Patent Application
20220211757
Embodiments of the disclosure concern at least the fields of immunology, cell biology, molecular biology, and medicine.
Gamma delta (γδ) T cells are a small subpopulation of T lymphocytes having the ability to bridge innate and adaptive immunity. The majority of γδ T cells in adult human blood exhibit Vγ9Vδ2 T cell receptors and respond to small phosphorylated nonpeptide antigens, called phosphoantigens (pAgs), which are commonly produced by malignant cells (see, e.g., Yang et al., Immunity 50, 1043-1053.e5 (2019)). Unlike conventional αβ T cells, γδ T cells do not recognize polymorphic classical major histocompatibility complex (MHC) molecules and are therefore free of graft versus host disease (GvHD) risk when adoptively transferred into an allogeneic host. Additionally, γδ T cells have several other unique features that make them ideal cellular carriers for developing off-the-shelf cellular therapy for cancer. These features include: 1) γδ T cells have roles in cancer immunosurveillance; 2) γδ T cells have the remarkable capacity to target tumors independent of tumor antigen- and major histocompatibility complex (MHC)-restrictions; 3) γδ T cells can employ multiple mechanisms to attack tumor cells through direct killing and adjuvant effects; and 4) γδ T cells express a surface receptor, FcγRIII (CD16), that is involved in antibody-dependent cellular cytotoxicity (ADCC) and can be potentially combined with monoclonal antibody for cancer therapy (see, e.g., Lepore et al., Front. Immunol. 9, 1-11 (2018), Harrer et al., Hum. Gene Ther. 29, 547-558 (2018) and Presti et al., Front. Immunol. 8, 1-11 (2017)). Unfortunately, however, the development of an allogeneic off-the-shelf γδ T cellular product is greatly hindered by their availability—these cells are of extremely low number and high variability in humans (˜1-5% T cells in human blood), making it very difficult to produce therapeutic numbers of γδ T cells using blood cells of allogeneic human donors (see, e.g., Silva-Santos et al., Nat. Rev. Immunol. 15, 683-691 (2015)).
The conventional method of generating γδ T cells, in particular the Vγ9Vδ2 subset, for adoptive therapy involves either in vitro or in vivo expansion of peripheral blood mononuclear cell (PBMC)-derived γδ T cells using aminobisphosphonates, such as Zoledronate (ZOL). However, this methodology generates highly variable yields of γδ T cells depending on PBMC donors; and most importantly, such a γδ T cell product will typically contain bystander αβ T cells and thereby incurring GvHD risk (see, e.g., Torikai et al., Mol. Ther. 24, 1178-1186 (2016)).
Novel methods and materials that can reliably generate a homogenous monoclonal population of γδ T cells at large quantities with a feeder-free differentiation system are pivotal to developing off-the-shelf γδ T cell therapies that are useful in the treatment of a wide variety of pathological conditions.
The ability to manufacture a therapeutic cell population or a cell population that can be used to create a therapeutic cell population “off-the-shelf” increases the availability and usefulness of new cellular therapies. Embodiments of the invention are provided to address the need for new cellular therapies, more particularly, the need for cellular therapies that are not hampered by the challenges posed in individualizing therapy using autologous cells.
As disclosed herein, we have discovered that engineered γδ T cells can be produced through γδ TCR gene-engineering of pluripotent cells (e.g., CD34+ stem and progenitor cells) followed by selectively differentiating the gene-engineered cells into transgenic γδ T cells in vivo or in vitro. As discussed below, such γδ T cells can further be engineered to co-express other disease-targeting molecules (e.g., chimeric antigen receptors, “CARs”) as well as immune regulatory molecules (e.g., cytokines, receptors/ligands and the like) to modulate their performance. Significantly, embodiments of these in vitro differentiated γδ T cells can be used for allogeneic “off-the-shelf” cell therapies for treating a broad range of diseases (e.g., cancers, autoimmune diseases, infections and the like).
