measured antitumor cytotoxicity by more than that numerical percentage. Therefore, a population of γδT cells containing antitumor cytotoxicity in which at least 50% of the antitumor cytotoxicity is unrelated to NKp30 activity will show a reduction of 50% or less in vitro antitumor cytotoxicity in the presence of an NKp30 antagonist compared to the absence of an NKp30 antagonist.

[0085] Overview In humans, γδT cells (plural) are a subset of T cells that provide a link between innate and adaptive immune responses. These cells produce antigen-specific γδT cell receptors (γδTCRs) through V-(D)-J segment rearrangement, and γδT cells (plural) can be directly activated by antigen recognition either by γδTCRs or by other non-TCR proteins that act independently or synergistically to activate γδT cell effector function. γδT cells constitute a small fraction of the overall T cell population in mammals, accounting for approximately 1–5% of T cells in peripheral blood and lymphoid organs, and appear to be primarily found in epithelial cell-rich compartments such as the skin, liver, gastrointestinal tract, respiratory tract, and reproductive tract. Unlike αβTCRs, which recognize antigens bound to major histocompatibility complex molecules (MHCs), γδTCRs can directly recognize bacterial antigens, viral antigens, stress antigens expressed on diseased cells, and tumor antigens in the form of complete proteins or non-peptide compounds.

[0086] TS-1, TS8.2, B6, and 15D can selectively activate γδT cells, including specific γδT cell subtypes. See, for example, PCT / US2015 / 061189, whose disclosure is expressly incorporated herein by reference. Without being bound by theory, different levels of activation and proliferation in cultures from different donors may be attributable to the donor's γδ variable TCR repertoire and antibody-binding epitopes. All agents that bind to specific γδT cell subsets can activate specific γδT cells, particularly specific γδT cell populations in enriched cultures to clinically reasonable levels, i.e., >10 8 It was discovered that it is not possible to activate individual target γδT cells. Similarly, not all binding epitopes in a population of γδT cells are activating epitopes, meaning that binding can activate a specific population of γδT cells.

[0087] Specific γδ variable TCR binding regions associated with potent activation of certain γδ T cell subtypes have been previously described in PCT / US2017 / 032530 and PCT / US2018 / 061384 for the in vitro activation and proliferation of specific γδ T cell subtypes. As demonstrated for the first time herein, in vitro activation and proliferation by conjugating soluble polyvalent agents to the identified TCR binding regions can produce highly enriched γδ T cell populations at levels close to those obtained with immobilized agents, resulting in improvements in ease of manufacture, consistency, reproducibility, and cost. Without being bound by theory, the remarkable utility of the soluble polyvalent activators provided herein stems, at least in part, from their ability to appropriately activate T cells when presented in solution. Solution-based activation depends, in part, on the distribution and / or location of the targeted epitope on the targeted protein (in this case, the γ and / or δ chains of the γδ TCR), as well as the ability of the soluble antibody to induce receptor clustering or otherwise activate the receptor. This principle is generally illustrated in Figures 6-8. In some cases, γδT cells grown in vitro can be stored, optionally manipulated, and / or administered to subjects who require them. Manipulation can be performed before or after in vitro growth.

[0088] In some embodiments, the soluble polyvalent agents used in the methods and compositions described herein include at least two or more antigen-binding sites derived from δ1 and / or δ2-specific activators described in PCT / US2017 / 32530. In some embodiments, the activators used in the methods and compositions described herein include at least two or more antigen-binding sites derived from δ3-specific activators described in PCT / US2018 / 061384. PCT / US17 / 32530 and PCT / US18 / 061384 are incorporated in whole by reference for all purposes including all disclosures of γδT cell activators, γδT cell compositions, and methods of γδT cell activation, γδT cell proliferation, treatment, administration, and drug delivery. In some embodiments, the soluble polyvalent agents used in the methods and compositions described herein include at least two or more antigen-binding sites derived from CDRs of antibodies such as TS-1, TS8.2, B6, and 15D.

[0089] Suitable antigen-binding sites for use in soluble polyvalent drugs provided herein can also be advantageously derived from monoclonal antibodies (MAbs) against γδTCR. In some embodiments, the antigen-binding site can bind to different epitopes on the constant or variable regions of δTCR and / or γTCR. In some embodiments, the antigen-binding site can include a CDR from a γδTCR pan-MAb. In some embodiments, the γδTCR pan-MAb can recognize domains shared by different γ and δTCRs on either or both of the γ or δ chain, including δ1, δ2, and δ3 T cell populations. In one embodiment, the antibody-binding site can be derived from a CDR of an antibody such as 5A6.E9 (Thermo Scientific), B1 (Biolegend), IMMU510, and / or 11F2 (11F2) (Beckman Coulter).

[0090] In some embodiments, methods are provided for the direct, selective activation and proliferation of general γδT cells from an isolated mixed cell population, or for the selective activation and proliferation of one or more γδT cell subtypes, without prior depletion of non-target cell types, providing an enriched γδT cell population(s) having clinically reasonable levels of cytotoxic properties. The present invention also provides a method of treatment with a composition comprising the enriched γδT cell population(s) of the present invention.

[0091] The substances described herein include one or more specific subsets of γδT cells at clinically reasonable levels (>10). 8 This is a method for producing or providing manipulated or unmanipulated γδT cells. Using such a method, it is possible to obtain such clinically valid levels from a single donor, including a single sample from a single donor. Also, using such a method, 10 8 It is possible to produce significantly more than 10 manipulated or unmanipulated γδT cells. For example, in some embodiments, about or at least about 10 cells containing one or more specific subsets of γδT cells are produced. 9 , 10 10 , 10 11 , or 10 12 Individual manipulated or unmanipulated γδT cells can be produced by the methods described herein. In some cases, such population sizes can be achieved in as little as 19–30 days and / or with a total volume of culture medium used of less than about 1 L.

[0092] In some embodiments, the present invention provides methods for the proliferation of manipulated or unmanipulated γδT cells. For example, γδT cells can be selectively proliferated in vitro and in vitro by contacting an isolated composite cell sample or isolated mixed cell population with one or more soluble polyvalent agents that selectively proliferate γδT cells or one or more subpopulations thereof, and can be optionally manipulated either before or after proliferation. In some cases, γδT cells are manipulated to stably express one or more tumor-recognizing moieties, and / or γδT cells are manipulated to contain a transgene encoding a secreted cytokine. In some cases, the in vitro proliferated γδT cells, whether manipulated or not, can be administered to a subject that requires them. In some cases, the in vitro proliferated γδT cells, or a portion thereof, are administered to the same subject from which the initial population was isolated. In some cases, the in vitro proliferated γδT cells, or a portion thereof, are administered to different subjects from which the initial population was isolated. In some cases, in vitro proliferated γδT cells can be further proliferated or maintained in vivo by administering one or more drugs that selectively promote the proliferation of γδT cells to the target.

[0093] Isolation of γδT cells In some embodiments, the present invention provides an in vitro method for producing an enriched γδT cell population from an isolated mixed cell population, comprising contacting a mixed cell population with one or more agents that selectively proliferate γδT cells; δ1T cells; δ2T cells; δ3T cells; δ1T cells and δ3T cells; δ1T cells and δ4T cells; or δ1, δ3, δ4, and δ5T cells by binding to epitopes specific to γδTCR; δ1TCR; δ2TCR; δ3TCR; δ1 and δ4TCR; or δ1, δ3, δ4, and δ5T cells, respectively, in order to obtain an enriched γδT cell population. In other embodiments, the present invention provides an in vitro method for producing an enriched γδ1T cell population from an isolated mixed cell population, comprising contacting a mixed cell population with one or more agents that selectively proliferate δ1T cells by binding to epitopes specific to δ1TCR, in order to obtain an enriched γδ2T cell population. In another embodiment, the present invention provides an in vitro method for producing an enriched γδ2T cell population from an isolated mixed cell population, comprising contacting the mixed cell population with one or more agents that selectively proliferate δ2T cells by binding to epitopes specific to δ2TCRs, thereby yielding an enriched γδ2T cell population. In another embodiment, the present invention provides an in vitro method for producing an enriched γδ3T cell population from an isolated mixed cell population, comprising contacting the mixed cell population with one or more agents that selectively proliferate δ3T cells by binding to epitopes specific to δ3TCRs, thereby yielding an enriched γδ3T cell population.

[0094] In other embodiments, the disclosure provides methods for genetically engineering γδT cells isolated from a subject. Methods of enrichment, proliferation, purification by, for example, positive and / or negative selection, or genetic engineering can be carried out individually or in combination, in any order. In one embodiment, γδT cells can be grown in vivo in a subject, isolated from the subject, genetically engineered, then grown in vitro, and optionally administered to the subject. In another embodiment, γδT cells can be isolated from a subject, genetically engineered, optionally activated and grown in vitro, administered to a subject, and then grown or maintained in vivo. In some cases, the subject from which γδT cells are isolated and the subject to which γδT cells are administered are the same subject. In some cases, the subject from which γδT cells are isolated and the subject to which γδT cells are administered are different subjects.

[0095] Manipulated or unmanipulated γδ T cell populations can be grown, for example, directly from a target composite sample. In some cases, the composite sample is isolated and grown in vitro by directly contacting the composite sample with one or more polyvalent agents that selectively grow the target γδ T cell population. In some cases, the composite sample is isolated and then purified by positive or negative selection before in vitro growth is carried out.

[0096] The composite sample may be derived from peripheral blood samples (e.g., PBL or PBMC), leukocyte apheresis samples, umbilical cord blood samples, tumors, stem cell precursors, tumor biopsy materials, tissues, lymph, or epithelial sites of the subject in direct contact with the external environment, or from stem progenitor cells. In some cases, this disclosure refers to Vδ1 + cell, Vδ2 + cells, Vδ3 + cell, Vδ1 + Cells and Vδ3 + cell, Vδ1 + Cells and Vδ4 + cell, Vδ1 + cells, Vδ3 + cells, Vδ4 + Cells and Vδ5 +The present invention provides a method for selectively growing cells, or any combination thereof.

[0097] Peripheral blood mononuclear cells can be collected from the subject using an apheresis machine, including, for example, the Ficoll-Paque® PLUS (GE Healthcare) system or another suitable device / system. From the collected sample, γδT cells(or more), or a desired subpopulation of γδT cells(or more), can be purified, for example, by flow cytometry. Umbilical cord blood cells can also be obtained from umbilical cord blood during the subject's birth. See WO2016 / 081518, incorporated herein by reference, for all purposes, including but not limited to, methods and compositions for PBMC isolation, γδT cell activation, and the manufacture and use of γδT cell activators.

[0098] γδT cells can be grown from an isolated composite sample or mixed cell population cultured in vitro by contacting the mixed cell population with one or more soluble polyvalent agents provided herein that selectively proliferate γδT cells by specifically binding to the epitope of the γδTCR in order to yield a richened γδT cell population, for example, in the first enrichment step. In some embodiments, γδT cells contained in a whole PBMC population can be activated and proliferated without prior depletion of one or more specific cell populations, e.g., one or more of the following non-γδT cell monocytes: αβT cells, B cells, and NK cells, yielding a richened γδT cell population. In some embodiments, the activation and proliferation of γδT cells are carried out without the presence of native or manipulated APCs. In some embodiments, the isolation and proliferation of γδT cells can be carried out using immobilized γδT cell mitogens provided herein that bind to the activating epitope of the γδTCR, e.g., other activators including antibodies and lectins specific to the activating epitope of the γδTCR.

[0099] In certain embodiments, an isolated mixed cell population is optionally purified by positive and / or negative selection and exposed to one or more agents that promote γδT cell proliferation for about or at least about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 17 days, about 19 days, about 21 days, about 25 days, about 29 days, about 30 days, or any range therein. For example, an isolated mixed cell population is exposed to one or more agents that promote γδT cell proliferation for about 1 to about 4 days, about 2 to about 4 days, about 2 to about 5 days, about 3 to about 5 days, about 5 to about 21 days, about 5 to about 19 days, about 5 to about 15 days, about 5 to about 10 days, or about 5 to about 7 days to obtain a first enriched γδT cell population. In another example, an isolated mixed cell population is exposed to one or more agents that promote γδT cell proliferation for approximately 7–21 days, 7–19 days, 7–23 days, or 7–15 days to yield a first enriched γδT cell population.

[0100] In some cases, a purification or isolation step is performed between the first and second growth steps. In some cases, the isolation step includes the removal of one or more activators. In some cases, the isolation step includes the specific isolation of γδT cells or their subtypes. In some cases, one or more (e.g., all) activators (e.g., all activators that are not common components of cell culture media, e.g., serum components and / or IL-2) are removed between the first and second growth steps, but γδT cells are not specifically isolated from other cell types (αβT cells).

[0101] In some embodiments, after activating and proliferating γδT cells using an activator that binds to the activation epitope of the γδTCR in a first enrichment step and optionally a second enrichment step, the first enriched γδT cell population(s) of the present invention may be further enriched or purified using techniques known in the art in second, third, fourth, fifth, etc. enrichment steps to obtain a second or further enriched γδT cell population(s). For example, the first enriched γδT cell population(s) may be depleted of αβT cells, B cells, and NK cells. By positive and / or negative selection of cell surface markers expressed on the collected γδT cells(s), a population of γδT cells or γδT cells(s) expressing similar cell surface markers can be directly isolated from, for example, the first enriched γδT cell population(s). For example, γδT cells can be isolated from an enriched γδT cell population (e.g., after the first and / or second proliferation step) based on the positive or negative expression of markers such as CD2, CD3, CD4, CD8, CD24, CD25, CD44, Kit, TCRα, TCRβ, TCRγ (including one or more TCRγ subtypes), TCRδ (including one or more TCRδ subtypes), NKG2D, CD70, CD27, CD28, CD30, CD16, OX40, CD46, CD161, CCR7, CCR4, NKp30, NKp44, NKp46, DNAM-1, CD242, JAML, and other suitable cell surface markers.

[0102] In some embodiments, after the first growth step (e.g., after the isolation step performed following the first growth step), the grown cells are optionally diluted and cultured in the second growth step. In preferred embodiments, the second growth step is carried out under conditions in which the culture medium is replenished every 1-2 days, 1-3 days, 1-4 days, 1-5 days, 2-5 days, 2-4 days, or 2-3 days during the second growth step. In some embodiments, the second growth step is carried out under conditions in which the cells are diluted or adjusted to a density that supports a further 1x, 2x, 3x, 4x, 5x, 6x or more proliferation of γδT cells. In some cases, the cell density adjustment is carried out concurrently with (i.e., on the same day or simultaneously with) the replenishment of the culture medium. For example, the cell density may be adjusted every 1-2 days, 1-3 days, 1-4 days, 1-5 days, 2-5 days, 2-4 days, or 2-3 days during the second growth step. A typical cell density that supports further proliferation of γδT cells is not limited to this, but for example, about 1 × 10⁶ cells in the culture. 5 , 2×10 5 , 3 x 10 5 , 4×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , 1 x 10 6 , 2×10 6 , 3 x 10 6 , 4×10 6 , 5×10 6 cells / mL, 10×10 6 cells / mL, 15×10 6 cells / mL, 20×10 6 cells / mL, or 30 × 10 6 Cells / mL is one example.

[0103] In some embodiments, the cell density is approximately 0.5 × 10⁻⁶. 6 ~Approx. 1×10 6 cells / mL, approximately 0.5×10 6 ~Approx. 1.5×10 6 cells / mL, approximately 0.5×10 6 ~about 2×10 6cells / mL, about 0.75×10 6 ~ about 1×10 6 cells / mL, about 0.75×10 6 ~ about 1.5×10 6 cells / mL, about 0.75×10 6 ~ about 2×10 6 cells / mL, about 1×10 6 ~ about 2×10 6 cells / mL, or about 1×10 6 ~ about 1.5×10 6 cells / mL, about 1×10 6 ~ about 2×10 6 cells / mL, about 1×10 6 ~ about 3×10 6 cells / mL, about 1×10 6 ~ about 4×10 6 cells / mL, about 1×10 6 ~ about 5×10 6 cells / mL, about 1×10 6 ~ about 10×10 6 cells / mL, about 1×10 6 ~ about 15×10 6 cells / mL, about 1×10 6 ~ about 20×10 6 cells / mL, or about 1×10 6 ~ about 30×10 6 is adjusted to a density of about 1×10

[0104] In some embodiments, the second growth step is performed under conditions that monitor the cells to maintain them at a predetermined cell density (or density range) and / or under conditions that maintain the cells in a culture medium having a predetermined glucose content. For example, the cells are about 0.5×10 6 ~ about 1×10 6 cells / mL, about 0.5×10 6 ~ about 1.5×10 6 cells / mL, about 0.5×10 6 ~ about 2×10 6 cells / mL, about 0.75×10 6 ~ about 1×10 6 cells / mL, about 0.75×10 6 ~ about 1.5×10 6 cells / mL, about 0.75×10 6 ~ about 2×106 cells / mL, approximately 1×10 6 ~about 2×10 6 cells / mL, or approximately 1 × 10⁶ 6 ~Approx. 1.5×10 6 cells / mL, approximately 1×10 6 ~Approx. 3×10 6 cells / mL, approximately 1×10 6 ~Approx. 4×10 6 cells / mL, approximately 1×10 6 ~Approx. 5×10 6 cells / mL, approximately 1×10 6 ~About 10×10 6 cells / mL, approximately 1×10 6 ~Approx. 15×10 6 cells / mL, approximately 1×10 6 ~About 20×10 6 cells / mL, approximately 1×10 6 ~Approx. 30×10 6 It can be maintained at a live cell density of cells / mL.

[0105] In some cases, cells can be maintained at a higher concentration over at least part of the proliferation. For example, cell viability may be enhanced at a higher cell concentration over the first part of the first or second proliferation. Another example is that the culture volume may be utilized most efficiently at a higher cell concentration over the final part of the first or second proliferation. Therefore, in some embodiments, cells may be maintained at approximately 1 × 10⁶ over at least part of or the entirety of the first or second proliferation culture. 6 ~About 20×10 6 It can be maintained at a live cell density of cells / mL.

[0106] For example, cells may be maintained in culture media having a glucose content of approximately 0.5 g / L to 1 g / L, approximately 0.5 g / L to 1.5 g / L, approximately 0.5 g / L to 2 g / L, approximately 0.75 g / L to 1 g / L, approximately 0.75 g / L to 1.5 g / L, approximately 0.75 g / L to 2 g / L, approximately 1 g / L to 1.5 g / L, approximately 1 g / L to 2 g / L, 1 g / L to 3 g / L, or 1 g / L to 4 g / L. In some embodiments, cells may be maintained in culture media having a glucose content of approximately 1.25 g / L. In some cases, for example, when maintaining high cell density culture, cells can be maintained in culture media having glucose content of approximately 1 g / L to 5 g / L, approximately 1 g / L to 4 g / L, approximately 2 g / L to 5 g / L, or approximately 2 g / L to 4 g / L.

[0107] Typically, glucose content is maintained by adding culture medium containing fresh serum or serum-free culture medium to the culture. In some embodiments, cells may be maintained in culture medium having a predetermined glucose content range at a predetermined viable cell density range by, for example, monitoring each parameter and adding fresh medium to maintain the parameters within predetermined limits. In some embodiments, glucose content is maintained by adding culture medium containing fresh serum or serum-free culture medium to the culture while removing used medium while retaining cells inside a perfusion bioreactor. In some embodiments, additional parameters, not limited to pH, partial pressure of O2, O2 saturation, partial pressure of CO2, CO2 saturation, lactate, glutamine, glutamate, ammonium, sodium, potassium, and calcium, are monitored and / or maintained during γδT cell proliferation (e.g., selective γδT cell proliferation) or during the first or second γδT cell proliferation (e.g., selective γδT cell proliferation) step described herein.

[0108] The γδ T cell subtype is, i) By specifically binding to the epitope of δ1TCR, selective proliferation of δ1T cells, ii) selectively expanding δ2 T cells by specifically binding to an epitope of δ2 TCR, or iii) selectively expanding δ1 and δ4 T cells by specifically binding to an epitope of δ1 and δ4 TCR, or iv) selectively expanding δ1, δ3, δ4 and δ5 T cells by specifically binding to an epitope of δ1, δ3, δ4 and δ5 TCR, or v) contacting a mixed cell population with one or more soluble multivalent agents that selectively expand δ3 T cells by specifically binding to an epitope of δ3 TCR, thereby selectively expanding from an isolated composite sample or mixed cell population cultured in vitro, For example, in a first enrichment step, an enriched γδ T cell population can be provided.

[0109] In some cases, the one or more multivalent agents specifically bind to δ1J1, δ1J2, or δ1J3 TCR, or two or all of them. In some embodiments, γδ cells in the total PBMC population can be activated and expanded without prior removal of specific cell populations, such as monocytes, αβ T cells, B cells, and NK cells, resulting in an enriched γδ T cell population. In some aspects, activation and expansion of γδ T cells are performed without the presence of natural or engineered APCs. In some aspects, isolation and expansion of γδ T cells from tumor specimens are specific to antibodies to activation epitopes unique to the δ1 TCR; δ1, δ3, δ4, and δ5 TCR; δ1 and δ4 TCR; δ3 TCR; or δ2 TCR provided herein, and other activators including lectins that bind to activation epitopes unique to the δ1 TCR; δ1, δ3, δ4, and δ5 TCR; δ1 and δ4 TCR; δ3 TCR; or δ2 TCR, using an immobilized γδ T cell mitogen.

[0110] In certain embodiments, an isolated mixed cell population is exposed to one or more polyvalent agents that selectively proliferate δ1, δ1 and δ4, δ2, δ3, δ1 and δ2, or δ1, δ2, and δ3 T cells for about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 days, or any range in between. For example, an isolated mixed cell population is exposed to one or more agents that selectively proliferate δ1 or δ2 T cells for about 1 to 3 days, 1 to 4 days, 1 to 5 days, 2 to 3 days, 2 to 4 days, 2 to 5 days, 3 to 4 days, 3 to 5 days, 4 to 5 days, 5 to 15 days, or 5 to 7 days to obtain a first enriched γδ T cell population. In some embodiments, selectively grown δ1, δ1 and δ3, δ1 and δ4, δ2, δ3, δ1 and δ2, or δ1, δ2 and δ3 T cells are further grown in a second growth step as described herein.

[0111] In certain embodiments, the isolated starting mixed cell population, e.g., peripheral blood sample, contains T lymphocytes in the range of about 20–80%. In certain embodiments, the percentages of residual αβ T cells and NK cells in the enriched γδ T cell population(s) of the present invention are about 2.5% or less and about 1% or less, respectively. The percentages of residual αβ T cells or NK cells in the enriched γδ T cell population(s) of the present invention are about 1% or less, about 0.5% or less, about 0.4% or less, about 0.2% or less, about 0.1% or less, or about 0.01% or less. In certain embodiments, the percentage of residual αβT cells in the enriched γδT cell population(s) of the present invention is about 0.4% or less, about 0.2% or less, about 0.1% or less, or about 0.01% or less (e.g., after a positive selection step of γδT cells or their subtypes, or after removal of αβT cells). In some embodiments, αβT cells are removed before or after the first and / or second γδT cell proliferation, but NK cells are not. In certain embodiments, the isolated mixed cell population is derived from a single donor. In other embodiments, the isolated mixed cell population is derived from more than one donor or a large number of donors (e.g., two, three, four, five, or two to five, two to ten, or five to ten, or more donors).

[0112] Thus, in some embodiments, the method of the present invention is clinically appropriate for a number (10 8 Super, 10 9 Super, 10 10 Super, 10 11 Over or 10 12 Over, or about 10 8 ~about 10 12 ) can be obtained from just one donor. In some cases, the method of the present invention can yield a clinically reasonable number (10) of proliferated γδT cells. 8 Super, 10 9 Super, 10 10 Super, 10 11 Over or 10 12 Over, or about 10 8 ~about 10 12Proliferated γδT cells can be delivered within 19 or 21 days of obtaining the donor sample.

[0113] Following the specific activation and proliferation of a specific γδT cell subset using a soluble polyvalent agent that binds to an activation epitope specific to γδTCR, δ1TCR, δ1 and δ3TCR, δ1 and δ4TCR, δ2TCR, or δ3TCR in the first enrichment step, the first enriched γδT cell population(s) of the present invention may be further enriched or purified in second, third, fourth, fifth, etc., using techniques known in the art, to obtain a second or further enriched γδT cell population(s). For example, the first enriched γδT cell population(s) may be depleted of αβT cells, B cells, and NK cells. By positive and / or negative selection of cell surface markers expressed on the collected γδT cells(s), a population of γδT cells or γδT cells(s) expressing similar cell surface markers can be directly isolated from the first enriched γδT cell population(s). For example, γδ T cells can be isolated from a first enriched γδ T cell population based on the positive or negative expression of markers such as CD2, CD3, CD4, CD8, CD24, CD25, CD44, Kit, TCRα, TCRβ, TCRγ (or one or more subtypes thereof), TCRδ (or one or more subtypes thereof), NKG2D, CD70, CD27, CD28, CD30, CD16, OX40, CD46, CD161, CCR7, CCR4, DNAM-1, JAML, and other appropriate cell surface markers.

[0114] In some embodiments, the enriched γδT cell population after the first γδT cell proliferation, first enrichment step, second γδT cell proliferation and / or second enrichment step of the present invention includes a clinically reasonable level of more than 10⁸ γδT cell subsets in culture volumes of less than 10 mL, less than 25 mL, less than 50 mL, less than 100 mL, less than 150 mL, less than 200 mL, less than 500 mL, less than 750 mL, less than 1 L, less than 2 L, less than 3 L, less than 4 L, less than 5 L, less than 10 L, less than 20 L, or less than 25 L. For example, the method of the present invention is for 10-100 mL, 25-100 mL, 50-100 mL, 75-100 mL, 10-150 mL, 25-150 mL, 50-150 mL, 75-150 mL, 100-150 mL, 10-200 mL, 25-200 mL, 50-200 mL, 75-200 mL, 100-200 mL, 10-250 mL, 25-250 mL, 50-250 mL, 75-2 >10 in growth cultures having a volume of 50 mL, 100-250 mL, 150-250 mL, 5-1,000 mL, 10-1,000 mL, or 100-1,000 mL, 150-1,000 mL, 200-1,000 mL, 250-1,000 mL, 400 mL-1 L, 1 L-2 L, 2 L-5 L, 2 L-10 L, 4 L-10 L, 4 L-15 L, 4 L-20 L, or 4 L-25 L 8 A clinically valid level of γδT cell subsets can be provided. In other embodiments, after the enrichment steps of the second, third, fourth, fifth, etc. of the present invention, the enriched γδT cell population is >10 8 It includes a clinically reasonable level of γδT cell subset.

[0115] In some embodiments, γδT cells can proliferate rapidly in response to contact with one or more antigens. Some γδT cells, such as Vγ9Vδ2+ γδT cells, proliferate rapidly in vitro in response to contact with certain antigens, such as prenyl pyrophosphate, alkylamines, and metabolites or microbial extracts in tissue culture. Furthermore, some wild-type γδT cells, such as Vγ2Vδ2+ γδT cells, proliferate rapidly in human vivo in response to specific types of vaccines. Stimulated γδT cells can exhibit adhesion molecules that facilitate various antigen presentations, co-stimulation, and isolation of γδT cells from composite samples. For example, γδT cells in a composite sample can be stimulated in vitro with at least one antigen for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, approximately 5–15 days, 5–10 days, or 5–7 days, or for another suitable period, either before or after proliferation with a selective γδT cell proliferation agent described herein, such as an antibody or immobilized antibody. Stimulation of γδT cells with a suitable antigen can cause a γδT cell population to proliferate in vitro.

[0116] Non-limiting examples of antigens that may be used to stimulate the proliferation of γδ T cells (or more) from a composite sample in vitro include prenyl pyrophosphate, e.g., isopentenyl pyrophosphate (IPP), alkylamines, metabolites of human microbial pathogens, metabolites of commensal bacteria, methyl-3-butenyl-1-pyrophosphate (2M3B1PP), (E)-4-hydroxy-3-methylbuta-2-enyl pyrophosphate (HMB-PP), ethyl pyrophosphate (EPP), farnesyl pyrophosphate (FPP), dimethylallyl phosphate (DMAP), dimethylallyl pyrophosphate (DMAPP), ethyl adenosine triphosphate (EPPPA), geranyl pyrophosphate (GPP), geranylgeranyl pyrophosphate (GGPP), isopentenyl Examples include yl-adenosine triphosphate (IPPPA), monoethyl phosphate (MEP), monoethyl pyrophosphate (MEPP), 3-formyl-1-butyl-pyrophosphate (TUBAg1), X-pyrophosphate (TUBAg2), 3-formyl-1-butyl-uridine triphosphate (TUBAg3), 3-formyl-1-butyl-deoxythymidine triphosphate (TUBAg4), monoethylalkylamine, allyl pyrophosphate, clotyl pyrophosphate, dimethylallyl-γ-uridine triphosphate, clotyl-γ-uridine triphosphate, allyl-γ-uridine triphosphate, ethylamine, isobutylamine, sec-butylamine, isoamylamine, and nitrogen-containing bisphosphonates (e.g., aminophosphonates).

[0117] Activation and proliferation of γδT cells can be performed using additional and / or alternative activators and costimulators to induce specific γδT cell proliferation and persistent populations. In some embodiments, activation and proliferation of γδT cells from different cultures can result in distinct clonal or mixed polyclonal population subsets. In some embodiments, various agonists can be used to identify agents that produce a specific γδ activation signal. In one embodiment, alternative agents that produce a specific γδ activation signal may be different monoclonal antibodies (MAbs) against the γδTCR.

[0118] In one embodiment, the MAb can bind to different epitopes on the constant or variable regions of the γTCR and / or δTCR. In one embodiment, the MAb may include a γδTCR panMAb. In one embodiment, the γδTCR panMAb may recognize domains common to various γ and δTCRs on either the γ chain, the δ chain, or both chains, including the δ3 cell population. In one embodiment, the antibody may be 5A6.E9 (Thermo Scientific), B1 (Biolegend), IMMU510, and / or 11F2 (11F2) (Beckman Coulter). In one embodiment, the MAb is a specific domain in the variable region of the γ chain (7A5 MAb (Thermo Scientific #TCR1720) that points to a Vγ9 TCR analog), or a domain on the Vδ1 variable region (Mab TS8.2 (Thermo Scientific #TCR1730); MAb TS-1 (ThermoFisher #TCR1055), MAb R9.12 (Beckman Coulter #IM1761)), or a Vδ2 chain (MAb 15D (Thermo Scientific #TCR1732 or Life Technologies #TCR2732), B6 ​​(Biolegend (#331402) It can be directed to one or more of the δ1-# antibodies described in Figures 1-2, one or more of the δ2-# antibodies described in Figures 3-4, or one or more of the δ3-08, δ3-20, δ3-23, δ3-31, δ3-42, δ3-47, and δ3-58 described in Figure 5.

[0119] In some embodiments, antibodies against various domains of the γδTCR (pan-antibodies and antibodies that recognize specific variable region epitopes on subset populations) can be combined and their ability to enhance γδT cell activation can be evaluated. In some embodiments, the γδT cell activator may include a γδTCR conjugate, e.g., MICA, an agonist antibody against NKG2D, a fusion protein of MICA with, for example, an Fc tag, ULBP1, or ULBP3 (R&D Systems Minneapolis, MN), ULBP2, or ULBP6 (Sino Biological Beijing, China). In some embodiments, companion costimulators can be identified that help induce specific γδT cell proliferation without inducing cellular anergy and apoptosis. These costimulators may include ligands for receptors expressed on γδ cells, such as ligands for one or more of the following: NKG2D, CD161, CD70, JAML, DNAX, CD81 accessory molecule-1 (DNAM-1), ICOS, CD27, CD196, CD137, CD30, HVEM, SLAM, CD122, DAP, and CD28. In some embodiments, the costimulator may be specific antibodies against distinctive epitopes on the CD2 and CD3 molecules. CD2 and CD3 may have different conformations when expressed on αβ or γδ T cells, and in some cases, specific antibodies against CD3 and CD2 can result in selective activation of γδ T cells.

[0120] A population of γδT cells(s) may be grown in vitro prior to the manipulation of the γδT cells(s). Non-limiting examples of reagents that can be used to promote the proliferation of γδ T cell populations in vitro include anti-CD3 or anti-CD2, anti-CD27, anti-CD30, anti-CD70, anti-OX40 antibodies, IL-2, IL-4, IL-7, IL-9, IL-12, IL-15, IL-18, IL-19, IL-21, IL-23, IL-33, IFNγ, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), CD70 (CD27 ligand), concavalin A (ConA), pokeweed (PWM), protein peanut agglutinin (PNA), soybean agglutinin (SBA), Les Culinaris agglutinin (LCA), Pisum Sativum agglutinin (PSA), Helix pomatia agglutinin (HPA), and Vicia Examples include graminea lectin (VGA), Phaseolus Vulgaris erythroaglutinin (PHA-E), Phaseolus Vulgaris leucoaglutinin (PHA-L), Sambucus Nigra lectin (SNA, EBL), Maackia Amurensis lectin II (MAL II), Sophora Japonica agglutinin (SJA), Dolichos Biflorus agglutinin (DBA), Lens Culinaris agglutinin (LCA), Wisteria Floribunda lectin (WFA, WFL), or other suitable mitogens that can stimulate T cell proliferation.

[0121] Genetic manipulation of γδT cells may involve stably incorporating constructs expressing tumor recognition regions such as αβTCRs, γδTCRs, antibodies, their antigen-binding fragments, or CARs encoding lymphocyte activation domains into the genome of isolated γδT cells, which may include cytokines (e.g., IL-15, IL-12, IL-2, IL-7, IL-21, IL-18, IL-19, IL-33, IL-4, IL-9, IL-23, or IL-1β) that enhance the proliferation, survival, and function of T cells in vitro and in vivo. In some cases, the cytokine is IL-2, IL-15, IL-12, or IL-21. In some cases, the cytokine is IL-2. In some cases, the cytokine is IL-15. In some cases, the cytokine is IL-4. In some cases, the cytokines are common gamma-chain cytokines selected from the group consisting of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, or combinations thereof. Genetic manipulation of isolated γδT cells may also involve deleting or interfering with the expression of one or more endogenous genes in the genome of isolated γδT cells, e.g., from MHC loci.

[0122] Ex vivo proliferation of γδT cells In other embodiments, the Disclosure provides methods for in vitro and in vitro proliferation of populations of unmanipulated or manipulated γδT cells for adoptive transfer therapy. The unmanipulated or manipulated γδT cells of the Disclosure may be proliferated in vitro. In vitro proliferation can be carried out in mixed cell populations, for example, by directly contacting an isolated sample containing γδT cells with one or more of the soluble polyvalent agents described herein. Additionally or alternatively, in vitro proliferation may be carried out after positive selection for γδT cells or one or more subtypes thereof, and / or negative selection to remove one or more of αβT cells, B cells, or NK cells.

[0123] In some embodiments, the method generally involves growing γδT cells. In some embodiments, the method involves growing various γδT cell subpopulations, for example, Vγ1 + , Vγ2 + , or Vγ3 + This method involves selectively proliferating a subpopulation of γδT cells in vivo. In some cases, the method of the present invention involves Vδ1 + T cell subpopulation; Vδ2 + T cell subpopulation; Vδ3 + T cell subpopulation; Vδ1 + and Vδ3 + T cell population; Vδ1 + and Vδ4 + T cell subpopulation; Vδ1 + and Vδ2 + T cell subpopulation; or Vδ1 + , Vδ3 + , Vδ4 + , and Vδ5 + It can proliferate T cell populations. Therefore, the soluble polyvalent activator of the present invention can specifically activate the growth of one or more types of γδT cells, for example, δ1;δ2;δ3;δ1 and δ3;δ1 and δ4;δ1 and δ5;δ1, δ3, and δ4; or δ1, δ3, δ4, and δ5 cell populations, or combinations thereof.

[0124] In some embodiments, the soluble polyvalent agent activates the growth of γδT cell populations to proliferate them. In some embodiments, the soluble polyvalent agent specifically activates the growth of δ1 cell populations to proliferate δ1T cell populations. In other embodiments, the soluble polyvalent agent specifically activates the growth of δ2 cell populations to proliferate δ2T cell populations. In other embodiments, the soluble polyvalent agent specifically activates the growth of δ3 cell populations to proliferate δ3T cell populations. In other embodiments, the soluble polyvalent agent specifically activates the growth of δ1 and δ3 cell populations to proliferate δ1 and δ3T cell populations. In other embodiments, the soluble polyvalent agent specifically activates the growth of δ1 and δ4 cell populations to proliferate δ1 and δ3T cell populations. In other embodiments, the soluble polyvalent agent specifically activates the growth of δ1 and δ5 cell populations to proliferate δ1 and δ5T cell populations.

[0125] In a preferred embodiment, the soluble polyvalent drug binds to a specific epitope(s) on the cell surface receptor of γδT cells. In some cases, the soluble polyvalent drug includes at least two antigen-binding sites that specifically bind to the same antigen, or the polyvalent drug includes at least two antigen-binding sites that specifically bind to the same epitope of the same antigen. In some cases, the soluble polyvalent drug includes at least three antigen-binding sites that specifically bind to the same antigen, or the polyvalent drug includes at least three antigen-binding sites that specifically bind to the same epitope of the same antigen. In some cases, the soluble polyvalent drug is at least bivalent, trivalent, tetravalent, or pentavalent and is optionally monospecific.

[0126] Suitable antigen-binding sites for use in soluble polyvalent drugs provided herein can be advantageously derived from monoclonal antibodies (MAbs) against γδTCR. In some embodiments, the antigen-binding site can bind to different epitopes on the constant or variable regions of δTCR and / or γTCR. In some embodiments, the antigen-binding site can include a CDR from a γδTCR pan-MAb. In some embodiments, the γδTCR pan-MAb can recognize domains shared by different γ and δTCRs on either or both of the γ or δ chain, including δ1, δ2, and δ3 T cell populations. In one embodiment, the antibody-binding site can be derived from a CDR of an antibody such as 5A6.E9 (Thermo Scientific), B1 (Biolegend), IMMU510, and / or 11F2 (11F2) (Beckman Coulter).

[0127] In some embodiments, the antigen-binding site in the soluble polyvalent drug of the present invention is directed to a specific domain in the variable region of the γ chain (7A5 MAb (Thermo Scientific #TCR1720) directed to the Vγ9 TCR), or a domain on the Vδ1 variable region (Mab TS8.2 (Thermo Scientific #TCR1730); MAb TS-1 (ThermoFisher #TCR1055), MAb R9.12 (Beckman Coulter #IM1761)), or the Vδ2 chain (MAb 15D (Thermo Scientific #TCR1732 or Life Technologies #TCR2732), B6 ​​(Biolegend #331402), one of the δ1-# antibodies described in Figures 1-2, one of the δ2-# antibodies described in Figures 3-4, or one of the δ3-# antibodies described in Figure 5).

[0128] In certain embodiments, the antigen-binding site in the soluble polyvalent drug binds to the same or essentially the same epitope as an antibody selected from the group consisting of 7A5, TS8.2, TS-1, R9.12, 15D, or B6. In certain embodiments, the antigen-binding domain in the soluble polyvalent drug competes with an antibody selected from the group consisting of 7A5, TS8.2, TS-1, R9.12, 15D, or B6. In certain embodiments, the antigen-binding domain in the soluble polyvalent drug contains a CDR of an antibody selected from the group consisting of 7A5, TS8.2, TS-1, R9.12, 15D, or B6.

[0129] In certain embodiments, the antigen-binding site in the soluble polyvalent drug binds to the same or essentially the same epitope as one of the δ1-# antibodies described in Figures 1-2, one of the δ2-# antibodies described in Figures 3-4, or one of the δ3-# antibodies described in Figure 5. In certain embodiments, the antigen-binding domain in the soluble polyvalent drug competes with one of the δ1-# antibodies described in Figures 1-2, one of the δ2-# antibodies described in Figures 3-4, or one of the δ3-# antibodies described in Figure 5. In certain embodiments, the antigen-binding domain in the soluble polyvalent drug contains a CDR of one of the δ1-# antibodies described in Figures 1-2, one of the δ2-# antibodies described in Figures 3-4, or one of the δ3-# antibodies described in Figure 5.