Embodiments of the invention include materials and methods relating to the gamma and delta chain polypeptides that are disclosed in Table 1 below. For example, embodiments of the invention include compositions of matter comprising a gamma chain polypeptide and/or a delta chain polypeptide having an amino acid sequence shown in Table 1 (SEQ ID NO: 1-SEQ ID NO: 52). Related embodiments of the invention include compositions of matter comprising polynucleotides encoding a gamma chain polypeptide and/or a delta chain polypeptide having an amino acid sequence shown in Table 1 (SEQ ID NO: 1-SEQ ID NO: 52). In certain embodiments of the invention, these polynucleotides are disposed in a vector, for example an expression vector designed to express these gamma and delta chain polypeptides in a cell. One such embodiment of the invention is a composition of matter comprising an immune cell that has been transduced with an expression vector comprising a polynucleotide encoding at least one T cell receptor gamma chain polypeptide and/or the T cell receptor delta chain polypeptide having an amino acid sequence shown in Table 1 (SEQ ID NO: 1-SEQ ID NO: 52).
Embodiments of the invention also include, for example, methods of making an engineered functional T cell modified to contain at least one exogenous nucleic acid molecule encoding a T cell receptor gamma chain polypeptide and/or a T cell receptor delta chain polypeptide (e.g., as disclosed in Table 1). Typically these methods comprise transducing a pluripotent cell (e.g. a human CD34+ hematopoietic stem or progenitor cell) with the at least one exogenous nucleic acid molecule encoding a T cell receptor gamma chain polypeptide and/or a T cell receptor delta chain polypeptide so that the cell transduced by the exogenous nucleic acid molecule expresses a T cell receptor comprising a gamma chain polypeptide and a delta chain polypeptide; and then differentiating the transduced human cell so as to generate the engineered functional gamma delta T cell.
The methodological embodiments of the invention can include, for example, differentiating transduced pluripotent cells in vitro. In illustrative methods, transduced CD34+ human hematopoietic stem or progenitor cells (HSPC) can be differentiated in vitro in the absence of feeder cells; and/or cultured in medium comprising a cytokine such as one or more of IL-3, IL-7, IL-6, SCF, EPO, TPO and FLT3L, and/or in the presence of an agent selected to facilitate nucleic acid transduction efficiency such as retronectin. In certain embodiments, the method further comprises contacting the transduced cell with an agonist antigen or other stimulatory agent. In some embodiments of the invention, the method further comprises co-culturing the transduced cells with peripheral blood mononuclear cells, antigen presenting cells, or artificial antigen presenting cells. Certain embodiments of the invention further comprise expanding the pluripotent cell transduced with the nucleic acid molecule encoding a T cell receptor gamma chain polypeptide or a T cell receptor delta chain polypeptide in vitro. Alternative methods of the invention can comprise engrafting the cell transduced with the nucleic acid molecule encoding a T cell receptor gamma chain polypeptide and a T cell receptor delta chain polypeptide into a subject to generate clonal populations of the engineered cells in vivo.
In some embodiments of the invention, the engineered T cell comprises a gene expression profile characterized as being at least one of: HLA-I-negative; HLA-II-negative; HLA-E-positive; and/or expressing a suicide gene. Optionally, the engineered T cell further comprises an exogenous T cell receptor nucleic acid molecule encoding a T cell receptor alpha chain polypeptide or a T cell receptor beta chain polypeptide; and/or an exogenous nucleic acid molecule encoding a cytokine; and/or suppressed endogenous TCRs. In certain embodiments of the invention, a T cell receptor gamma chain polypeptide and/or the T cell receptor delta chain polypeptide expressed by these engineered cells comprises an amino acid sequence shown in Table 1 (SEQ ID NO: 1-SEQ ID NO: 52).