[0130] In some embodiments, the activation and proliferation of the non-engineered or engineered γδ T cells of the present disclosure can be carried out without using an aminophosphonate or a prenyl phosphate. In some embodiments, the activation and proliferation of the non-engineered or engineered γδ T cells of the present disclosure can be carried out, at least in part, by using an aminophosphonate or a prenyl phosphate. For example, activation and / or proliferation can be achieved by contacting an isolated mixed cell population with one or more soluble multivalent agents that selectively expand the non-engineered or engineered γδ T cells of the present disclosure by binding to epitopes specific for δ1, δ2, or δ3 γδ T cells, or combinations thereof, and the method further includes adding an aminophosphonate or a prenyl phosphate to the culture.

[0131] Non-limiting alternative activators and costimulatory molecules include any one or more antibodies that are selective for the δ or γ chains or subtypes thereof described herein, antibodies such as 5A6.E9, B1, TS8.2, 15D, B6, B3, TS-1, γ3.20, 7A5, IMMU510, R9.12, 11F2, or combinations thereof. Other examples of activators and costimulatory molecules include zoledronate, phorbol 12-myristate-13-acetate (TPA), mezerein, staphylococcal enterotoxin A (SEA), streptococcal protein A, or combinations thereof.

[0132] In certain embodiments, in vitro activation and / or proliferation may be further supported by co-culturing or sequentially with cytokines or other stimulants such as IL-2, IL-4, IL-7, IL-9, IL-12, IL-15, IL-18, IL-19, IL-21, IL-23, IL-33, IFNγ, granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte colony-stimulating factor (G-CSF). In some cases, the cytokine is IL-2, IL-15, IL-12, or IL-21. In some cases, the cytokine is IL-2. In some cases, the cytokine is IL-15. In some cases, the cytokine is IL-4. In some cases, the cytokine is not IL-4. In some cases, the cytokine is a general gamma chain cytokine selected from the group consisting of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, or combinations thereof.

[0133] In some embodiments, the method further comprises culturing a population of γδT cells simultaneously or sequentially with cytokines, preferably common gamma chain cytokines. In some embodiments, the cytokines are selected from the group consisting of IL-2, IL-7, IL-9, IL-12, IL-15, IL-18, IL-21, and IL-33, preferably from the group consisting of IL-2, IL-7, IL-15, or IL-21, and even more preferably from the group consisting of IL-2, IL-7, and IL-15. In some embodiments, the culture conditions are IL-4-free, and the cells are not exposed to IL-4 before proliferation.

[0134] The unmanipulated or manipulated γδT cells of this disclosure can be grown in vitro without activation with APC or without co-culture with APC and / or aminophosphonates. Furthermore, or otherwise, the unmanipulated or manipulated γδT cells of this disclosure can be grown in vitro by at least one growth step including activation with APC and / or one or more aminophosphonates or co-culture with them.

[0135] In some embodiments, the unmanipulated or manipulated γδT cells of the Disclosure can be grown in vitro without activation by APC in a first γδT cell proliferation, and then grown in vitro with activation by APC in a second γδT cell proliferation. In some cases, the first γδT cell proliferation involves (a) growing γδT cells, or (b) selectively growing δ1T cells; δ2T cells; δ3T cells; δ1T cells and δ3T cells; δ1T cells and δ4T cells; or δ1, δ3, δ4, and δ5T cells by binding to activation epitopes specific to δ1TCR; δ2TCR; δ3TCR; δ1 and δ4TCR; or δ1, δ3, δ4, and δ5TCR.

[0136] In some cases, secondary γδT cell proliferation is performed in culture media that does not contain one or more of the agents used in primary γδT cell proliferation. In some cases, secondary γδT cell proliferation is performed in culture media that (a) promotes T cell proliferation, (b) promotes γδT cell proliferation, or (c) selectively promotes δ1T cells; δ2T cells; δ3T cells; δ1T cells and δ3T cells; δ1T cells and δ4T cells; or δ1, δ3, δ4, and δ5T cells by binding to activation epitopes specific to δ1TCR; δ2TCR; δ3TCR; δ1 and δ4TCR; or δ1, δ3, δ4, and δ5T cells.

[0137] In some cases, the second agent is different from the agent used for first-stage γδT cell proliferation (e.g., it has a different primary amino acid sequence and / or binds to a structurally different γδTCR epitope). In some cases, the second agent binds to a γδTCR epitope that overlaps with or is the same as the agent used for first-stage γδT cell proliferation, or it can compete with the agent for binding to the γδTCR. In some cases, the second agent is expressed on the cell surface of the APC. In some cases, the second agent is bound to the surface of the APC, for example, by a binding interaction between the constant region of the second agent and an Fc receptor on the surface of the APC. In some cases, the second agent is soluble. In some cases, second-stage γδT cell proliferation is carried out in a culture medium containing a soluble second agent and the APC, and the APC may express or bind to an agent on its cell surface that proliferates or selectively proliferates the γδT cell population.

[0138] In some cases, first γδ T cell proliferation is performed without APC, while second γδ T cell proliferation is performed with APC. In some cases, second γδ T cell proliferation is performed with APC and one or more second agents that either (a) proliferate T cells, (b) proliferate γδ T cells, or (c) selectively proliferate δ1 T cells, δ2 T cells, δ3 T cells, δ1 and δ4 T cells, or δ1, δ3, δ4 and δ5 T cells by binding to activating epitopes specific to each of the δ1, δ3, δ4 and δ5 TCRs.

[0139] Those skilled in the art will understand that in certain embodiments, the method of the second growth step described herein may be carried out as the first growth step, and the method of the first step described herein may be carried out as the second growth step. To give an example without limitation, in some embodiments, a mixed population of cells (e.g., PBMCs) can be grown by contacting them with APC in the first step, and then grown in the absence of APC by contacting the grown population from the first growth step with an immobilized agent that selectively grows δ1 T cells; δ2 T cells; δ1 and δ4 T cells; δ1 T cells and δ3 T cells; δ1 T cells and δ4 T cells; or δ1, δ3, δ4 and δ5 T cells by binding to an activation epitope specific to δ1 TCR; δ2 TCR; δ1 and δ4 TCR; or δ1, δ5 TCR, respectively.

[0140] The method of the present invention relates to various γδT cell populations, for example, Vγ1 + , Vγ2 + , or Vγ3 + The γδT cell population can be proliferated. In some cases, the method of the present invention can be used to proliferate Vδ1 + T cell population; Vδ1 + and Vδ3 + T cell population; Vδ1 + and Vδ4 + T cell population; Vδ1 + and Vδ2 + T cell population; or Vδ1 + , Vδ3 + , Vδ4 + , and Vδ5 + It is possible to increase the T cell population.

[0141] In some cases, a population of γδT cells can be grown in vitro for less than 36 days, less than 35 days, less than 34 days, less than 33 days, less than 32 days, less than 31 days, less than 30 days, less than 29 days, less than 28 days, less than 27 days, less than 26 days, less than 25 days, less than 24 days, less than 23 days, less than 22 days, less than 21 days, less than 20 days, less than 19 days, less than 18 days, less than 17 days, less than 16 days, less than 15 days, less than 14 days, less than 13 days, less than 12 days, less than 11 days, less than 10 days, less than 9 days, less than 8 days, less than 7 days, less than 6 days, less than 5 days, less than 4 days, or less than 3 days.

[0142] In some embodiments, methods are provided for selectively proliferating various γδT cells, including manipulated and unmanipulated γδT cells, by contacting γδT cells from a mixed cell population with a soluble multivalent activator, preferably one that binds to specific epitopes on the cell surface receptors of γδT cells. In some embodiments, the multivalent agent can specifically activate the growth of one or more γδT cell populations, for example, δ1, δ2, δ1 and δ3, or δ1 and δ4 cell populations. In some embodiments, the multivalent agent specifically activates the growth of the δ1 cell population to result in a fermented δ1T cell population. In other embodiments, the multivalent agent specifically activates the growth of the δ2 ​​cell population to result in a fermented δ2T cell population. In other embodiments, the multivalent agent specifically activates the growth of the δ3 cell population to result in a fermented δ3T cell population.

[0143] Polyvalent drugs can stimulate the proliferation of manipulated and unmanipulated γδT cells at rapid growth rates. For example, the drug can induce cell division at intervals of less than 30 hours, less than 29 hours, less than 28 hours, less than 27 hours, less than 26 hours, less than 25 hours, less than 24 hours, less than 23 hours, less than 22 hours, less than 21 hours, less than 20 hours, less than 19 hours, less than 18 hours, less than 17 hours, and less than 16 hours. It stimulates the proliferation of γδT cell populations at an average rate of cell division: one cell division in less than 15 hours, one cell division in less than 14 hours, one cell division in less than 13 hours, one cell division in less than 12 hours, one cell division in less than 11 hours, one cell division in less than 10 hours, one cell division in less than 9 hours, one cell division in less than 8 hours, one cell division in less than 7 hours, one cell division in less than 6 hours, one cell division in less than 5 hours, one cell division in less than 4 hours, one cell division in less than 3 hours, and one cell division in less than 2 hours.

[0144] In some cases, polyvalent drugs divide at an average rate of approximately 1 division every 4 hours, 1 division every 5 hours, 1 division every 6 hours, 1 division every 7 hours, 1 division every 8 hours, 1 division every 9 hours, 1 division every 10 hours, 1 division every 11 hours, 1 division every 12 hours, 1 division every 13 hours, 1 division every 14 hours, 1 division every 15 hours, 1 division every 16 hours, 1 division every 17 hours, 1 division every 18 hours, 1 division every 19 hours, and 1 division every 20 hours. The proliferation of manipulated and unmanipulated γδ T cells can be stimulated at the following average division rates: approximately once every 21 hours, approximately once every 22 hours, approximately once every 23 hours, approximately once every 24 hours, approximately once every 25 hours, approximately once every 26 hours, approximately once every 27 hours, approximately once every 28 hours, approximately once every 29 hours, approximately once every 30 hours, approximately once every 31 hours, approximately once every 32 hours, approximately once every 33 hours, approximately once every 34 hours, approximately once every 35 hours, and approximately once every 36 hours.

[0145] In some cases, polyvalent agents may stimulate the rapid proliferation of manipulated and / or unmanipulated γδT cells in γδT cell proliferation cultures, where the rapid proliferation is one of the aforementioned average rates of cell division and is maintained over approximately 1 to 19 consecutive days, approximately 1 to 14 consecutive days, approximately 1 to 7 consecutive days, approximately 1 to 5 consecutive days, approximately 2 to 19 consecutive days, approximately 2 to 14 consecutive days, approximately 2 to 7 consecutive days, approximately 2 to 5 consecutive days, approximately 4 to 19 consecutive days, approximately 4 to 14 consecutive days, approximately 4 to 7 consecutive days, or approximately 4 to 5 consecutive days.

[0146] In some cases, polyvalent drugs were observed in γδ T cell proliferation cultures maintained for approximately 2 to 7 consecutive days, or approximately 2 to 5 consecutive days, with an average rate of division of approximately 10:1 every 12 hours (e.g., 10-12 hours), approximately 10:1 every 13 hours (e.g., 10-13 hours), approximately 10:1 every 14 hours (e.g., 10-14 hours), approximately 10:1 every 15 hours (e.g., 10-15 hours), approximately 10:1 every 16 hours (e.g., 10-16 hours), approximately 10:1 every 17 hours (e.g., 10-17 hours or 12-17 hours), approximately 10:1 every 18 hours (e.g., 10-18 hours or 12-18 hours), and approximately 19 hours (e.g., For example, the proliferation of manipulated and / or unmanipulated γδ T cells can be stimulated at an average rate of approximately one division every 10-19 hours or 12-19 hours, an average rate of approximately one division every 20 hours (e.g., 12-20 hours, 16-20 hours, or 18-20 hours), an average rate of approximately one division every 21 hours (e.g., 12-21 hours, 16-21 hours, or 18-21 hours), an average rate of approximately one division every 22 hours (e.g., 12-22 hours, 16-22 hours, or 18-22 hours), an average rate of approximately one division every 23 hours or less (e.g., 12-23 hours, 16-23 hours, or 18-23 hours), and an average rate of approximately one division every 24 hours (e.g., 12-24 hours, 16-24 hours, or 18-24 hours).

[0147] In some cases, the polyvalent drug was maintained in γδ T cell proliferation cultures for approximately 2 to 7 consecutive days, or approximately 2 to 5 consecutive days, with an average rate of approximately one division every 25 hours (e.g., 12-25 hours, 16-25 hours, 18-25 hours, or 20-25 hours), an average rate of approximately one division every 26 hours (e.g., 12-26 hours, 16-26 hours, 18-26 hours, or 20-26 hours), and approximately 27 hours (e.g., 12-27 hours, 16-2 The average rate of division is approximately once every 7 hours, 18-27 hours, or 20-27 hours; the rate of division is approximately once every 28 hours (e.g., 12-28 hours, 16-28 hours, 18-28 hours, 20-28 hours, or 22-28 hours); the rate of division is approximately once every 29 hours (e.g., 16-29 hours, 18-29 hours, 20-29 hours, or 22-29 hours); the rate of division is approximately once every 30 hours (e.g., 16-30 hours, 18-30 hours, 20-30 hours, or 22-30 hours) The average rate of division is approximately once every 31 hours (e.g., 16-31 hours, 18-31 hours, 20-31 hours, 22-31 hours, or 24-31 hours), the average rate of division is approximately once every 32 hours (e.g., 18-32 hours, 20-32 hours, 22-32 hours, or 24-32 hours), and the average rate of division is approximately once every 33 hours (e.g., 18-33 hours, 20-33 hours, 22-33 hours, or 24-33 hours). The proliferation of manipulated and / or unmanipulated γδ T cells can be stimulated at a rate of approximately one division every 34 hours (e.g., 18-34 hours, 20-34 hours, 22-34 hours, or 24-34 hours), a rate of approximately one division every 35 hours (e.g., 18-35 hours, 20-35 hours, 22-35 hours, or 24-35 hours), and an average rate of approximately one division every 36 hours (e.g., 18-36 hours, 20-36 hours, 22-36 hours, or 24-36 hours).

[0148] In some cases, polyvalent drugs increased the average rate of cell division approximately every 12 hours (e.g., 10-12 hours), approximately every 13 hours (e.g., 10-13 hours), approximately every 14 hours (e.g., 10-14 hours), approximately every 15 hours (e.g., 10-15 hours), approximately every 16 hours (e.g., 10-16 hours), approximately every 17 hours (e.g., 10-17 hours or 12-17 hours), approximately every 18 hours (e.g., 10-18 hours or 12-18 hours), and approximately every 19 hours (e.g., 10-17 hours) in γδ T cell proliferation cultures maintained for at least 14 consecutive days. The proliferation of manipulated and / or unmanipulated γδ T cells can be stimulated at an average rate of approximately one division every 19 hours or 12-19 hours, an average rate of approximately one division every 20 hours (e.g., 12-20 hours, 16-20 hours, or 18-20 hours), an average rate of approximately one division every 21 hours (e.g., 12-21 hours, 16-21 hours, or 18-21 hours), an average rate of approximately one division every 22 hours (e.g., 12-22 hours, 16-22 hours, or 18-22 hours), an average rate of approximately one division every 23 hours or less (e.g., 12-23 hours, 16-23 hours, or 18-23 hours), and an average rate of approximately one division every 24 hours (e.g., 12-24 hours, 16-24 hours, or 18-24 hours).

[0149] In some cases, the polyvalent drug increased the average rate of cell division approximately once every 25 hours (e.g., 12–25 hours, 16–25 hours, 18–25 hours, or 20–25 hours) in γδ T cell proliferation cultures maintained for at least 14 consecutive days, approximately once every 26 hours (e.g., 12–26 hours, 16–26 hours, 18–26 hours, or 20–26 hours), and approximately once every 27 hours (e.g., 12–27 hours, 16–27 hours, 18–2 The average rate of division is approximately once every 7 hours, or 20-27 hours; the rate of division is approximately once every 28 hours (e.g., 12-28 hours, 16-28 hours, 18-28 hours, 20-28 hours, or 22-28 hours); the rate of division is approximately once every 29 hours (e.g., 16-29 hours, 18-29 hours, 20-29 hours, or 22-29 hours); the rate of division is approximately once every 30 hours (e.g., 16-30 hours, 18-30 hours, 20-30 hours, or 22-30 hours) The average rate of one division approximately every 31 hours (e.g., 16-31 hours, 18-31 hours, 20-31 hours, 22-31 hours, or 24-31 hours), the average rate of one division approximately every 32 hours (e.g., 18-32 hours, 20-32 hours, 22-32 hours, or 24-32 hours), the average rate of one division approximately every 33 hours (e.g., 18-33 hours, 20-33 hours, 22-33 hours, or 24-33 hours), approximately 3 The proliferation of manipulated and / or unmanipulated γδ T cells can be stimulated at a rate of approximately one division every 4 hours (e.g., 18-34 hours, 20-34 hours, 22-34 hours, or 24-34 hours), a rate of approximately one division every 35 hours (e.g., 18-35 hours, 20-35 hours, 22-35 hours, or 24-35 hours), and an average rate of approximately one division every 36 hours (e.g., 18-36 hours, 20-36 hours, 22-36 hours, or 24-36 hours).

[0150] In some cases, polyvalent drugs were observed in γδ T cell proliferation cultures maintained for at least 19 consecutive days, with an average rate of approximately one division every 12 hours (e.g., 10-12 hours), approximately one division every 13 hours (e.g., 10-13 hours), approximately one division every 14 hours (e.g., 10-14 hours), approximately one division every 15 hours (e.g., 10-15 hours), approximately one division every 16 hours (e.g., 10-16 hours), approximately one division every 17 hours (e.g., 10-17 hours or 12-17 hours), approximately one division every 18 hours (e.g., 10-18 hours or 12-18 hours), and approximately one division every 19 hours (e.g., 10- The proliferation of manipulated and / or unmanipulated γδ T cells can be stimulated at an average rate of approximately one division every 19 hours or 12-19 hours, an average rate of approximately one division every 20 hours (e.g., 12-20 hours, 16-20 hours, or 18-20 hours), an average rate of approximately one division every 21 hours (e.g., 12-21 hours, 16-21 hours, or 18-21 hours), an average rate of approximately one division every 22 hours (e.g., 12-22 hours, 16-22 hours, or 18-22 hours), an average rate of approximately one division every 23 hours or less (e.g., 12-23 hours, 16-23 hours, or 18-23 hours), and an average rate of approximately one division every 24 hours (e.g., 12-24 hours, 16-24 hours, or 18-24 hours).

[0151] In some cases, the polyvalent drug increased the average rate of cell division approximately once every 25 hours (e.g., 12–25 hours, 16–25 hours, 18–25 hours, or 20–25 hours) in γδ T cell proliferation cultures maintained for at least 19 consecutive days, approximately once every 26 hours (e.g., 12–26 hours, 16–26 hours, 18–26 hours, or 20–26 hours), and approximately once every 27 hours (e.g., 12–27 hours, 16–27 hours, 18–2 The average rate of division is approximately once every 7 hours, or 20-27 hours; the rate of division is approximately once every 28 hours (e.g., 12-28 hours, 16-28 hours, 18-28 hours, 20-28 hours, or 22-28 hours); the rate of division is approximately once every 29 hours (e.g., 16-29 hours, 18-29 hours, 20-29 hours, or 22-29 hours); the rate of division is approximately once every 30 hours (e.g., 16-30 hours, 18-30 hours, 20-30 hours, or 22-30 hours) The average rate of one division approximately every 31 hours (e.g., 16-31 hours, 18-31 hours, 20-31 hours, 22-31 hours, or 24-31 hours), the average rate of one division approximately every 32 hours (e.g., 18-32 hours, 20-32 hours, 22-32 hours, or 24-32 hours), the average rate of one division approximately every 33 hours (e.g., 18-33 hours, 20-33 hours, 22-33 hours, or 24-33 hours), approximately 3 The proliferation of manipulated and / or unmanipulated γδ T cells can be stimulated at a rate of approximately one division every 4 hours (e.g., 18-34 hours, 20-34 hours, 22-34 hours, or 24-34 hours), a rate of approximately one division every 35 hours (e.g., 18-35 hours, 20-35 hours, 22-35 hours, or 24-35 hours), and an average rate of approximately one division every 36 hours (e.g., 18-36 hours, 20-36 hours, 22-36 hours, or 24-36 hours).

[0152] Polyvalent drugs can stimulate the proliferation of manipulated or unmanipulated γδT cell subpopulations at different growth rates. For example, a drug may stimulate the growth of a δ1 cell population over a period of 1 to 90 days of culture (e.g., approximately 1 to approximately 19, 21, or 23 days) to be greater than that of another γδT cell population, e.g., a δ2 or δ3 population; greater than the initial number of γδT cells before proliferation; greater than the initial number of γδ1T cells before proliferation; or greater than approximately 10 times, 100 times, or 200 times greater than that of an αβT cell population in the culture. It can be stimulated at a faster rate that results in growth of over 300 times, over 400 times, over 500 times, over 600 times, over 700 times, over 800 times, over 900 times, over 1,000 times, over 10,000 times, over 20,000 times, over 30,000 times, over 50,000 times, over 70,000 times, over 100,000 times, or over 1,000,000 times greater.

[0153] In other cases, the drug has shown that the growth of δ1 and δ4 populations over a period of 1 to 90 days of culture (e.g., approximately 1 to 19, 21, or 23 days) is greater than that of the δ2 ​​T cell population; greater than that of another γδ T cell subpopulation; greater than the initial number of γδ T cells before proliferation; greater than the initial number of γδ1 T cells before proliferation; greater than the initial number of γδ1 and γδ3 T cells before proliferation; or greater than that of αβ T cells in the culture. They can be stimulated at a faster rate, resulting in growth that is 10 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, 1,000 times, 10,000 times, 20,000 times, 30,000 times, 50,000 times, 70,000 times, 100,000 times, or 1,000,000 times greater than that of the cell population.

[0154] In other cases, the drug has shown that the growth of δ1 and δ4 populations over a period of 1 to 90 days of culture (e.g., approximately 1 to 19, 21, or 23 days) is greater than that of the δ2 ​​T cell population; greater than that of another γδ T cell subpopulation; greater than the initial number of γδ T cells before proliferation; greater than the initial number of γδ1 T cells before proliferation; greater than the initial number of γδ1 and γδ4 T cells before proliferation; or greater than that of αβ T cells in the culture. They can be stimulated at a faster rate, resulting in growth that is 10 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, 1,000 times, 10,000 times, 20,000 times, 30,000 times, 50,000 times, 70,000 times, 100,000 times, or 1,000,000 times greater than that of the cell population.

[0155] In other cases, the drug promotes the growth of δ1, δ3, δ4, and δ5 populations over a period of 1 to 90 days of culture (e.g., approximately 1 to 19, 21, or 23 days of culture) compared to the δ2 ​​T cell population; compared to another γδ T cell subpopulation; compared to the initial number of γδ T cells before proliferation; compared to the initial number of γδ1 T cells before proliferation; compared to the initial number of γδ1 and γδ3 T cells before proliferation; and compared to the initial number of γδ1, γδ3, γδ4, and γδ5 T cells before proliferation. They can be stimulated at a faster rate that results in proliferation more than 10 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, 1,000 times, 10,000 times, 20,000 times, 30,000 times, 50,000 times, 70,000 times, 100,000 times, or 1,000,000 times greater than the number of cells at time; or more than 10 times, 100,000 times, 20,000 times, 30,000 times, 50,000 times, 70,000 times, 100,000 times, or 1,000,000 times greater than the αβT cell population in the culture.

[0156] In other cases, the drug may stimulate the growth of the δ2 ​​population at a faster rate, resulting in proliferation of more than 10-fold, more than 100-fold, more than 200-fold, more than 300-fold, more than 400-fold, more than 600-fold, more than 700-fold, more than 800-fold, more than 900-fold, more than 1,000-fold, more than 10,000-fold, more than 20,000-fold, more than 30,000-fold, more than 50,000-fold, more than 70,000-fold, more than 100,000-fold, or more than 1,000,000-fold over a culture period of 1 to 90 days (e.g., about 1 to about 19, 21, or 23 days) relative to the δ1 T cell population; the δ3 T cell population; another γδ T cell subpopulation; relative to the number of γδ T cells initiated before proliferation; relative to the number of γδ2 T cells initiated before proliferation; or more than 1,000,000-fold relative to αβ T cells.

[0157] In some embodiments, the disclosure provides manipulated or unmanipulated γδT cell populations that are in contact with a multivalent agent that stimulates the proliferation of the γδT cell population at a rapid rate, e.g., about one cell division every 30 hours, or faster. In some cases, the multivalent agent selectively stimulates the proliferation of any of the δ1;δ2;δ3;δ1 and δ4; or δ1, δ3, δ4, and δ5 T cells. The γδT cell population may consist of some amount of unmanipulated γδT cells and some amount of manipulated γδT cells. In some cases, the γδT cell population may consist of δ1, δ2, δ3, and δ4 T cells in different percentages. A manipulated or unmanipulated γδT cell population may include, for example, less than 90% δ1T cells, less than 80% δ1T cells, less than 70% δ1T cells, less than 60% δ1T cells, less than 50% δ1T cells, less than 40% δ1T cells, less than 30% δ1T cells, less than 20% δ1T cells, less than 10% δ1T cells, or less than 5% δ1T cells. Alternatively, a manipulated or unmanipulated γδT cell population may include more than 5% δ1T cells, more than 10% δ1T cells, more than 20% δ1T cells, more than 30% δ1T cells, more than 40% δ1T cells, more than 50% δ1T cells, more than 60% δ1T cells, more than 70% δ1T cells, more than 80% δ1T cells, or more than 90% δ1T cells. In some cases, the agent is one of the selective proliferative agents described herein. In some cases, the drug is immobilized on a surface such as the cell culture surface or the surface of an APC (for example, expressed on the surface of an APC or bound to an Fc receptor expressed on the surface of an APC).

[0158] A manipulated or unmanipulated γδT cell population may include, for example, less than 90% δ2T cells, less than 80% δ2T cells, less than 70% δ2T cells, less than 60% δ2T cells, less than 50% δ2T cells, less than 40% δ2T cells, less than 30% δ2T cells, less than 20% δ2T cells, less than 10% δ2T cells, or less than 5% δ2T cells. Alternatively, a manipulated or unmanipulated γδT cell population may include more than 5% δ2T cells, more than 10% δ2T cells, more than 20% δ2T cells, more than 30% δ2T cells, more than 40% δ2T cells, more than 50% δ2T cells, more than 60% δ2T cells, more than 70% δ2T cells, more than 80% δ2T cells, or more than 90% δ2T cells.

[0159] Manipulated or unmanipulated γδ T cell populations may include, for example, less than 90% δ1 and δ4 T cells, less than 80% δ1 and δ4 T cells, less than 70% δ1 and δ4 T cells, less than 60% δ1 and δ4 T cells, less than 50% δ1 and δ4 T cells, less than 40% δ1 and δ4 T cells, less than 30% δ1 and δ4 T cells, less than 20% δ1 and δ4 T cells, less than 10% δ1 and δ4 T cells, or less than 5% δ1 and δ4 T cells. Alternatively, a manipulated or unmanipulated γδT cell population may include more than 5% δ1 and δ4 T cells, more than 10% δ1 and δ4 T cells, more than 20% δ1 and δ4 T cells, more than 30% δ1 and δ4 T cells, more than 40% δ1 and δ4 T cells, more than 50% δ1 and δ4 T cells, more than 60% δ1 and δ4 T cells, more than 70% δ1 and δ4 T cells, more than 80% δ1 and δ4 T cells, or more than 90% δ1 and δ4 T cells.

[0160] A manipulated or unmanipulated γδT cell population may include, for example, less than 90% δ4T cells, less than 80% δ4T cells, less than 70% δ4T cells, less than 60% δ4T cells, less than 50% δ4T cells, less than 40% δ4T cells, less than 30% δ4T cells, less than 20% δ4T cells, less than 10% δ4T cells, or less than 5% δ4T cells. Alternatively, a manipulated or unmanipulated γδT cell population may include more than 5% δ1 and δ4 T cells, more than 10% δ1 and δ4 T cells, more than 20% δ1 and δ4 T cells, more than 30% δ1 and δ4 T cells, more than 40% δ1 and δ4 T cells, more than 50% δ1 and δ4 T cells, more than 60% δ1 and δ4 T cells, more than 70% δ1 and δ4 T cells, more than 80% δ1 and δ4 T cells, or more than 90% δ1 and δ4 T cells. Manipulated or unmanipulated γδ T cell populations may include, for example, less than 90% δ1 and δ4 T cells, less than 80% δ1 and δ4 T cells, less than 70% δ1 and δ4 T cells, less than 60% δ1 and δ4 T cells, less than 50% δ1 and δ4 T cells, less than 40% δ1 and δ4 T cells, less than 30% δ1 and δ4 T cells, less than 20% δ1 and δ4 T cells, less than 10% δ1 and δ4 T cells, or less than 5% δ1 and δ4 T cells.

[0161] In certain embodiments, the present invention provides a mixture of proliferated γδT cell populations containing 10-90% δ1 T cells and 90-10% δ2 T cells. In certain embodiments, the present invention provides a mixture of proliferated γδT cell populations containing 10-90% δ1 and δ3 T cells and 90-10% δ2 T cells. In certain embodiments, the present invention provides a mixture of proliferated γδT cell populations containing 10-90% δ1 and δ4 T cells and 90-10% δ2 T cells. In certain embodiments, the present invention provides a mixture of proliferated γδT cell populations containing 10-90% δ1, δ3, δ4 and δ5 T cells and 90-10% δ2 T cells.

[0162] One or more polyvalent agents can come into contact with γδT cells (e.g., activators of the γδT cell innate receptor), after which a costimulatory molecule can come into contact with the γδT cells to provide further stimulation and promote γδT cell proliferation. In some embodiments, the activator and / or costimulator may be lectins derived from plants and non-plants, monoclonal antibodies that activate γδT cells, and other non-lectin / non-antibody agents. In other cases, the plant lectin may be concanavalin A (ConA), but other plant lectins, etc., may be used. Other examples of lectins include peanut protein agglutinin (PNA), soybean agglutinin (SBA), les culinaris agglutinin (LCA), pisum sativum agglutinin (PSA), Helix pomatia agglutinin (HPA), Vicia graminea lectin (VGA), Phaseolus Vulgaris erythroaglutinin (PHA-E), Phaseolus Vulgaris leucoaglutinin (PHA-L), Sambucus Nigra lectin (SNA, EBL), Maackia Amurensis lectin II (MAL II), Sophora Japonica agglutinin (SJA), Dolichos Biflorus agglutinin (DBA), Lens Culinaris agglutinin (LCA), and Wisteria Floribunda lectin (WFA, WFL).

[0163] Non-limiting examples of alternative activators and costimulatory molecules include any one or more antibodies selective for the δ or γ chains or their subtypes described herein, such as antibodies 5A6.E9, B1, TS8.2, 15D, B6, B3, TS-1, γ3.20, 7A5, IMMU510, R9.12, 11F2, or combinations thereof. Other examples of activators and costimulatory molecules include zoledronate, phorbol 12-myristate-13-acetate (TPA), mezelein, Staphylococcus enterotoxin A (SEA), Streptococcus protein A, or combinations thereof.

[0164] In other cases, alternative activators and / or co-stimulants may be antibodies or ligands against αTCR, βTCR, γTCR, δTCR, CD277, CD28, CD46, CD81, CTLA4, ICOS, PD-1, CD30, NKG2D, NKG2A, HVEM, 4-1BB (CD137), OX40 (CD134), CD70, CD80, CD86, DAP, CD122, GITR, FcεRIγ, CD1, CD16, CD161, DNAX, accessory molecule-1 (DNAM-1), one or more NCRs (e.g., NKp30, NKp44, NKp46), SLAM, coxsackievirus and adenovirus receptors, or combinations thereof.

[0165] Manipulated γδT cells Manipulated γδT cells can be generated by various methods known in the art. Manipulated γδT cells can be designed to stably express specific tumor-recognizing regions. Polynucleotides encoding expression cassettes containing tumor-recognizing regions or other types of recognition regions can be stably introduced into γδT cells by transposon / transposase systems or virus-based gene transfer systems, e.g., lentivirus or retrovirus systems, or by other suitable methods, e.g., transfection, electroporation, transduction, lipofection, calcium phosphate (CaPO4), nano-manipulated materials, e.g., Ormosil, adenovirus, retrovirus, lentivirus, adeno-associated virus, and other viral delivery methods, or by other suitable methods. Antigen-specific TCRs, which are either αβ or γδ, can be introduced into manipulated γδT cells by stably inserting polynucleotides containing the genetic code of the antigen-specific TCR into the genome of the γδT cells. Polynucleotides encoding CARs with tumor-recognizing regions can be introduced into manipulated γδT cells by stably inserting polynucleotides into the genome of the γδT cells. In some cases, the engineered tumor recognition portion is an engineered T cell receptor, and the expression cassette incorporated into the genome of engineered γδ T cells contains polynucleotides encoding an engineered TCRα (TCR alpha) gene, an engineered TCRβ (TCR beta) gene, a TCRδ (TCR delta) gene, or an engineered TCRγ (TCR gamma) gene. In some cases, the expression cassette incorporated into the genome of engineered γδ T cells contains polynucleotides encoding an antibody fragment or its antigen-binding portion. In some cases, the antibody fragment or its antigen-binding fragment is a polynucleotide encoding a whole antibody, antibody fragment, single-chain variable fragment (scFv), single-domain antibody (sdAb), Fab, F(ab)2, Fc, light or heavy chain on an antibody, variable or constant region of an antibody, or any combination thereof, which binds to a cell surface tumor antigen as part of a chimeric antigen receptor (CAR) construct, or a bispecific construct containing a CAR and a T cell receptor (TCR) or an antibody that points to a different antigen.In some cases, the polynucleotides are of human origin or originate from another species. Polynucleotides of antibody or antigen-binding fragments derived from non-human species can be modified to improve their similarity to naturally produced antibody variants in humans, and the antibody or antigen-binding fragments can be partially or completely humanized. Polynucleotides of antibody or antigen-binding fragments can also be chimeric, for example, mouse-human antibody chimeras. Manipulated γδ T cells expressing CARs can also be manipulated to express ligands for antigens recognized by the tumor-recognition moiety.

[0166] Using various techniques known in the art, cloned or synthetically engineered nucleic acids containing the genetic code for tumor recognition regions can be introduced to specific locations within the genome of engineered γδT cells. Efficient genomic manipulation in γδT cells can be achieved using techniques of RNA-guided Cas9 nucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and meganucleases from microbial clustered short repeat palindromic sequence (CRISPR) systems, as described in WO201409370, WO2003087341, WO2014134412, and WO2011090804 (each of which is incorporated herein by reference). The techniques described herein can also be used to insert expression cassettes into genomic locations that simultaneously result in the knockout of one gene and the knock-in of another. For example, polynucleotides containing the expression cassettes disclosed herein can be inserted into genomic regions encoding MHC genes. Such operations can simultaneously result in the knock-in of one or more genes, such as a gene contained within an expression cassette, and the knock-out of another gene, such as an MHC locus.

[0167] In one example, a Sleeping Beauty transposon containing a nucleic acid encoding a tumor-recognition portion is introduced into the γδT cells to be manipulated. Alternatively, a mutant Sleeping Beauty transposase, such as the one described in US7,985,739 (which is incorporated herein by reference in its entirety), which provides enhanced integration compared to wild-type Sleeping Beauty, may be used to introduce the polynucleotide into the manipulated γδT cells.

[0168] In some cases, viral methods are used to introduce polynucleotides containing tumor-recognizing regions into the genome of manipulated γδT cells. Many viral methods, such as those described in WO1993020221 (which is incorporated herein by reference in its entirety), have been used for human gene therapy. Non-exclusive examples of viral methods that can be used to manipulate γδT cells include methods using retroviruses, adenoviruses, lentiviruses, herpes simplex viruses, vaccinia viruses, poxviruses, or adenovirus-associated viruses.

[0169] Polynucleotides containing the genetic code for the tumor recognition region may include mutations or other transgenes that affect the growth, proliferation, and activation state of manipulated γδT cells, or tumor cell-specific antigens such as testicular cancer antigens. The γδT cells of this disclosure may be manipulated to express polynucleotides containing an activation domain linked to the antigen recognition region, for example, a molecule in the TCR-CD3 complex, or a costimulatory factor. Manipulated γδT cells may express an intracellular signaling domain, which is a T lymphocyte activation domain. γδT cells may be manipulated to express an intracellular activation domain gene or an intracellular signaling domain. The intracellular signaling domain gene may be, for example, CD3ζ, CD28, CD2, ICOS, JAML, CD27, CD30, OX40, NKG2D, CD4, OX40 / CD134, 4-1BB / CD137, FcεRIγ, IL-2RB / CD122, IL-2RG / CD132, DAP molecule, CD70, cytokine receptor, CD40, or any combination thereof. In some cases, manipulated γδ T cells are further engineered to express cytokines, antigens, cell receptors, or other immunomodulatory molecules.

[0170] The appropriate tumor recognition moiety to be expressed in manipulated γδ T cells can be selected based on the disease to be treated. For example, in some cases, the tumor recognition moiety is the TCR. In other cases, the tumor recognition moiety is a receptor for a ligand expressed on cancer cells. Non-limiting examples of appropriate receptors include NKG2D, NKG2A, NKG2C, NKG2F, LLT1, AICL, CD26, NKRP1, CD244(2B4), DNAM-1, NKp30, NKp44, NKp46, and NKp80. In some cases, the tumor recognition moiety can include a ligand, such as an IL-13 ligand, or a ligand mimetic for a tumor antigen, such as an IL-13 mimetic for IL13R.

[0171] γδT cells can be engineered to express a chimeric tumor recognition moiety comprising a ligand-binding domain derived from NKG2D, NKG2A, NKG2C, NKG2F, LLT1, AICL, CD26, NKRP1, CD244(2B4), DNAM-1, or an antitumor antibody, such as anti-Her2neu or anti-EGFR, and a signaling domain obtained from CD3-ζ, Dap10, Dap12, CD28, 41BB, and CD40L. In some examples, chimeric receptors include MICA, MICB, Her2neu, EGFR, EGFRvIII, mesothelin, CD38, CD20, CD19, BCMA, PSA, RON, CD30, CD22, CD37, CD38, CD56, CD33, CD138, CD123, CD79b, CD70, CD75, CA6, GD2, alpha-fetoprotein (AFP), CS1, carcinoembryonic antigen (CEA), CEACAM5, CA-125, MUC-16, 5T4, NaPi2b, ROR1, ROR2, PLIF, Her2 / Neu, EGFRvIII, GPMNB, LIV-1, glycolipid F77, fibroblast-activating protein (FAP), PSMA, STEAP-1, STEAP-2, c-Met It binds to CSPG4, CD44v6, PVRL-4, VEGFR2, C4.4a, PSCA, folate-binding protein / receptor, SLC44A4, Cripto, CTAG1B, AXL, IL-13Rα2, IL-3R, EPHA3, SLTRK6, gp100, MART1, tyrosinase, SSX2, SSX4, NYESO-1, epithelial tumor antigen (ETA), MAGEA family genes (e.g., MAGEA3, MAGEA4), KKLC1, mutant ras (H, N, K), BRaf, p53, β-catenin, EGFRT790, MHC class I chain-related molecule A (MICA) or MHC class I chain-related molecule B (MICB), or one or more antigens from HPV, CMV, or EBV.