Embodiments of the invention include engineered functional gamma delta T cells produced by the methods disclosed herein. For example, embodiments of the invention include compositions of matter comprising an engineered T cell comprising a gene expression profile characterized as: HLA-I-negative; HLA-II-negative; HLA-E-positive; expressing a suicide gene; and expressing at least one exogenous T cell receptor gamma chain polypeptide and at least one exogenous T cell receptor delta chain polypeptide. In certain embodiments, a T cell receptor gamma chain polypeptide or a T cell receptor delta chain polypeptide comprises at least one amino acid sequence shown in SEQ ID NO: 1-SEQ ID NO: 52. In some embodiments of the invention, a CD34+ HSPCs can be isolated from cord blood (CB) or peripheral blood. In such embodiments of the invention, CB CD34+ HSCs can be obtained from commercial providers (e.g., HemaCare) or from established CB banks.
As the γδ T gamma/delta cellular product is an off-the-shelf product that can be used to treat patients independent of MHC restrictions, once commercialized, this cellular product has broad applications in a variety of potentially life-saving therapies. In this context, yet another embodiment of the invention is a method of treating a subject in need of gamma delta T cells (e.g., to fight a disease such as an autoimmune disease or a cancer or an infection such as COVID-19) which comprises administering to the subject an engineered functional T cell disclosed herein.
Other objects, features and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. It is to be understood, however, that the detailed description and specific examples, while indicating some embodiments of the present invention, are given by way of illustration and not limitation. Many changes and modifications within the scope of the present invention may be made without departing from the spirit thereof, and the invention includes all such modifications.
In the description of embodiments, reference may be made to the accompanying figures which form a part hereof, and in which is shown by way of illustration a specific embodiment in which the invention may be practiced. It is to be understood that other embodiments may be utilized, and structural changes may be made without departing from the scope of the present invention.
Gamma delta (γδ) T cells normally account for 1 to 5% of peripheral blood lymphocytes in healthy individuals. Unlike classical αβ T cells that recognize specific peptide antigens presented by major histocompatibility complex (MHC) molecules, γδ T cells can recognize generic determinants expressed by cells that have become dysregulated as a result of either malignant transformation or viral infection. Consequently, γδ-T cells have the innate ability to recognize and kill a broad spectrum of tumor cell types, in a manner that does not require the existence of conventional tumor-specific antigens.
There is a need in the art for methods and materials that can reliably generate a homogenous monoclonal population of various engineered human T cells such as engineered γδ T cells in large quantities. These technologies are pivotal to developing off-the-shelf T cell therapies. Such methods and materials can, for example, provide γδ T cells that can be used in allogeneic or autologous recipient subjects for the treatment of a variety of pathological conditions including, for example, viral infections, fungal infections, protozoal infections and cancers.
As discussed below, we have discovered that engineered γδ T cells can be produced through γδ TCR gene-engineering of pluripotent human cells such as CD 34+ stem and progenitor cells (e.g., HSCs, iPSCs, ESCs) followed by selectively differentiating the gene-engineered stem and progenitor cells into transgenic γδ T cells in vivo and/or in vitro. As is known in the art, hematopoietic stem or progenitor cells possess multipotentiality, enabling them to self-renew and also to produce mature blood cells, such as erythrocytes, leukocytes, platelets, and lymphocytes. CD34 is a marker of human HSC, and all colony-forming activity of human bone marrow (BM) cells is found in the CD34+ fraction. See e.g., Mata et al., Transfusion. 2019 December; 59(12):3560-3569. doi: 10.1111/trf.15597.
This discovery is unexpected because developmental path of gamma delta T cells is unique and unlike the developmental paths of other T cells such as iNKT cells and αβ T cells (see, e.g., Dolens et al., EMBO Rep. 2020 May 6; 21(5):e49006. doi: 10.15252/embr.201949006. Epub 2020 and Shissier et al., Mol. Immunol. 2019; 105: 116-130). Importantly, the in vitro differentiated γδ T cells disclosed herein can be used for allogeneic “off-the-shelf” cell therapies for treating a broad range of diseases (e.g., cancer, infection, autoimmunity, etc.). Moreover, the γδ T cells can also be engineered to co-express other disease-targeting molecules (e.g., CARs) as well as immune regulatory molecules (e.g., cytokines, receptors/ligands) to enhance their performance.