[0172] In some cases, the tumor recognition moiety targets MHC class I molecules (HLA-A, HLA-B, or HLA-C) complexed with tumor-associated peptides. Methods and compositions for generating and using tumor recognition moieties that target tumor-associated peptides complexed with MHC class I molecules are described, for example, in Weidanz et al., Int. Rev. Immunol. 30:328-40, 2011; Scheinberg et al, Oncotarget. 4(5):647-8, 2013; Cheever et al, Clin. Cancer Res. 15(17):5323-37, 2009; Dohan & Reiter Expert Rev Mol Med. 14:e6, 2012; Dao et al., Sci Transl Med. 2013 Mar 13; 5(176):176ra33, US9, 540, 448; and WO2017 / 011804. In some embodiments, the tumor-associated peptides targeted by the peptide MHC complex are peptides of Wilms tumor protein 1 (WT1), human telomerase reverse transcriptase (hTERT), Survivin, mouse double microchromosome 2 homolog (MDM2), cytochrome P450 (CYP1B), KRAS, or BRAF.

[0173] Two or more tumor-recognition moieties may be expressed in manipulated γδT cells from αβTCR polynucleotides stably expressed from manipulated γδT cells, or from genetically distinct αβTCR polynucleotides stably incorporated into manipulated γδT cells, which are genetically distinct, substantially distinct, or substantially identical. In cases of genetically distinct αβTCRs, αβTCRs that recognize different antigens associated with the same symptom may be utilized. In one preferred embodiment, γδT cells are engineered to express different TCRs of human or mouse origin from one or more expression cassettes that recognize the same antigen in association with different MHC haplotypes. In another preferred embodiment, γδT cells are engineered to express one TCR and two or more antibodies that point to the same or different peptides from a given antigen complexed with different MHC haplotypes. In some cases, expression of a single TCR by manipulated γδT cells facilitates appropriate TCR pairing. Engineered γδT cells expressing different TCRs can provide a universal allogeneic engineered γδT cells. In another preferred embodiment, γδT cells are engineered to express one or more different antibodies that target peptide-MHC complexes, each antibody targeting the same or different peptides complexed with the same or different MHC haplotypes. In some cases, the tumor recognition moiety may be an antibody that binds to the peptide-MHC complex.

[0174] γδT cells can be engineered to express TCRs from one or more expression cassettes that recognize the same antigen in association with different MHC haplotypes. In some cases, engineered γδT cells are designed to express a single TCR or a TCR combined with a CAR to minimize the possibility of TCR mispairing within the engineered cells. Tumor recognition moieties expressed from two or more expression cassettes preferably have different polynucleotide sequences and encode tumor recognition moieties that recognize different epitopes of the same target in association with different HLA haplotypes, for example. Engineered γδT cells expressing such different TCRs or CARs can provide universal allogeneic engineered γδT cells.

[0175] In some cases, γδT cells are designed to express one or more tumor-recognition moieties. Two or more tumor-recognition moieties may be expressed from genetically identical or substantially identical antigen-specific chimeric (CAR) polynucleotides engineered in γδT cells. Two or more tumor-recognition moieties may be expressed from genetically distinct CAR polynucleotides engineered in γδT cells. Genetically distinct CARs may be designed to recognize different antigens associated with the same symptom.

[0176] γδT cells may be bispecific. Bispecific manipulated γδT cells may express two or more tumor-recognizing moieties. Bispecific manipulated γδT cells may express both TCR tumor-recognizing moieties and CAR tumor-recognizing moieties. Bispecific manipulated γδT cells may be designed to recognize different antigens associated with the same symptom. Manipulated γδT cells may express two or more CAR / TCR(or more) bispecific polynucleotides that recognize the same or substantially the same antigen. Manipulated γδT cells may express two or more CAR / TCR(or more) bispecific constructs that recognize distinct antigens. In some cases, the bispecific constructs of this disclosure bind to activation and inactivation domains of target cells, thereby providing enhanced target specificity. γδT cells can be engineered to express at least one tumor recognition region, at least two tumor recognition regions, at least three tumor recognition regions, at least four tumor recognition regions, at least five tumor recognition regions, at least six tumor recognition regions, at least seven tumor recognition regions, at least eight tumor recognition regions, at least nine tumor recognition regions, at least ten tumor recognition regions, at least eleven tumor recognition regions, at least twelve tumor recognition regions, or any other appropriate number of tumor recognition regions.

[0177] Appropriate TCR function can be enhanced by two functional zeta proteins containing the ITAM motif. Appropriate TCR function can also be enhanced by αβ or γδ activation domains, such as CD3ζ, CD28, CD2, CTLA4, ICOS, JAML, PD-1, CD27, CD30, 41-BB, OX40, NKG2D, HVEM, CD46, CD4, FcεRIγ, IL-2RB / CD122, IL-2RG / CD132, DAP molecules, and CD70. The expressed polynucleotide may contain the genetic code for the tumor recognition moiety, linker moiety, and activation domain. Translation of the polynucleotide by manipulated γδ T cells may provide the tumor recognition moiety and activation domain linked by a protein linker. Typically, the linker contains amino acids that do not interfere with the folding of the tumor recognition moiety and activation domain. The linker molecule may have a length of at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some cases, at least 50%, 70%, or 90% of the amino acids in the linker are serine or glycine.

[0178] In some cases, the activation domain may contain one or more mutations. Suitable mutations may, for example, be those that constitutively activate the activation domain. Altering the identity of one or more nucleic acids alters the amino acid sequence of the translated amino acids. Nucleic acid mutations can result in altering the encoded amino acids to polar, nonpolar, basic, or acidic amino acids. Nucleic acid mutations can result in optimizing the tumor recognition portion to recognize epitopes from tumors. The manipulated tumor recognition region, the manipulated activation domain, or another manipulated component of the γδT cell may have more than one amino acid mutation, two amino acid mutations, three amino acid mutations, four amino acid mutations, five amino acid mutations, six amino acid mutations, seven amino acid mutations, eight amino acid mutations, nine amino acid mutations, ten amino acid mutations, eleven amino acid mutations, twelve amino acid mutations, thirteen amino acid mutations, fourteen amino acid mutations, fifteen amino acid mutations, sixteen amino acid mutations, seventeen amino acid mutations, eighteen amino acid mutations, nineteen amino acid mutations, twenty amino acid mutations, twenty-one amino acid mutations, twenty-two amino acid mutations, twenty-three amino acid mutations, or twenty-four amino acid mutations. May include natural mutations, 25 amino acid mutations, 26 amino acid mutations, 27 amino acid mutations, 28 amino acid mutations, 29 amino acid mutations, 30 amino acid mutations, 31 amino acid mutations, 32 amino acid mutations, 33 amino acid mutations, 34 amino acid mutations, 35 amino acid mutations, 36 amino acid mutations, 37 amino acid mutations, 38 amino acid mutations, 39 amino acid mutations, 40 amino acid mutations, 41 amino acid mutations, 42 amino acid mutations, 43 amino acid mutations, 44 amino acid mutations, 45 amino acid mutations, 46 amino acid mutations, 47 amino acid mutations, 48 ​​amino acid mutations, 49 amino acid mutations, or 50 amino acid mutations.

[0179] In some cases, the γδT cells of this disclosure do not express one or more MHC molecules. Deletion of one or more MHC loci in manipulated γδT cells can reduce the likelihood of the manipulated γδT cells being recognized by the host immune system. The human major histocompatibility complex (MHC) loci, known as the human leukocyte antigen (HLA) system, constitute a large family of genes expressed in antigen-presenting cells, including γδT cells. HLA-A, HLA-B, and HLA-C molecules function to present intracellular peptides as antigens to antigen-presenting cells. HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR molecules function to present extracellular peptides as antigens to antigen-presenting cells. Several alleles of HLA genes have been associated with GVHD, autoimmune disorders, and cancer. The manipulated γδT cells described herein can be further manipulated to lack or disrupt the gene expression of one or more HLA genes. The manipulated γδT cells described herein can be further manipulated to eliminate or disrupt the gene expression of one or more components of the MHC complex, for example, by completely deleting one or more MHC genes, deleting specific exons, or deleting β2-microglobulin (B2m). Gene excision or disruption of at least one HLA gene can result in clinically therapeutic γδT cells that can be administered to subjects having any HLA haplotype without causing host-versus-graft disease. The manipulated γδT cells described herein can serve as universal donors for human subjects having any HLA haplotype.

[0180] γδT cells can be engineered to lack one or more HLA loci. Engineered γδT cells can be engineered to lack the HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ, or HLA-DP alleles. In some cases, HLA alleles are associated with human conditions, such as autoimmune conditions. For example, the HLA-B27 allele is associated with arthritis and uveitis, the HLA-DR2 allele is associated with systemic lupus erythematosus and multiple sclerosis, the HLA-DR3 allele is associated with 21-hydroxylase deficiency, and the HLA-DR4 allele is associated with rheumatoid arthritis and type 1 diabetes. Engineered γδT cells lacking the HLA-B27 allele, for example, can be administered to a patient with arthritis without being immediately recognized by the target immune system. In some cases, deletion of one or more HLA loci results in manipulated γδT cells, which are universal donors for any subject with any HLA haplotype.

[0181] In some cases, manipulating γδT cells requires the deletion of a portion of the γδT cell genome. In some cases, the genomic deletion includes a portion of an MHC locus. In some cases, the manipulated γδT cells are derived from wild-type human γδT cells, and the MHC locus is an HLA locus. In some cases, the genomic deletion includes a portion of a gene corresponding to a protein in the MHC complex. In some cases, the genomic deletion includes the β2 microglobulin gene. In some cases, the genomic deletion includes immune checkpoint genes, such as PD-1, CTLA-4, LAG3, ICOS, BTLA, KIR, TIM3, A2aR, B7-H3, B7-H4, and CECAM-1. In some cases, the manipulated γδT cells can be designed to express an activating domain that enhances T cell activation and cytotoxicity. Non-limiting examples of activation domains that can be expressed in manipulated γδT cells include CD2, ICOS, 4-1BB (CD137), OX40 (CD134), CD27, CD70, CD80, CD86, DAP molecules, CD122, GITR, and FcεRIγ.

[0182] Any portion of the genome of manipulated γδT cells can be deleted to disrupt the expression of endogenous γδT cell genes. Non-limited examples of genomic regions that can be deleted or disrupted in the γδT cell genome include promoters, activators, enhancers, exons, introns, non-coding RNAs, microRNAs, nuclear small RNAs, variable number tangent repeats (VNTRs), short tangent repeats (STRs), SNP patterns, hypervariable regions, minisatellites, dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats, or simple repeats. In some cases, the genomic deletion region can range from 1 to approximately 10 nucleic acids, 1 to approximately 100 nucleic acids, 1 to approximately 1,000 nucleic acids, 1 to approximately 10,000 nucleic acids, 1 to approximately 100,000 nucleic acids, 1 to approximately 1,000,000 nucleic acids, or other appropriate ranges.

[0183] HLA gene expression in manipulated γδT cells can also be interfered with using various techniques known in the art. In some cases, broad locus gene editing techniques are used to excise genes from the manipulated γδT cell genome or to interfere with the gene expression of at least one HLA locus in manipulated γδT cells. Non-limiting examples of gene editing techniques that can be used to edit desired loci on the genome of manipulated γδT cells include the techniques of regularly spaced clustered short repeat palindromic sequences (CRISPR)-Cas, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and meganucleases, as described in WO201409370, WO2003087341, WO2014134412, and WO2011090804 (each of which is incorporated herein by reference in its entirety), respectively.

[0184] γδT cells may be engineered from isolated, unengineered γδT cells that already express a tumor-recognition region. Engineered γδT cells may retain the tumor-recognition region endogenously expressed in isolated wild-type γδT cells, for example, isolated from tumor-infiltrating lymphocytes in a tumor sample. In some cases, the wild-type γδTCR is replaced by the tumor-recognition region of the engineered γδT cells.

[0185] γδT cells can be engineered to express one or more homing molecules, such as lymphocyte homing molecules. These homing molecules may be, for example, lymphocyte homing receptors or cell adhesion molecules. Homing molecules can help engineered γδT cells migrate and infiltrate solid tumors, including targeted solid tumors, when administered to a target. Non-exclusive examples of homing receptors include members of the CCR family, e.g., CCR2, CCR4, CCR7, CCR8, CCR9, CCR10, CLA, CD44, CD103, CD62L, E-selectin, P-selectin, L-selectin, integrins, e.g., VLA-4 and LFA-1. Non-exclusive examples of cell adhesion molecules include ICAM, N-CAM, VCAM, PE-CAM, L1-CAM, nectin (PVRL1, PVRL2, PVRL3), LFA-1, integrin alpha-X beta-2, alpha-v beta-7, macrophage-1 antigen, CLA-4, and glycoprotein IIb / IIIa. Further examples of cell adhesion molecules include calcium-dependent molecules such as T-cadherin and antibodies against matrix metalloproteinases (MMPs) such as MMP9 or MMP2.

[0186] Steps related to T cell maturation, activation, proliferation, and function can be regulated by costimulatory and inhibitory signals mediated by immune checkpoint proteins. Immune checkpoints are inherent costimulatory and inhibitory elements of the immune system. They help maintain self-tolerance to prevent tissue damage when the immune system responds to disease symptoms, such as cell transformation or infection, and regulate the duration and magnitude of the physiological immune response. The balance between costimulatory and inhibitory signals used to control the immune response from either γδ T cells or αβ T cells can be regulated by immune checkpoint proteins. Immune checkpoint proteins, such as PD1 and CTLA4, are present on the surface of T cells and can be used to switch the immune response "on" or "off." Tumors can dysregulate checkpoint protein function as an immune resistance mechanism, particularly with respect to tumor antigen-specific T cells. The manipulated γδT cells of this disclosure can be further manipulated to lack one or more immune checkpoint loci, such as PD-1, CTLA-4, LAG3, ICOS, BTLA, KIR, TIM3, A2aR, CEACAM1, B7-H3, and B7-H4. Alternatively, the expression of endogenous immune checkpoint genes in the manipulated γδT cells of this disclosure can be inhibited by gene editing techniques.

[0187] Immunological checkpoints may be molecules that modulate repressive signaling pathways (e.g., CTLA4, PD1, and LAG3) or stimulatory signaling pathways (e.g., ICOS) in manipulated γδT cells of this disclosure. Several proteins within the broad immunoglobulin superfamily can serve as ligands for immunological checkpoints. Non-limiting examples of immune checkpoint ligand proteins include B7-H4, ICOSL, PD-L1, PD-L2, MegaCD40L, MegaOX40L, and CD137L. In some cases, the immune checkpoint protein is an antigen expressed in a tumor. In some cases, the immune checkpoint gene is the CTLA-4 gene. In some cases, the immune checkpoint gene is the PD-1 gene.

[0188] PD1 is an inhibitory receptor belonging to the CD28 / CTLA4 family and is expressed in activated T lymphocytes, B cells, monocytes, DCs, and T-regs. Two known ligands for PD1, PD-L1 and PD-L2, exist and are expressed in T cells, APCs, and malignant cells. They function to suppress autoreactive lymphocytes and inhibit the effector function of TAA-specific cytotoxic T lymphocytes (CTLs). Therefore, manipulated γδ T cells lacking PD1 can retain their cytotoxic activity regardless of the expression of PD-L1 and PD-L2 by tumor cells. In some cases, the manipulated γδ T cells in this disclosure lack the locus of the PD-1 gene. In some cases, the expression of the PD-1 gene in manipulated γδ T cells is disrupted by gene editing techniques.

[0189] CTLA4 (cytotoxic T lymphocyte antigen 4) is also known as CD152 (differentiation antigen 152). While CTLA4 shares sequence homology and ligands (CD80 / B7-1 and CD86 / B7-2) with the costimulatory molecule CD28, it differs in that it delivers inhibitory signals to T cells expressing CTLA4 as a receptor. CTLA4 has a much higher overall affinity for both ligands and can outperform CD28 in terms of binding when ligand density is limited. CTLA4 is also associated with CD8 + CTLA4 is frequently expressed on the surface of effector T cells and plays a functional role in the initial activation phase of both naive and memory T cells. During the initial stages of T cell activation, CTLA4 counteracts CD28 activity through increased affinity for CD80 and CD86. Key functions of CTLA4 include downregulation of helper T cells and enhancement of the immunosuppressive activity of regulatory T cells. In some cases, the manipulated γδ T cells in this disclosure lack the CTLA4 gene. In several cases, CTLA4 gene expression in manipulated γδ T cells is disrupted by gene editing techniques.

[0190] LAG3 (Lymphocyte Activation Gene 3) is expressed on activated antigen-specific cytotoxic T cells and can enhance the function of regulatory T cells, independently of CD8 + Effector T cell activity can be suppressed. LAG3 is a CD4-like negative regulatory protein with high binding affinity to MHC class II proteins, and is upregulated in some epithelial cancers, leading to tolerance of T cell proliferation and homeostasis. Mitigation of LAG-3 / class II interactions using LAG-3-IG fusion proteins may enhance the anti-tumor immune response. In some cases, the LAG3 gene locus is absent in the manipulated γδ T cells of this disclosure. In some cases, LAG3 gene expression in manipulated γδ T cells is disrupted by gene editing techniques.

[0191] Phenotypes of unmanipulated and manipulated γδT cells Manipulated γδ T cells can migrate to specific locations within the target body. The migration and homing of manipulated γδ T cells may depend on a combination of the expression and action of specific chemokines and / or adhesion molecules. The homing of manipulated γδ T cells can be controlled by the interaction of chemokines with their receptors. For example, cytokines, including but not limited to CXCR3 (whose ligands are represented by IP-10 / CXCL10 and 6Ckine / SLC / CCL21), CCR4+CXCR5+ (receptors for RANTES, MIP-1α, and MIP-1β), CCR6+, and CCR7, can influence the homing of manipulated γδ T cells. In some cases, manipulated γδ T cells can homing to sites of inflammation and injury, as well as diseased cells, to exert repair functions. In some cases, manipulated γδ T cells can homing to cancer cells. In some cases, manipulated γδ T cells may homing in the thymus, bone marrow, skin, larynx, trachea, pleura, lungs, esophagus, abdomen, stomach, small intestine, large intestine, liver, pancreas, kidneys, urethra, bladder, testes, prostate, vas deferens, ovaries, ureters, mammary glands, parathyroid glands, spleen, or another part of the body of the subject. Manipulated γδ T cells may express one or more homing regions, such as specific TCR alleles and / or lymphocyte homing molecules.

[0192] Manipulated γδT cells may have a specific phenotype, which can be expressed in terms of cell surface marker expression. Various types of γδT cells can be manipulated as described herein. In preferred embodiments, manipulated γδT cells are derived from humans, but they may also be derived from other sources, such as mammalian or synthetic cells.

[0193] The immunophenotype of activated and / or proliferated cell populations can be determined using markers including, but not limited to, CD137, CD27, CD45RA, CD45RO, CCR7, and CD62L (Klebanoff et al., Immunol Rev. 211:214 2006). CD137, or 4-1BB, is an activation-inducible costimulatory molecule and an important regulator of the immune response. Pollok et al., J.Immunol. 150, 771-81 (1993). CD45RA is expressed on naive T lymphocytes and replaced by CD45RO after antigen encounter, but is reexpressed in late effector cells (Michie et al., Nature 360, 264-265 (1992)). CD62L is a cell adhesion molecule that acts as a homing molecule for entering secondary lymphoid tissue and is lost after T cell activation when T cells acquire effector function (Sallusto et al., Nature 401:708 (1999)). CD27 is a co-stimulatory marker that is lost during T cell differentiation (Appay et al., Nat Med. 8:379 (2002), Klebanoff et al., Immunol Rev. 211:214 2006). Additional or alternative activation markers include, but are not limited to, one or more of CD25, PD-1, and CD69.

[0194] antigen The present invention, as disclosed herein, provides manipulated γδT cells expressing an antigen-recognizing moiety, in which case the antigen-recognizing moiety recognizes disease-specific epitopes. The antigen may be a molecule that elicits an immune response. This immune response may involve either antibody production, activation of specific cells with immune responsiveness, or both. The antigen may be, for example, a peptide, protein, hapten, lipid, carbohydrate, bacterium, pathogen, or virus. The antigen may be a tumor antigen. Tumor epitopes may be presented on the surface of tumor cells by MHCI or MHCII complexes. The epitope may be part of an antigen that is expressed on the cell surface and recognized by the tumor-recognizing moiety.

[0195] Non-exclusive examples of antigens recognized by manipulated γδT cells include 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-activating protein (FAP), PSMA, STEAP-1, STEAP-2, mesothelin, c-Met, CSPG4, and PVRL-4. Examples include VEGFR2, PSCA, CLEC12a, L1CAM, GPC2, GPC3, folate-binding protein / receptor, SLC44A4, Cripto, CTAG1B, AXL, IL-13R, IL-3Rα2, SLTRK6, gp100, MART1, tyrosinase, SSX2, SSX4, NYESO-1, WT-1, PRAME, epithelial tumor antigen (ETA), MAGEA family genes (e.g., MAGEA3, MAGEA4), KKLC1, mutant ras, VRaf, p53, MHC class I chain-related molecule A (MICA) or MHC class I chain-related molecule B (MICB), or one or more antigens of HPV, CMV or EBV.

[0196] Antigens can be expressed in the intracellular or extracellular compartment of a cell, and manipulated γδT cells can recognize intracellular or extracellular tumor antigens. In some cases, the αβTCR of manipulated γδT cells recognizes peptides derived from either intracellular or extracellular tumor antigens. For example, the antigen may be a protein produced intracellularly or extracellularly by a virus-infected cell, such as an HIV, EBV, CMV, or HPV protein. The antigen may also be a protein expressed intracellularly or extracellularly in cancer cells.

[0197] The antigen-recognition region can recognize antigens from cells under stress, such as cancer cells or virus-infected cells. For example, human MHC class I chain-related genes (MICA and MICB) are located within the HLA class I region of chromosome 6. MICA and MICB proteins are thought to be markers of "stress" in human epithelium and act as ligands for cells expressing the common natural killer cell receptor (NKG2D). MICA and MICB can be highly expressed as stress markers in cancer cells. Manipulated γδ T cells can recognize MICA or MICB tumor epitopes.

[0198] The tumor recognition portion can be engineered to recognize the antigen at a specific avidity. For example, the tumor recognition portion encoded by the TCR or CAR construct may be at least 10 fM, at least 100 fM, at least 1 picomole (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 nanomol (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 n The antigen can be recognized with a dissociation constant of M, 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 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 μM, at least 2 μM, at least 3 μM, at least 4 μM, at least 5 μM, at least 6 μM, at least 7 μM, at least 8 μM, at least 9 μM, at least 10 μM, at least 20 μM, at least 30 μM, at least 40 μM, at least 50 μM, at least 60 μM, at least 70 μM, at least 80 μM, at least 90 μM, or at least 100 μM.

[0199] In some cases, the tumor recognition portion is ≤10 fM, ≤100 fM, ≤1 picomole (pM), ≤10 pM, ≤20 pM, ≤30 pM, ≤40 pM, ≤50 pM, ≤60 pM, ≤7 pM, ≤80 pM, ≤90 pM, ≤100 pM, ≤200 pM, ≤300 pM, ≤400 pM, ≤500 pM, ≤600 pM, ≤700 pM, ≤800 pM, ≤900 pM, ≤1 nanomoles (nM), ≤2 nM, ≤3 nM, ≤4 nM, ≤5 nM, ≤6 nM, ≤7 nM, ≤8 nM, ≤9 nM, ≤10 nM, ≤20 nM, ≤30 nM, ≤40 n The device can be manipulated to recognize the antigen with dissociation constants of M or less, 50 nM or less, 60 nM or less, 70 nM or less, 80 nM or less, 90 nM or less, 100 nM or less, 200 nM or less, 300 nM or less, 400 nM or less, 500 nM or less, 600 nM or less, 700 nM or less, 800 nM or less, 900 nM or less, 1 μM or less, 2 μM or less, 3 μM or less, 4 μM or less, 5 μM or less, 6 μM or less, 7 μM or less, 8 μM or less, 9 μM or less, 10 μM or less, 20 μM or less, 30 μM or less, 40 μM or less, 50 μM or less, 60 μM or less, 70 μM or less, 80 μM or less, 90 μM or less, or 100 μM or less.

[0200] Treatment method The pharmaceutical compositions described herein, containing unmanipulated enriched gamma-δ T cell populations, manipulated enriched gamma-δ T cell populations, and / or mixtures thereof, may be administered for prophylactic and / or therapeutic purposes. For therapeutic use, a subject already suffering from a disease or condition may be administered an amount of the composition sufficient to cure or at least partially inhibit the symptoms of the disease or condition. Unmanipulated, enriched gamma-δ T cell populations, manipulated, enriched gamma-δ T cell populations, and / or mixtures thereof may also be administered to reduce the likelihood of developing, contracting, or worsening a condition. For therapeutic use, the effective dose of a population of unmanipulated, enriched gamma-δ T cell populations, manipulated, enriched gamma-δ T cell populations, and / or mixtures thereof may vary based on the severity and course of the disease or condition, previous therapies, the subject's health status, weight, and / or response to the drug, and / or the judgment of the treating physician.

[0201] The unmanipulated, enriched gamma-δ T cell populations, manipulated, enriched gamma-δ T cell populations, and / or mixtures thereof of this disclosure can be used to treat subjects requiring treatment of a condition. Examples of conditions include cancer, infectious diseases, autoimmune disorders, and sepsis. Subjects may be humans, non-human primates, e.g., chimpanzees and other apes and monkey species; livestock, e.g., cattle, horses, sheep, goats, pigs; domestic animals, e.g., rabbits, dogs, and cats; laboratory animals, e.g., rodents, e.g., rats, mice, and guinea pigs. Subjects may be of any age. Subjects may be, for example, elderly adults, adults, adolescents, boys, children, young children, and infants.

[0202] A method for treating a condition in a subject (e.g., a disease) with the enriched γδT cell population of the present invention may involve administering a therapeutically effective amount of unmanipulated, enriched γδT cell populations, manipulated, enriched γδT cell populations, and / or mixtures thereof to the subject. The enriched γδT cell populations and / or mixtures thereof of this disclosure may be administered in various regimens (e.g., timing, concentration, dosage, treatment interval, and / or dosage form). The subject may also be pre-treated with, for example, chemotherapy, radiation, or a combination of both, before receiving the enriched γδT cell populations and / or mixtures thereof of this disclosure. As part of the treatment, the unmanipulated, enriched γδT cell populations, manipulated, enriched γδT cell populations, and / or mixtures thereof may be administered to the subject in a first regimen, and the subject may be monitored to determine whether the treatment meets a given level of therapeutic efficacy in the first regimen. In some embodiments, at least one other manipulated γδT cell may be administered to the subject in a second regimen. The second regimen may be the same as the first regimen or different from the first regimen. In some situations, for example, if the administration of manipulated γδ T cells in the first regimen has proven effective, the second regimen may not be performed (e.g., a single dose may be sufficient to treat the condition). Due to their allogeneic and universal donor characteristics, the population of manipulated γδ T cells can be administered to a variety of subjects with different MHC haplotypes. Manipulated γδ T cells may be frozen or cryopreserved before being administered to the subject.

[0203] Enriched populations of γδT cells (i.e., manipulated or unmanipulated) and / or mixtures thereof may be frozen or cryopreserved before administration to a subject and may be further activated, proliferated, and / or maintained in vivo by administration of one or more agents that selectively proliferate the administered γδT cells. In certain embodiments, a population of manipulated enriched γδT cells may comprise two or more cells expressing the same tumor-recognizing moiety, different tumor-recognizing moieties, or a combination of the same tumor-recognizing moiety and different tumor-recognizing moieties.

[0204] For example, a population of manipulated γδ T cells may include several distinct manipulated γδ T cells designed to recognize different antigens, or different epitopes of the same antigen. For instance, human cells affected by melanoma may express the NY-ESO-1 oncogene. Infected cells in humans may process the NY-ESO-1 oncoprotein into smaller fragments, allowing them to present different portions of the NY-ESO-1 protein for antigen recognition. A population of manipulated γδ T cells may include various manipulated γδ T cells expressing different tumor recognition regions designed to recognize different parts of the NY-ESO-1 protein.

[0205] In some embodiments, the present invention provides a method for treating a subject with a population of manipulated γδT cells that recognize different epitopes of the melanoma antigen NY-ESO-1. In a first step, a population of manipulated γδT cells that recognize different epitopes of the same antigen is selected. For example, the population of manipulated γδT cells may include two or more cells expressing different tumor-recognition regions that recognize different parts of the NY-ESO-1 protein. In a second step, the population of manipulated γδT cells may be administered in the first regimen. In the second step, the subject may be monitored, for example, by a healthcare provider (e.g., the treating physician or nurse). In a third step, the subject may be administered one or more agents that selectively proliferate the administered γδT cells in vivo, thereby proliferating and / or maintaining the administered γδT cell population in vivo. In a fourth step, the subject may be monitored to determine the effectiveness of in vivo proliferation and / or maintenance. In some embodiments, the second step is omitted. In some embodiments, the fourth step is omitted.

[0206] One or more compositions of this disclosure may be used to treat a variety of conditions. In some cases, compositions of this disclosure may be used to treat cancer, including solid tumors and hematological malignancies. Non-limiting examples of cancer include acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendiceal cancer, astrocytoma, neuroblastoma, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, brain tumors, e.g., cerebellar astrocytoma, cerebral astrocytoma / gliomas, ependymoma, medulloblastoma, supratentorial primordial neuroectoderm tumor, optic tract hypothalamic glioma, breast cancer, bronchial adenoma, Burkitt lymphoma, carcinoma of unknown primary origin, central nervous system lymphoma, cerebellar astrocytoma, cervical cancer, childhood cancer, and chronic lymphocytic leukemia. Diseases, chronic myeloid leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, fibrinogenic round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, glioma, 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, e.g., non-small cell and small cell lung cancer, lymphoma, leukemia Macroglobulinemia, malignant fibrohistiocytoma / osteosarcoma of bone, medulloblastoma, melanoma, mesothelioma, metastatic squamous neck cancer of unknown primary origin, oral cancer, multiple endocrine neoplasia 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 / malignant fibrohistiocytoma of bone, ovarian cancer, epithelial ovarian cancer, ovarian germ cell tumor, pancreatic cancer, islet cell carcinoma, paranasal and nasal sinus cancer, parathyroid cancer, penile cancer, pharyngeal cancer, chromaffin cell tumor, pineal astrocytoma, pineal These include somatic tumors, pituitary adenomas, pleuroblastomas, plasmacytic neoplasms, primary central nervous system lymphomas, prostate cancer, rectal cancer, renal cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcomas, skin cancer, cutaneous Merkel cell carcinoma, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, T-cell lymphoma, pharyngeal cancer, thymoma, thymic carcinoma, thyroid cancer, trophoblastoma (pregnancy-related), cancer of unknown primary site, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, and Wilms' tumor.

[0207] In some cases, the compositions of this disclosure may be used to treat infectious diseases. Infectious diseases may be caused, for example, by pathogenic bacteria or viruses. Various pathogenic proteins, nucleic acids, lipids or fragments thereof may be expressed in diseased cells. Antigen-presenting cells can internalize such pathogenic molecules, for example, by phagocytosis or receptor-mediated endocytosis, and can present antigen fragments bound to appropriate MHC molecules. For example, various 9-mer fragments of pathogenic proteins may be presented by APCs. Manipulated enriched γδT cells of this disclosure may recognize various antigens and antigen fragments of pathogenic bacteria or viruses. Non-limiting examples of pathogenic bacteria include: a) the genus Bordetella, e.g., Bordetella pertussis; b) the genus Borrelia, e.g., Borrelia burgdorferi; c) the genus Brucelia, e.g., Brucella abortus, Brucella canis, Brucella meliterisis, and / or Brucella suis; d) the genus Campylobacter, e.g., Campylobacter jejuni; e) the genera Chlamydia and Chlamydophila, e.g., Chlamydia pneumonia, Chlamydia trachomatis, and / or Chlamydophila psittaci; f) the genus Clostridium, e.g., Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani; g) the genus Corynebacterium, e.g., Corynebacterium diphtheria species; h) Enterococcus genus, e.g., Enterococcus faecalis and / or Enterococcus faecium species; i) Escherichia genus, e.g., Escherichia coli species; j) Francisella genus, e.g., Francisella tularensis species; k) Haemophilus genus, e.g., Haemophilus influenzae species; l) Helicobacter genus, e.g., Helicobacter pylori species;m) Legionella genus, e.g., Legionella pneumophila; n) Leptospira genus, e.g., Leptospira interrogans; o) Listeria genus, e.g., Listeria monocytogenes; p) Mycobacterium genus, e.g., Mycobacterium leprae, mycobacterium tuberculosis, and / or mycobacterium ulcerans; q) Mycoplasma genus, e.g., Mycoplasma pneumonia; r) Neisseria genus, e.g., Neisseria gonorrhoeae and / or Neisseria meningitidia; s) Pseudomonas genus, e.g., Pseudomonas aeruginosa; t) Rickettsia genus, e.g., Rickettsia rickettsii; u) Salmonella genus, e.g., Salmonella typhi and / or Salmonella typhimurium; v) Shigella genus, e.g., Shigella It can be found in the species sonnei; w) the genus Staphylococcus, e.g., Staphylococcus aureus, Staphylococcus epidermidis, and / or the species Staphylococcus saprophyticus; x) the genus Streptococcus, e.g., Streptococcus agalactiae, Streptococcus pneumonia, and / or the species Streptococcus pyogenes; y) the genus Treponema, e.g., the species Treponema pallidum; z) the genus Vibrio, e.g., Vibrio cholera; and / or aa) the genus Yersinia, e.g., the species Yersinia pestis.

[0208] In some cases, the compositions of this disclosure may be used to treat infectious diseases, which may be caused by viruses. Non-limiting examples of viruses can be found in the following families of viruses, shown with typical species: a) Adenoviridae, e.g., adenovirus species; b) Herpesviridae, e.g., herpes simplex virus type 1, herpes simplex virus type 2, varicella-zoster virus, Epstein-Barr virus, human cytomegalovirus, human herpesvirus type 8 species; c) Papillomaviridae, e.g., human papillomavirus species; d) Polyomaviridae, e.g., BK virus , JC virus species; e) Poxviridae family, e.g. smallpox virus; f) Hepadnaviridae family, e.g. hepatitis B virus species; g) Parvoviridae family, e.g. human bocavirus, parvovirus B19 species; h) Astroviridae family, e.g. human astrovirus species; i) Caliciviridae family, e.g. Norwalk virus species; j) Flaviviridae family, e.g. hepatitis C virus (HCV), yellow fever virus, dengue virus, West Nile virus species; k) Togaviridae family, e.g., rubella virus species; l) Hepeviridae family, e.g., hepatitis E virus species; m) Retroviridae family, e.g., human immunodeficiency virus (HIV) species; n) Orthomyxoviridae family, e.g., influenza virus species; o) Arenaviridae family, e.g., guanalithovirus, Junin virus, lassa virus, Machupovirus, and / or Sabia virus species; p) Bunyaviridae family, e.g., Crimean-Congo hemorrhagic fever virus s) Species of the family Filoviridae, e.g., Ebola virus and / or Marburg virus species; species of the family Paramyxoviridae, e.g., measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, human metapneumovirus, Hendra virus and / or Nipah virus species; r) Genus Rhabdoviridae, e.g., rabies virus species; s) Family Reoviridae, e.g., rotavirus, orbivirus, cortivirus and / or vannavirus species.In some cases, the viruses, such as those causing hepatitis D, are not assigned to the virological family.

[0209] In some cases, the compositions of this disclosure may be used to treat immune diseases, such as autoimmune diseases. Inflammatory diseases, including autoimmune diseases, are also a category of diseases associated with B cell damage. Examples of immune diseases or conditions, including autoimmune states, include rheumatoid arthritis, rheumatic fever, multiple sclerosis, experimental autoimmune encephalomyelitis, psoriasis, uveitis, diabetes mellitus, systemic lupus erythematosus (SLE), lupus nephritis, eczema, scleroderma, polymyositis / scleroderma, polymyositis / dermatomyositis, ulcerative proctitis, ulcerative colitis, severe combined immunodeficiency (SCID), DiGeorge syndrome, ataxia telangiectasia, seasonal allergies, perennial allergies, food allergies, anaphylaxis, mastocytosis, allergic rhinitis, atopic dermatitis, Parkinson's disease, Alzheimer's disease, hypersplenicity, leukocyte adhesion deficiency, X-linked lymphoproliferative disorder, X-linked agammaglobulinemia, selective immunoglobulin A deficiency, hyper-IgM syndrome, HIV, and autoimmune lymphoma. Polygonum thrombocytosis, Wiscott-Aldrich syndrome, chronic granulomatous disease, unclassified immunodeficiency (CVID), hyperimmune globulin E syndrome, Hashimoto's thyroiditis, acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham's chorea, myasthenia gravis, polyglandular syndrome, bullous pemphigoid, Henoch-Schönlein purpura, post-streptococcal nephritis, erythema nodosum, erythema multiforme, gA nephropathy, Takayasu's arteritis, Addison's disease, sarcoidosis, ulcerative colitis, polyarteritis nodosa, ankylosing spondylitis, Goodpasture syndrome, thromboangiitis obliterans, Sjögren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, chronic active hepatitis, polychondritis, pemphigus vulgaris Examples include vulgaris, Wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant cell arteritis / polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis, psoriasis, fibrotic alveolitis, and cancer.

[0210] Treatment with the compositions of this disclosure may be provided to subjects before, during, and after the clinical onset of symptoms. Treatment may be provided to subjects 1 day, 1 week, 6 months, 12 months, or 2 years after the clinical onset of the disease. Treatment may be provided to subjects over a period of 1 day, 1 week, 1 month, 6 months, 12 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or longer after the clinical onset of the disease. Treatment may be provided to subjects less than 1 day, less than 1 week, less than 1 month, less than 6 months, less than 12 months, or less than 2 years after the clinical onset of the disease. Treatment may also include treating humans in clinical trials. Treatment may include administering to subjects a pharmaceutical composition comprising the unmanipulated enriched γδ T cell population, the manipulated enriched γδ T cell population, and / or mixtures thereof of this disclosure. In some cases, the pharmaceutical composition comprises one or more agents of the Disclosure that selectively grow the γδT cell population and the unmanipulated enriched γδT cell population, the manipulated enriched γδT cell population, and / or mixtures thereof.

[0211] In some cases, administration of the compositions of this disclosure to subjects modulates the activity of endogenous lymphocytes in the subjects' bodies. In some cases, administration of the compositions of this disclosure to subjects may provide antigens to endogenous T cells and enhance the immune response. In some cases, memory T cells are CD4 + These are T cells. In some cases, memory T cells are CD8 +These are T cells. In some cases, administration to subjects of the compositions of this disclosure activates the cytotoxicity of another immune cell. In some cases, the other immune cell is a CD8+ T cell. In some cases, the other immune cell is a natural killer T cell. In some cases, administration to subjects of the compositions suppresses regulatory T cells. In some cases, the regulatory T cells are Fox3+ Treg cells. In some cases, the regulatory T cells are Fox3-Treg cells. Non-limiting examples of cells whose activity can be regulated by the γδ T cell population include hematopoietic stem cells, B cells, CD4, CD8, erythrocytes, white blood cells, dendritic cells, e.g., dendritic antigen-presenting cells, leukocytes, macrophages, memory B cells, memory T cells, monocytes, natural killer cells, neutrophils, T helper cells, and T killer cells.