Embodiments of the invention include, for example, methods of making an engineered functional T cell modified to contain at least one exogenous nucleic acid molecule (e.g., one disposed in an expression vector such as a lentiviral vector as discussed below) encoding a T cell receptor gamma chain polypeptide and/or a T cell receptor delta chain polypeptide such as a gamma chain polypeptide and/or a delta chain polypeptide having an amino acid sequence shown in Table 1 (SEQ ID NO: 1-SEQ ID NO: 52). Typically these methods comprise transducing a pluripotent human cell such as a hematopoietic stem/progenitor cell (i.e., a pluripotent stem cell, a hematopoietic stem cell, or a hematopoietic progenitor cell) with the at least one exogenous nucleic acid molecule encoding a T cell receptor gamma chain polypeptide and/or a T cell receptor delta chain polypeptide so that the human cell transduced by the exogenous nucleic acid molecule expresses a T cell receptor comprising a gamma chain polypeptide and a delta chain polypeptide; and then differentiating the transduced human cell (e.g. a hematopoietic stem/progenitor cell) so as to generate the engineered functional gamma delta T cell. In certain methodological embodiments of the invention, the T cell receptor gamma chain polypeptide and T cell receptor delta chain polypeptide encoded by the exogenous nucleic acid are selected as ones known to form a γδ T cell receptor that has been previously observed to target cancer cells or cells infected with a virus, bacteria, fungi or protozoan. Certain methods of the invention include the steps of differentiating the transduced human cell in an in vitro culture; and then further expanding these differentiated cells in an in vitro culture. In some methodological embodiments of the invention, expanding these differentiated cells in an in vitro culture is performed under conditions selected to expand the differentiated population of transduced cells by at least 2-fold, 5-fold, 10-fold or 100-fold. In some embodiments of the invention, the engineered functional gamma delta T cell is exposed to zoledronic acid.
The methodological embodiments of the invention include differentiating the transduced pluripotent human cells (e.g., human hematopoietic stem or progenitor cells) in vitro or in vivo and then expanding this differentiated population of cells. In certain embodiments, the method further comprises contacting the transduced cell with a stimulatory agent such as an agonist antigen. In some methodological embodiments of the invention, a population of γδ T cells is made by the methods disclosed herein wherein such methods do not include a cell sorting step (e.g., FACS or magnetic bead sorting) following transduction of the nuclei acids encoding the γ and δ polypeptides into the human cells. In some embodiments of the invention, the method further comprises co-culturing the transduced cells with peripheral blood mononuclear cells, antigen presenting cells, or artificial antigen presenting cells. Typically in these methods, the transduced human cell is differentiated in vitro in the absence of feeder cells; and/or the transduced hematopoietic stem or progenitor cell is cultured in medium comprising a cytokine such as one or more of IL-3, IL-7, IL-6, SCF, MCP-4, EPO, TPO, FLT3L, and/or an agent selected to facilitate nucleic acid transduction efficiency such as retronectin. Alternative methods of the invention can comprise engrafting the cell transduced with the nucleic acid molecule encoding a T cell receptor gamma chain polypeptide or a T cell receptor delta chain polypeptide into a subject (i.e., in vivo) to generate clonal populations of the engineered cell.
In some methodological embodiments of the invention, the engineered T cell is selected to comprise a certain gene expression profile, for example one characterized as being at least one of: HLA-I-negative; HLA-II-negative; HLA-E-positive; and/or expressing a suicide gene. Typically, the engineered T cell further comprises one or more exogenous T cell receptor nucleic acid molecules encoding a T cell receptor alpha chain polypeptide and a T cell receptor beta chain polypeptide; and/or one or more exogenous nucleic acid molecules encoding a cytokine; and/or suppressed endogenous TCRs. In some embodiments of the invention disclosed herein, the T cell receptor gamma chain polypeptide and the T cell receptor delta chain polypeptide comprises an amino acid sequence shown in Table 1 below. In particular embodiments, the one or more additional nucleic acids encode one or more therapeutic gene products. Examples of therapeutic gene products include at least the following: 1. Antigen recognition molecules, e.g. a CAR (chimeric antigen receptor) and/or an αβ TCR (T cell receptor), a γδ T receptor and the like; 2. Co-stimulatory molecules, e.g. CD28, 4-1BB, 4-1BBL, CD40, CD40L, ICOS; and/or 3. Cytokines, e.g. IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-7, IL-9, IL-15, IL-12, IL-17, IL-21, IL-23, IFN-γ, TNF-α, TGF-β, G-CSF, GM-CSF; 4. Transcription factors, e.g. T-bet, GATA-3, RORγt, FOXP3, and Bcl-6. Therapeutic antibodies are included, as are chimeric antigen receptors, single chain antibodies, monobodies, humanized, antibodies, bi-specific antibodies, single chain FV antibodies or combinations thereof.
Embodiments of the invention also include materials and methods relating to the gamma and delta chain polypeptides that are disclosed in Table 1 below. For example, embodiments of the invention include compositions of matter comprising a gamma chain polypeptide and/or a delta chain polypeptide having an amino acid sequence shown in Table 1 (SEQ ID NO: 1-SEQ ID NO: 52). Related embodiments of the invention include compositions of matter comprising polynucleotides encoding a gamma chain polypeptide and/or a delta chain polypeptide having an amino acid sequence shown in Table 1 (SEQ ID NO: 1-SEQ ID NO: 52). In certain embodiments of the invention, these polynucleotides are disposed in a vector, for example an expression vector designed to express these gamma and delta chain polypeptides in a cell (e.g. a mammalian cell). The compositions of the invention may contain preservatives and/or antimicrobial agents as well as pharmaceutically acceptable excipient substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, detergents and the like. For such compositions, the term “excipient” is meant to include, but is not limited to, those ingredients described in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21st ed. (2006).
Embodiments of the invention further include engineered functional gamma delta T cells and populations of these cell produced by the methods disclosed herein. Typically, these populations consist essentially of functional gamma delta T cells (e.g., do not include conventional αβ T cells). Embodiments of the invention include compositions of matter comprising an engineered γδ T cell or T cell population disclosed herein such as one comprising a gene expression profile characterized as: HLA-I-negative; HLA-II-negative; HLA-E-positive; expressing a suicide gene; and expressing an exogenous T cell receptor gamma chain polypeptide and an exogenous T cell receptor delta chain polypeptide. Optionally, the engineered T cell further comprises an exogenous nucleic acid molecule encoding another polypeptide such as a T cell receptor alpha chain polypeptide and/or a T cell receptor beta chain polypeptide and/or an iNKT receptor polypeptide; and/or a cytokine; and/or comprises suppressed endogenous TCRs. Embodiments of the invention also include composition of matter comprising an immune cell that has been transduced with an expression vector comprising a polynucleotide encoding at least one exogenous T cell receptor gamma chain polypeptide and/or the T cell receptor delta chain polypeptide having an amino acid sequence shown in Table 1 (SEQ ID NO: 1-SEQ ID NO: 52).
Methods of treating patients with an γδ T cell or cell population as disclosed herein are also provided. Embodiments of the invention include methods of treating a subject in need of gamma delta T cells (e.g., to fight a disease such as an autoimmune disease or a cancer or an infection such as COVID-19) which comprises administering to the subject an engineered functional gamma delta T cell disclosed herein. In this way, engineered gamma delta T cells may be used to treat patients in need of therapeutic intervention. In some therapeutic embodiments of the invention, the methods include introducing one or more additional nucleic acids into the gamma delta T cells, which may or may not have been previously frozen and thawed. This use provides one of the advantages of creating an off-the-shelf gamma delta T cell.