[0212] During most bone marrow transplants, a combination of cyclophosphamide and total body irradiation is conventionally employed to prevent rejection of hematopoietic stem cells (HSCs) in the graft by the recipient's immune system. In some cases, in vitro incubation of donor bone marrow with interleukin-2 (IL-2) is performed to enhance killer lymphocyte production in the bone marrow. Interleukin-2 (IL-2) is a cytokine required for the growth, proliferation, and differentiation of wild-type lymphocytes. Current research on adoptive transfer of γδT cells into humans may require co-administration of γδT cells and interleukin-2. However, both low and high doses of IL-2 can have toxic side effects. IL-2 toxicity can manifest in numerous organs / systems, most notably the heart, lungs, kidneys, and central nervous system. In some cases, this disclosure provides methods for administering unmanipulated enriched gamma-δ T cell populations, manipulated enriched gamma-δ T cell populations, and / or mixtures thereof to subjects without co-administration of cytokines, such as IL-2, IL-15, IL-12, or IL-21. In some cases, unmanipulated enriched gamma-δ T cell populations, manipulated enriched gamma-δ T cell populations, and / or mixtures thereof can be administered to subjects without co-administration of IL-2. In some cases, unmanipulated enriched gamma-δ T cell populations, manipulated enriched gamma-δ T cell populations, and / or mixtures thereof are administered to subjects without co-administration of IL-2 during procedures such as bone marrow transplantation.

[0213] In some cases, the present disclosure provides a method for administering unmanipulated enriched γδT cell populations, manipulated enriched γδT cell populations, and / or mixtures thereof to a target with simultaneous or sequential co-administration of cytokines or other stimulants, such as IL-2, IL-4, IL-7, IL-9, IL-12, IL-15, IL-18, IL-19, IL-21, IL-23, IL-33, IFNγ, granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte colony-stimulating factor (G-CSF). In some cases, the cytokine is IL-2, IL-15, IL-12, or IL-21. In some cases, the cytokine is IL-2. In some cases, the cytokine is IL-15. In some cases, the cytokine is IL-4. In some cases, the cytokines are common gamma-chain cytokines selected from the group consisting of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, or combinations thereof.

[0214] Method of administration The compositions of the present invention, comprising unmanipulated enriched γδT cell populations, manipulated enriched γδT cell populations, and / or mixtures thereof, can be administered to a subject in any order or simultaneously. If administered simultaneously, the compositions can be provided in a single, integrated form such as intravenous injection, or in a multi-dose form, for example, as multiple intravenous infusions. The compositions can be packaged together or separately in a single package or multiple packages. One or all of the compositions of the present invention can be given in multi-dose doses. If not administered simultaneously, the timing between multi-dose doses can vary to lengths of about one week, one month, two months, three months, four months, five months, six months, or even about one year. In some cases, the administered γδT cell populations, manipulated enriched γδT cell populations, and / or mixtures thereof can proliferate in vivo, within the subject's body, after administration to the subject. Pharmaceutical compositions comprising γδT cells and / or polyvalent agents can be packaged as kits. The kits may include one or more of the compositions described herein, in addition to instructions for use of the compositions (e.g., instructions).

[0215] In some cases, a method of treating cancer comprises administering a composition described herein, thereby treating the cancer. In some embodiments, a therapeutically effective amount of the composition is administered over a period of at least about 10 seconds, 30 seconds, 1 minute, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or 1 year.

[0216] One or more compositions described herein may be administered before, during, or after the onset of a disease or symptom, and the timing of administration of the pharmaceutical compositions may vary. For example, one or more compositions may be used as a prophylactic agent and may be continuously administered to subjects prone to a condition or disease in order to reduce the likelihood of the onset of the disease or condition. One or more compositions may be administered to a subject during or as soon as possible after the onset of symptoms. Administration of one or more compositions may be started promptly at the time of symptom onset, within the first three hours after symptom onset, within the first six hours after symptom onset, within the first 24 hours after symptom onset, within 48 hours after symptom onset, or at any time after symptom onset. The initial dose may be administered via any practical route, for example, by any route described herein using any formulation described herein. In some examples, administration of one or more compositions of this disclosure is intravenous. A single or multiple dose of one or more compositions may be administered as soon as possible after the onset of cancer, infectious disease, immune disorder, or sepsis, or in conjunction with a bone marrow transplant, and over a period of time necessary for the treatment of the immune disorder, for example, over a period of about 24 to 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. For the treatment of cancer, a single or multiple dose of one or more compositions may be administered several years after the onset of cancer, and before or after other treatments. In some cases, one or more compositions described herein may be administered 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 length of treatment may vary for each subject.

[0217] Dosage The unmanipulated enriched γδ T cell populations, manipulated enriched γδ T cell populations, and / or mixtures thereof disclosed herein may be formulated as unit dosage forms suitable for single-dose administration of precise dosages. In some cases, the unit dosage forms contain further lymphocytes. In unit dosage forms, the formulation is divided into unit doses containing appropriate amounts of one or more compounds. Unit doses may be in the form of packages containing individual amounts of the formulation. Non-limiting examples include packaged tablets or capsules, and powders in vials or ampoules. Aqueous suspension compositions may be placed in single-dose, non-resealable containers. Multi-dose, resealable containers may be used, for example, with or without preservatives. In some cases, the pharmaceutical compositions do not contain preservatives. Parenteral injection formulations may be provided in unit dosage forms, e.g., ampoules, or in multi-dose containers with preservatives.

[0218] The unmanipulated enriched γδT cell populations, manipulated enriched γδT cell populations, and / or mixtures thereof described herein contain at least 5 cells, at least 10 cells, at least 20 cells, at least 30 cells, at least 40 cells, at least 50 cells, at least 60 cells, at least 70 cells, at least 80 cells, at least 90 cells, at least 100 cells, at least 200 cells, at least 300 cells, at least 400 cells, at least 500 cells, at least 600 cells, at least 700 cells, at least 800 cells, at least 900 cells, and at least 1 × 10⁶ cells. 3 Cells, at least 2 × 10 3 Cells, at least 3 × 10 3 Cells, at least 4 × 10 3 Cells, at least 5 × 10 3 Cells, at least 6 × 10 3 Cells, at least 7 × 10 3 Cells, at least 8 × 10 3 Cells, at least 9 × 10 3 Cells, at least 1 × 10 4 Cells, at least 2 × 10 4 Cells, at least 3 × 104 Cells, at least 4 × 10 4 Cells, at least 5 × 10 4 Cells, at least 6 × 10 4 Cells, at least 7 × 10 4 Cells, at least 8 × 10 4 Cells, at least 9 × 10 4 Cells, at least 1 × 10 5 Cells, at least 2 × 10 5 Cells, at least 3 × 10 5 Cells, at least 4 × 10 5 Cells, at least 5 × 10 5 Cells, at least 6 × 10 5 Cells, at least 7 × 10 5 Cells, at least 8 × 10 5 Cells, at least 9 × 10 5 Cells, at least 1 × 10 6 Cells, at least 2 × 10 6 Cells, at least 3 × 10 6 Cells, at least 4 × 10 6 Cells, at least 5 × 10 6 Cells, at least 6 × 10 6 Cells, at least 7 × 10 6 Cells, at least 8 × 10 6 Cells, at least 9 × 10 6 Cells, at least 1 × 10 7 Cells, at least 2 × 10 7 Cells, at least 3 × 10 7 Cells, at least 4 × 10 7 Cells, at least 5 × 10 7 Cells, at least 6 × 10 7 Cells, at least 7 × 10 7 Cells, at least 8 × 10 7 Cells, at least 9 × 10 7 Cells, at least 1 × 10 8 Cells, at least 2 × 10 8 Cells, at least 3 × 10 8 Cells, at least 4 × 10 8 Cells, at least 5 × 10 8 Cells, at least 6 × 10 8 Cells, at least 7 × 108 Cells, at least 8 × 10 8 Cells, at least 9 × 10 8 Cells, at least 1 × 10 9 Cells, or even larger amounts, may be present in the composition.

[0219] The therapeutically effective doses of the unmanipulated enriched γδT cell population, the manipulated enriched γδT cell population, and / or mixtures thereof of the present invention are approximately 1 to 10 cells, approximately 1 to 100 cells, approximately 1 to 10 cells, approximately 1 to 20 cells, approximately 1 to 30 cells, approximately 1 to 40 cells, approximately 1 to 50 cells, approximately 1 to 60 cells, approximately 1 to 70 cells, approximately 1 to 80 cells, approximately 1 to 90 cells, approximately 1 to 100 cells, and approximately 1 to 1 × 10 3 Cells, about 1 cell ~ about 2 x 10 3 Cells, about 1 cell ~ about 3 x 10 3 Cells, about 1 cell ~ about 4 x 10 3 Cells, about 1 cell ~ about 5 x 10 3 Cells, about 1 cell ~ about 6 x 10 3 Cells, about 1 cell ~ about 7 x 10 3 Cells, about 1 cell ~ about 8 x 10 3 Cells, about 1 cell ~ about 9 x 10 3 Cells, about 1 cell ~ about 1 x 10 4 Cells, about 1 cell ~ about 2 x 10 4 Cells, about 1 cell ~ about 3 x 10 4 Cells, about 1 cell ~ about 4 x 10 4 Cells, about 1 cell ~ about 5 x 10 4 Cells, about 1 cell ~ about 6 x 10 4 Cells, about 1 cell ~ about 7 x 10 4 Cells, about 1 cell ~ about 8 x 10 4 Cells, about 1 cell ~ about 9 x 10 4 Cells, about 1 cell ~ about 1 x 10 5 Cells, about 1 cell ~ about 2 x 10 5 Cells, about 1 cell ~ about 3 x 10 5 Cells, about 1 cell ~ about 4 x 10 5 Cells, about 1 cell ~ about 5 x 10 5 Cells, about 1 cell ~ about 6 x 10 5 Cells, about 1 cell ~ about 7 x 10 5 Cells, about 1 cell ~ about 8 x 105 Cells, about 1 cell ~ about 9 x 10 5 Cells, about 1 cell ~ about 1 x 10 6 Cells, about 1 cell ~ about 2 x 10 6 Cells, about 1 cell ~ about 3 x 10 6 Cells, about 1 cell ~ about 4 x 10 6 Cells, about 1 cell ~ about 5 x 10 6 Cells, about 1 cell ~ about 6 x 10 6 Cells, about 1 cell ~ about 7 x 10 6 Cells, about 1 cell ~ about 8 x 10 6 Cells, about 1 cell ~ about 9 x 10 6 Cells, about 1 cell ~ about 1 x 10 7 Cells, about 1 cell ~ about 2 x 10 7 Cells, about 1 cell ~ about 3 x 10 7 Cells, about 1 cell ~ about 4 x 10 7 Cells, about 1 cell ~ about 5 x 10 7 Cells, about 1 cell ~ about 6 x 10 7 Cells, about 1 cell ~ about 7 x 10 7 Cells, about 1 cell ~ about 8 x 10 7 Cells, about 1 cell ~ about 9 x 10 7 Cells, about 1 cell ~ about 1 x 10 8 Cells, about 1 cell ~ about 2 x 10 8 Cells, about 1 cell ~ about 3 x 10 8 Cells, about 1 cell ~ about 4 x 10 8 Cells, about 1 cell ~ about 5 x 10 8 Cells, about 1 cell ~ about 6 x 10 8 Cells, about 1 cell ~ about 7 x 10 8 Cells, about 1 cell ~ about 8 x 10 8 Cells, about 1 cell ~ about 9 x 10 8 Cells, or approximately 1 cell to approximately 1 × 10⁻⁶ cells 9 It could be a cell.

[0220] In some cases, the therapeutically effective dose of the unmanipulated enriched γδT cell population, the manipulated enriched γδT cell population, and / or mixture thereof of the present invention is approximately 1 × 10⁻⁶ 3 cells ~ approx. 2 x 10 3 cells, approximately 1 x 10 3 cells ~ approx. 3 x 10 3 cells, approximately 1 x 10 3 cells ~ approx. 4 x 10 3Cells, approximately 1 × 10 3 Cells ~ approximately 5 × 10 3 Cells, approximately 1 × 10 3 Cells ~ Approximately 6 × 10 3 Cells, approximately 1 × 10 3 Cells ~ Approximately 7 × 10 3 Cells, approximately 1 × 10 3 Cells ~ Approximately 8 × 10 3 Cells, approximately 1 × 10 3 Cells ~ approximately 9 × 10 3 Cells, approximately 1 × 10 3 Cells ~ Approximately 1×10 4 Cells, approximately 1 × 10 3 Cells ~ approximately 2 × 10 4 Cells, approximately 1 × 10 3 Cells ~ Approximately 3 × 10 4 Cells, approximately 1 × 10 3 Cells ~ approximately 4 × 10 4 Cells, approximately 1 × 10 3 Cells ~ approximately 5 × 10 4 Cells, approximately 1 × 10 3 Cells ~ Approximately 6 × 10 4 Cells, approximately 1 × 10 3 Cells ~ Approximately 7 × 10 4 Cells, approximately 1 × 10 3 Cells ~ Approximately 8 × 10 4 Cells, approximately 1 × 10 3 Cells ~ approximately 9 × 10 4 Cells, approximately 1 × 10 3 Cells ~ Approximately 1×10 5 Cells, approximately 1 × 10 3 Cells ~ approximately 2 × 10 5 Cells, approximately 1 × 10 3 Cells ~ Approximately 3 × 10 5 Cells, approximately 1 × 10 3 Cells ~ approximately 4 × 10 5 Cells, approximately 1 × 10 3 Cells ~ approximately 5 × 10 5 Cells, approximately 1 × 10 3 Cells ~ Approximately 6 × 10 5 Cells, approximately 1 × 10 3 Cells ~ Approximately 7 × 10 5 Cells, approximately 1 × 10 3 Cells ~ Approximately 8 × 10 5 Cells, approximately 1 × 10 3 Cells ~ approximately 9 × 10 5 Cells, approximately 1 × 10 3 Cells ~ Approximately 1×106 Cells, approximately 1 × 10 3 Cells ~ approximately 2 × 10 6 Cells, approximately 1 × 10 3 Cells ~ Approximately 3 × 10 6 Cells, approximately 1 × 10 3 Cells ~ approximately 4 × 10 6 Cells, approximately 1 × 10 3 Cells ~ approximately 5 × 10 6 Cells, approximately 1 × 10 3 Cells ~ Approximately 6 × 10 6 Cells, approximately 1 × 10 3 Cells ~ Approximately 7 × 10 6 Cells, approximately 1 × 10 3 Cells ~ Approximately 8 × 10 6 Cells, approximately 1 × 10 3 Cells ~ approximately 9 × 10 6 Cells, approximately 1 × 10 3 Cells ~ Approximately 1×10 7 Cells, approximately 1 × 10 3 Cells ~ approximately 2 × 10 7 Cells, approximately 1 × 10 3 Cells ~ Approximately 3 × 10 7 Cells, approximately 1 × 10 3 Cells ~ approximately 4 × 10 7 Cells, approximately 1 × 10 3 Cells ~ approximately 5 × 10 7 Cells, approximately 1 × 10 3 Cells ~ Approximately 6 × 10 7 Cells, approximately 1 × 10 3 Cells ~ Approximately 7 × 10 7 Cells, approximately 1 × 10 3 Cells ~ Approximately 8 × 10 7 Cells, approximately 1 × 10 3 Cells ~ approximately 9 × 10 7 Cells, approximately 1 × 10 3 Cells ~ Approximately 1×10 8 Cells, approximately 1 × 10 3 Cells ~ approximately 2 × 10 8 Cells, approximately 1 × 10 3 Cells ~ Approximately 3 × 10 8 Cells, approximately 1 × 10 3 Cells ~ approximately 4 × 10 8 Cells, approximately 1 × 10 3 Cells ~ approximately 5 × 10 8 Cells, approximately 1 × 10 3 Cells ~ Approximately 6 × 10 8 Cells, approximately 1 × 10 3cells ~ approx. 7 x 10 8 cells, approximately 1 x 10 3 cells ~ approx. 8 x 10 8 cells, approximately 1 x 10 3 cells ~ approx. 9 x 10 8 cells, approximately 1 x 10 3 cells ~ approx. 1×10 9 It could be a cell.

[0221] In some cases, the therapeutically effective dose of the unmanipulated enriched γδT cell population, the manipulated enriched γδT cell population, and / or mixture thereof of the present invention is approximately 1 × 10⁻⁶ 6 cells ~ approx. 2 x 10 6 cells, approximately 1 x 10 6 cells ~ approx. 3 x 10 6 cells, approximately 1 x 10 6 cells ~ approx. 4 x 10 6 cells, approximately 1 x 10 6 Cells ~ approx. 5 x 10 6 cells, approximately 1 x 10 6 cells ~ approx. 6 x 10 6 cells, approximately 1 x 10 6 cells ~ approx. 7 x 10 6 cells, approximately 1 x 10 6 cells ~ approx. 8 x 10 6 cells, approximately 1 x 10 6 cells ~ approx. 9 x 10 6 cells, approximately 1 x 10 6 cells ~ approx. 1×10 7 cells, approximately 1 x 10 6 cells ~ approx. 2 x 10 7 cells, approximately 1 x 10 6 cells ~ approx. 3 x 10 7 cells, approximately 1 x 10 6 cells ~ approx. 4 x 10 7 cells, approximately 1 x 10 6 Cells ~ approx. 5 x 10 7 cells, approximately 1 x 10 6 cells ~ approx. 6 x 10 7 cells, approximately 1 x 10 6 cells ~ approx. 7 x 10 7 cells, approximately 1 x 10 6 cells ~ approx. 8 x 10 7 cells, approximately 1 x 10 6 cells ~ approx. 9 x 10 7 cells, approximately 1 x 10 6 cells ~ approx. 1×108 Cells, approximately 1 × 10 6 Cells ~ approximately 2 × 10 8 Cells, approximately 1 × 10 6 Cells ~ Approximately 3 × 10 8 Cells, approximately 1 × 10 6 Cells ~ approximately 4 × 10 8 Cells, approximately 1 × 10 6 Cells ~ approximately 5 × 10 8 Cells, approximately 1 × 10 6 Cells ~ Approximately 6 × 10 8 Cells, approximately 1 × 10 6 Cells ~ Approximately 7 × 10 8 Cells, approximately 1 × 10 6 Cells ~ Approximately 8 × 10 8 Cells, approximately 1 × 10 6 Cells ~ approximately 9 × 10 8 Cells, approximately 1 × 10 6 Cells ~ Approximately 1×10 9 Cells, approximately 1 × 10 6 Cells ~ approximately 2 × 10 9 Cells, approximately 1 × 10 6 Cells ~ Approximately 3 × 10 9 Cells, approximately 1 × 10 6 Cells ~ approximately 4 × 10 9 Cells, approximately 1 × 10 6 Cells ~ approximately 5 × 10 9 Cells, approximately 1 × 10 6 Cells ~ Approximately 6 × 10 9 Cells, approximately 1 × 10 6 Cells ~ Approximately 7 × 10 9 Cells, approximately 1 × 10 6 Cells ~ Approximately 8 × 10 9 Cells, approximately 1 × 10 6 Cells ~ approximately 9 × 10 9 Cells, approximately 1 × 10 7 Cells ~ Approximately 1×10 9 Cells, approximately 1 × 10 7 Cells ~ approximately 2 × 10 9 Cells, approximately 1 × 10 7 Cells ~ Approximately 3 × 10 9 Cells, approximately 1 × 10 7 Cells ~ approximately 4 × 10 9 Cells, approximately 1 × 10 7 Cells ~ approximately 5 × 10 9 Cells, approximately 1 × 10 7 Cells ~ Approximately 6 × 10 9 Cells, approximately 1 × 10 7cells ~ approx. 7 x 10 9 cells, approximately 1 x 10 7 cells ~ approx. 8 x 10 9 cells, approximately 1 x 10 7 cells ~ approx. 9 x 10 9 cells, approximately 1 x 10 8 cells ~ approx. 1×10 9 cells, approximately 1 x 10 8 cells ~ approx. 2 x 10 9 cells, approximately 1 x 10 8 cells ~ approx. 3 x 10 9 cells, approximately 1 x 10 8 cells ~ approx. 4 x 10 9 cells, approximately 1 x 10 8 Cells ~ approx. 5 x 10 9 cells, approximately 1 x 10 8 cells ~ approx. 6 x 10 9 cells, approximately 1 x 10 8 cells ~ approx. 7 x 10 9 cells, approximately 1 x 10 8 cells ~ approx. 8 x 10 9 cells, approximately 1 x 10 8 cells ~ approx. 9 x 10 9 Cells, or approximately 1 × 10⁻⁶ 9 cells ~ approx. 1×10 10 It could be a cell.

[0222] When antibodies or other polyvalent drugs, such as those that bind to or compete with the same or essentially the same epitope as the antibody shown in any one of Figures 1-5, are administered, the usual dosage may vary depending on the route of administration from approximately 10 ng / kg to a maximum of 100 mg / kg or more per day of mammalian body weight, preferably from approximately 1 μg / kg / day to 10 mg / kg / day. Guidance on specific dosages and delivery methods is provided in the literature; see, for example, U.S. Patents 4,657,760, 5,206,344, or 5,225,212. Different formulations are expected to be effective for different treatment compounds and different disorders, and for example, administration targeting one organ or tissue may require delivery to another organ or tissue in a different manner.

[0223] For the treatment or reduction of the severity of immune-related diseases, the appropriate dosage of the compositions of the present invention will depend, as defined above, on the type of disease being treated, the severity and course of the disease, whether the drug is administered for preventive or therapeutic purposes, previous therapies, the patient's clinical history and response to the compound, and the discretion of the attending physician. The compositions can be administered to the subject in a single dose or over a series of treatments.

[0224] For example, depending on the type and severity of the disease, a polyvalent drug (e.g., polypeptide or antibody) of approximately 1 mg / kg to 15 mg / kg (e.g., 0.1 to 20 mg / kg) is an initial candidate dosage for administration to the subject, whether by one or more separate doses or by continuous infusion. A typical daily dose may range from approximately 1 mg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administrations over several days or more, the treatment should be maintained until the desired suppression of disease symptoms occurs, depending on the patient's condition. However, other drug regimens may be useful. The progress of this therapy can be easily monitored using conventional techniques and assays.

[0225] keep In some embodiments, enriched γδT cell populations and / or mixtures thereof, obtained by in vitro proliferation of γδT cell populations, may be formulated in cryogenic medium and placed in cryogenic storage units such as liquid nitrogen freezers (-195°C) or ultra-low temperature freezers (-65°C, -80°C, or -120°C) for long-term storage of at least about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, or at least 5 years. The cryogenic medium may contain dimethyl sulfoxide (DMSO) and / or sodium chloride (NaCl), and / or dextrose, and / or dextran sulfate, and / or hydroxyethyl starch (HES) together with a physiological pH buffer to maintain a pH of about 6.0 to about 6.5, about 6.5 to about 7.0, about 7.0 to about 7.5, about 7.5 to about 8.0, or about 6.5 to about 7.5. Cryopreserved γδT cells can be thawed and further treated with stimulation by antibodies, proteins, peptides, and / or cytokines as described herein. Cryopreserved γδT cells can be thawed and genetically modified by viral vectors (including retroviral and lentiviral vectors) or nonviral means (including RNA, DNA, and proteins) as described herein. In some cases, unmodified γδT cells can be grown by the methods described herein, which include the steps of in vitro or in vitro growth, genetic modification, and cryopreservation.

[0226] In this way, genetically modified and / or non-modified γδT cells are further cryopreserved, with at least about 10 per 1 mL of freezing solvent. 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 or at least about 10 10Cell banks can be generated in quantities of at least 1, 5, 10, 100, 150, 200, or 500 vials of cells. The cryopreserved cell banks can retain their functionality and can be thawed, further stimulated, and proliferated. In some embodiments, thawed cells can be stimulated and proliferated in appropriate closed containers such as cell culture bags and / or bioreactors to produce large quantities of cells as allogeneic cell products. Cryopreserved γδT cells can maintain their biological function under cryopreservation conditions for at least approximately 6, 7, 8, 9, 10, 11, 12, 13, 15, 18, 20, 24, 30, 36, 40, 50, or at least approximately 60 months. In some embodiments, no preservatives are used in the formulation. Cryopreserved γδT cells can be thawed and administered (e.g., by infusion) to multiple patients as a commercially available allogeneic cell product. By administering one or more of the agents described herein that selectively proliferate γδT cells, the injected cells can be proliferated and / or maintained in the administered subject(s).

[0227] All publications and patents referenced herein are incorporated herein by reference for the purpose of describing and disclosing, for example, the constructs and methodologies described in such publications that may be used in connection with the inventions described herein. The publications mentioned herein are provided solely for the purpose of their disclosure prior to the filing date of this application. Nothing herein should be construed as an acknowledgment that the inventors described herein have no prior rights to such disclosure for prior art or for any other reason. [Examples]

[0228] Example 1: Use of polyvalent soluble activators in the proliferation of γδT cell populations in vitro. Construction of plasmid PL426 pCI-D1-08-chimeric scorpion Mammalian expression vector pCI containing the mammalian selectable marker neomycin and the bacterial selectable marker ampicillin was linearized using restriction enzymes EcoRI and XhoI. Full-length chimera D1-08 was assembled using the Gibson Assembly protocol with fragments synthesized as either g blocks or PCR amplification. The assembled products were transformed into appropriate E. coli strains and seeded on carbenicillin. Colonies were screened for correct aggregates using colony PCR and / or restriction digestion. Restriction digestion analysis was performed to first screen for positive clones, and then the construct was confirmed using Sanger sequences. After confirmation, the plasmid was scaled up and rearranged to prevent errors from being generated during scale-up. Expi293 cells were transfected with purified endotoxin-free plasmids, and soluble activators were generated in serum-free medium according to the vendor protocol (Thermo Fisher).

[0229] The supernatant was collected 5 days after transfection, and the protein was purified using a Protein A or Protein L column. The eluted protein was characterized using reducing gel and size exclusion chromatography, and aggregation and monomer percentage were determined. The protein was polished on SEC, and this material was subsequently used to stimulate gamma delta T cell proliferation.

[0230] PBMCs were obtained from previously screened donors that were shown to have a favorable Vδ1 percentage. PBMCs were seeded at 1e6 cells / ml in XVIVO15 medium supplemented with 10% FBS and 100 RJ / ml IL2. After 48 hours of quiescence, the cells were transferred to new plates containing two concentrations of soluble stimulating molecules (50 ng and 5 ug), and the cells were allowed to grow for a further 2 days. Cell doubling and proliferation were assessed using CFSE cell tracking dye. Cells were harvested on day 5 and analyzed for γδ1 and αβ T cell percentages.

[0231] The data obtained support the use of expensive molecules for soluble γδ T cell activation as an alternative to conventional antibody immobilization techniques. Pan05 and Pan07 show the greatest potential for pan-γδ T cell activation and high yield of γδ T cells. In particular, Pan05 and Pan07 showed signs of improved proliferation compared to soluble D1-35_mIgG2a and approach those of plate-bound D1-35_mIgG2a. In addition, soluble D1-08_hIgG1 mini scorpions, and to a lesser extent D1-08 scorpions, showed selective ability to proliferate γδ1 T cells. In particular, soluble D1-08_hIgG1 mini-scorpions showed signs of improved proliferation compared to soluble D1-35_mIgG2a and approach those of plate-bound D1-35_mIgG2a.

[0232] Therefore, an exemplary embodiment of a soluble polyvalent drug is D1-08 hIgG1 Scorpion, which is tetravalent (mAb with scFv on each CH3) and monospecific to Vd1 TCR (derived from D1-08) and exhibits the desired properties as a soluble activator of Vd1 T cells from PBMCs on day 0. Similarly, Pan-05 Scorpion and Pan-07 Scorpion are tetravalent (mAb with scFv on each CH3) and monospecific to Pan γδ TCR (derived from Pan-05 or Pan-07), and also exhibit the desired properties as soluble activators of Vd1 T cells from PBMCs on day 0.

[0233] Preferred embodiments of the present invention have been shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided merely as examples. Those skilled in the art will now be able to conceive of numerical variations, changes, and substitutions without departing from the present invention. It should be understood that various modifications to the embodiments of the present invention described herein may be adopted in the practice of the present invention. The scope of the present invention is defined by the following claims and is intended to encompass the methods and structures within the scope of these claims and their equivalents.

Claims

1. An in vitro method for selectively activating and proliferating γδT cells in an isolated mixed cell population, the method comprising contacting the isolated mixed cell population with one or more soluble polyvalent agents that selectively activate and proliferate γδT cells by binding to at least one epitope of γδTCR, wherein the soluble polyvalent agent is a polyvalent monospecific antibody that is at least tetravalent, A method comprising the above-mentioned one or more soluble polyvalent agents selectively activating and proliferating δ1 T cells, δ2 T cells, or δ3 T cells, or a combination thereof, wherein one or more of the agents that selectively activate and proliferate δ1 T cells bind to the γ and / or δ chains of δ1 TCRs, one or more of the agents that selectively activate and proliferate δ2 T cells bind to the γ and / or δ chains of δ2 TCRs, and one or more of the agents that selectively activate and proliferate δ3 T cells bind to the γ and / or δ chains of δ3 TCRs, thereby activating and proliferating one or more γδ T cell subtypes in the mixed cell population.

2. An in vitro and / or in vitro method for producing a population of enriched γδ T cells, comprising directly contacting an isolated mixed cell population containing γδ T cells or a purified fraction thereof with one or more soluble polyvalent agents, wherein the soluble polyvalent agents are at least tetravalent polyvalent monospecific antibodies, and A method comprising the above-mentioned one or more soluble polyvalent agents i) selectively activating and proliferating δ1 T cells by binding to the γ chain and / or δ chain of δ1 TCR, ii) selectively activating and proliferating δ2 T cells by binding to the γ chain and / or δ chain of δ2 TCR, and / or iii) selectively activating and proliferating δ3 T cells by binding to the γ chain and / or δ chain of δ3 TCR, thereby resulting in a fermented γδ T cell subpopulation.

3. The method according to claim 1 or 2, wherein the soluble polyvalent drug includes at least two antigen-binding sites that specifically bind to the same antigen, or the polyvalent drug includes at least two antigen-binding sites that specifically bind to the same epitope of the same antigen.

4. The method according to any one of claims 1 to 3, wherein the antibody comprises two CH3 domains and one scFv domain on each CH3 domain.

5. (a) The antigen-binding site binds to different epitopes on the constant or variable regions of the δTCR and / or γTCR, or (b) The method according to claim 3, wherein the antigen-binding site comprises a CDR from a γδTCR panMAb, and the antigen-binding site specifically binds to a domain shared by different γ and δTCRs on either or both of the γ or δ chain, comprising a population of δ1, δ2, and δ3 T cells.

6. (a) The soluble polyvalent drug contains at least two or more antigen-binding sites that selectively proliferate δ1 T cells and specifically bind to epitopes containing the δ1 variable region, (b) The soluble polyvalent drug contains at least two or more antigen-binding sites that specifically bind to the δ1TCR bin1δ1 epitope, bin1bδ1 epitope, bin2δ1 epitope, bin2bδ1 epitope, bin2cδ1 epitope, bin3δ1 epitope, bin4δ1 epitope, bin5δ1 epitope, bin6δ1 epitope, bin7δ1 epitope, bin8δ1 epitope, or bin9δ1 epitope of human δ1TCR, (c) The soluble polyvalent drug contains at least two or more antigen-binding sites that specifically bind to the same or essentially the same epitope as the antibody selected from the group consisting of δ1-05, δ1-08, δ1-18, δ1-22, δ1-23, δ1-26, δ1-35, δ1-37, δ1-39, δ1-113, δ1-143, δ1-149, δ1-155, δ1-182, δ1-183, δ1-191, δ1-192, δ1-195, δ1-197, δ1-199, δ1-201, δ1-203, δ1-239, δ1-253, δ1-257, δ1-278, δ1-282, and δ1-285, or competes with the antibody. (d) The soluble polyvalent drug contains a CDR of an antibody selected from the group consisting of δ1-05, δ1-08, δ1-18, δ1-22, δ1-23, δ1-26, δ1-35, δ1-37, δ1-39, δ1-113, δ1-143, δ1-149, δ1-155, δ1-182, δ1-183, δ1-191, δ1-192, δ1-195, δ1-197, δ1-199, δ1-201, δ1-203, δ1-239, δ1-253, δ1-257, δ1-278, δ1-282, and δ1-285, (e) The soluble polyvalent drug contains a CDR of antibody δ1-35 or δ1-08, or binds to the same epitope as antibody δ1-08 or δ1-35. (f) The soluble polyvalent drug contains at least two or more antigen-binding sites that specifically bind to the same epitopes as the antibody selected from TS-1 and TS8.2, (g) The soluble polyvalent drug contains a CDR of TS-1 or TS8.2 and / or is humanized TS-1 or TS8.2, (h) The soluble polyvalent drug contains at least two or more antigen-binding sites that specifically bind to the epitope containing the residues Arg71, Asp72, and Lys120 of the δ1 variable region, or (i) The method according to claim 3, wherein the soluble polyvalent drug comprises at least two or more antigen-binding sites having reduced binding to mutant δ1TCR polypeptides containing mutations in K120 of delta J1 and delta J2.

7. The method according to claim 6, wherein the soluble polyvalent agent selectively proliferates δ1 T cells and δ3 T cells, or the soluble polyvalent agent selectively proliferates δ1, δ3, δ4, and δ5γδ T cells.

8. (a) The soluble polyvalent drug contains at least two or more antigen-binding sites that selectively proliferate δ2 T cells and specifically bind to epitopes containing the δ2 ​​variable region, (b) The soluble polyvalent drug contains at least two or more antigen-binding sites that specifically bind to the δ2TCR bin1δ2 epitope, bin2δ2 epitope, bin3δ2 epitope, or bin4δ2 epitope of human δ2TCR, (c) The soluble polyvalent drug contains at least two or more antigen-binding sites that specifically bind to the same or essentially the same epitope as the antibody selected from the group consisting of δ2-14, δ2-17, δ2-22, δ2-30, δ2-31, δ2-32, δ2-33, δ2-35, δ2-36, and δ2-37, or competes with the antibody. (d) The soluble polyvalent drug contains a CDR of an antibody selected from the group consisting of δ2-14, δ2-17, δ2-22, δ2-30, δ2-31, δ2-32, δ2-33, δ2-35, δ2-36, and δ2-37, (e) The soluble polyvalent drug contains the CDR of antibody δ2-37, or binds to the same epitope as antibody δ2-37, (f) The soluble polyvalent drug contains at least two or more antigen-binding sites that specifically bind to the same epitope as the antibody selected from 15D and B6, (g) The soluble polyvalent drug comprises a CDR of antibody 15D or B6 and / or is a humanized 15D and B6 antibody, (h) The method according to claim 3, wherein the soluble polyvalent drug comprises at least two or more antigen-binding sites having reduced binding to a mutant δ2TCR polypeptide having a mutation in G35 of the δ2 ​​variable region.

9. (a) The soluble polyvalent drug contains at least two or more antigen-binding sites that selectively proliferate δ3 T cells and specifically bind to epitopes containing the δ3 variable region, (b) The soluble polyvalent drug contains at least two or more antigen-binding sites that specifically bind to the same or essentially the same epitope as the antibody selected from the group consisting of δ3-08, δ3-20, δ3-23, δ3-31, δ3-42, δ3-47, and δ3-58, or competes with the antibody, or (c) The method according to claim 3, wherein the soluble polyvalent agent comprises a CDR of an antibody selected from the group consisting of δ3-08, δ3-20, δ3-23, δ3-31, δ3-42, δ3-47, and δ3-58.

10. (a) The method further comprises culturing the isolated mixed cell population with gamma chain cytokines simultaneously or sequentially, and / or (b) The method according to any one of claims 1 to 9, wherein the γδ T cell population is manipulated before and / or after activation and / or proliferation.

11. The method according to claim 10, wherein the cytokine is selected from the group consisting of IL-2, IL-7, IL-9, IL-12, IL-15, IL-18, IL-19, IL-21, IL-23, and IL-33, or the cytokine is IL-2, IL-15, IL-12, or IL-21.

12. The method according to claim 10, wherein the γδ T cell population is engineered to stably express one or more tumor recognition moieties encoded by an expression cassette, and / or the γδ T cell population is engineered to stably express a transgene encoding at least one secreted cytokine.

13. The method according to claim 12, wherein the at least one secreted cytokine is a common gamma chain cytokine, or the cytokine is IL-2, IL-15, or IL-4.

14. The method according to any one of claims 1 to 13, wherein a proliferated / enriched manipulated and / or unmanipulated γδ T cell population is formulated for administration to a subject in need thereof, the γδ T cell population comprising at least 60% γδ T cells.

15. The method of claim 14, further comprising performing a depletion step for αβT cells in a γδT cell population before administering the proliferated / enriched manipulated and / or unmanipulated γδT cells to the subject.

16. The method according to claim 14, wherein the manipulated and / or unmanipulated γδ T cells are autologous cells of the subject.

17. The method according to claim 14, wherein the manipulated and / or unmanipulated γδ T cells are allogeneic cells with respect to the subject.

18. The method according to claim 1 or 2, wherein the soluble polyvalent drug is tetravalent.

19. The method according to claim 18, wherein the soluble polyvalent agent comprises a polypeptide containing SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 99, SEQ ID NO: 101, or SEQ ID NO:

105.

20. A composition for selectively activating and proliferating γδ T cells, their subtypes, or combinations thereof, comprising a proliferated population of γδ T cells and one or more soluble polyvalent agents, wherein the polyvalent agent comprises at least two antigen-binding sites that specifically bind to the same epitope of a γδ TCR, and the polyvalent agent is at least tetravalent.

21. The composition according to claim 20, wherein the soluble polyvalent agent is monospecific.

22. (a) The soluble polyvalent drug comprises at least two or more antigen-binding sites that selectively proliferate δ1 T cells and specifically bind to epitopes including the δ1 variable region, (b) The soluble polyvalent drug contains at least two or more antigen-binding sites that specifically bind to the δ1TCR bin1δ1 epitope, bin1bδ1 epitope, bin2δ1 epitope, bin2bδ1 epitope, bin2cδ1 epitope, bin3δ1 epitope, bin4δ1 epitope, bin5δ1 epitope, bin6δ1 epitope, bin7δ1 epitope, bin8δ1 epitope, or bin9δ1 epitope of human δ1TCR, (c) The soluble polyvalent drug contains at least two or more antigen-binding sites that specifically bind to the same or essentially the same epitope as the antibody selected from the group consisting of δ1-05, δ1-08, δ1-18, δ1-22, δ1-23, δ1-26, δ1-35, δ1-37, δ1-39, δ1-113, δ1-143, δ1-149, δ1-155, δ1-182, δ1-183, δ1-191, δ1-192, δ1-195, δ1-197, δ1-199, δ1-201, δ1-203, δ1-239, δ1-253, δ1-257, δ1-278, δ1-282, and δ1-285, or competes with the antibody. (d) The soluble polyvalent drug contains a CDR of an antibody selected from the group consisting of δ1-05, δ1-08, δ1-18, δ1-22, δ1-23, δ1-26, δ1-35, δ1-37, δ1-39, δ1-113, δ1-143, δ1-149, δ1-155, δ1-182, δ1-183, δ1-191, δ1-192, δ1-195, δ1-197, δ1-199, δ1-201, δ1-203, δ1-239, δ1-253, δ1-257, δ1-278, δ1-282, and δ1-285, (e) The soluble polyvalent drug contains a CDR of antibody δ1-35 or δ1-08, or binds to the same epitope as antibody δ1-08 or δ1-35. (f) The soluble polyvalent drug contains at least two or more antigen-binding sites that specifically bind to the same epitopes as the antibody selected from TS-1 and TS8.2, (g) The soluble polyvalent drug contains a CDR of TS-1 or TS8.2 and / or is humanized TS-1 or TS8.2, (h) The soluble polyvalent drug contains at least two or more antigen-binding sites that specifically bind to the epitope containing the residues Arg71, Asp72, and Lys120 of the δ1 variable region, or (i) The composition according to claim 20 or 21, wherein the soluble polyvalent drug comprises at least two or more antigen-binding sites having reduced binding to mutant δ1TCR polypeptides containing mutations in K120 of delta J1 and delta J2.

23. The composition according to claim 20, wherein the antigen-binding site comprises a CDR from γδTCR panMAb, and the soluble polyvalent drug comprises the amino acid sequence of SEQ ID NO: 95 or 101.

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