In certain therapeutic methods of the invention, the patient has been diagnosed with a cancer. In some embodiments, the patient has a disease or condition involving inflammation, which, in some embodiments, excludes cancer. In specific embodiments, the patient has an autoimmune disease or condition. In particular aspects, the cells or cell population is allogeneic with respect to the patient. In additional embodiments, the patient does not exhibit signs of rejection or depletion of the cells or cell population. Some therapeutic methods further include administering to the patient a stimulatory molecule (e.g., alone or loaded onto APCs) that activates γδ T cells, or a compound that initiates the suicide gene product.
Treatment of a cancer patient with the γδ T cells may result in tumor cells of the cancer patient being killed after administering the cells or cell population to the patient. Treatment of an inflammatory disease or condition may result in reducing inflammation. In other embodiments, a patient with an autoimmune disease or condition may experience an improvement in symptoms of the disease or condition or may experience other therapeutic benefits from the γδ T cells. Combination treatments with γδ T cells and standard therapeutic regimens or another immunotherapy regimen(s) may be employed.
As discussed below, the figures included herewith provide examples of a number of illustrative working embodiments of the invention as well as data obtained from such embodiments of the invention.
For the convenience of expression in this disclosure, we refer to a pair of γ9δ2 TCR genes as a γδTCR gene. As shown in
We established an in vitro SARS-CoV-2 infection model (
It is specifically noted that any embodiment discussed in the context of a particular cell or cell population embodiment may be employed with respect to any other cell or cell population embodiment. Moreover, any embodiment employed in the context of a specific method may be implemented in the context of any other methods described herein. Furthermore, aspects of different methods described herein may be combined so as to achieve other methods, as well as to create or describe the use of any cells or cell populations. It is specifically contemplated that aspects of one or more embodiments may be combined with aspects of one or more other embodiments described herein. Furthermore, any method described herein may be phrased to set forth one or more uses of cells or cell populations described herein. For instance, use of engineered γδ T cells or a γδ T cell population can be set forth from any method described herein.
In a particular embodiment, there is an engineered γδ T cell that expresses at least one γδ T-cell receptor (γδ TCR) and an exogenous suicide gene product, wherein the at least one γδ TCR is expressed from an exogenous nucleic acid and/or from an endogenous γδ TCR gene that is under the transcriptional control of a recombinantly modified promoter region. Methods in the art for suicide gene usage may be employed, such as in U.S. Pat. No. 8,628,767, U.S. Patent Application Publication 20140369979, U.S. 20140242033, and U.S. 20040014191, all of which are incorporated by reference in their entirety. In further embodiments, a TK gene is a viral TK gene, i.e., a TK gene from a virus. In particular embodiments, the TK gene is a herpes simplex virus TK gene. In some embodiments, the suicide gene product is activated by a substrate. Thymidine kinase is a suicide gene product that is activated by ganciclovir, penciclovir, or a derivative thereof. In certain embodiments, the substrate activating the suicide gene product is labeled in order to be detected. In some instances, the substrate that may be labeled for imaging. In some embodiments, the suicide gene product may be encoded by the same or a different nucleic acid molecule encoding one or both of TCR-gamma or TCR-delta. In certain embodiments, the suicide gene is sr39TK or inducible caspase 9. In alternative embodiments, the cell does not express an exogenous suicide gene.
In additional embodiments, an engineered γδ T cell is lacking or has reduced surface expression of at least one HLA-I or HLA-II molecule. In some embodiments, the lack of surface expression of HLA-I and/or HLA-II molecules is achieved by disrupting the genes encoding individual HLA-I/II molecules, or by disrupting the gene encoding B2M (beta 2 microglobulin) that is a common component of all HLA-I complex molecules, or by disrupting the genes encoding CIITA (the class II major histocompatibility complex transactivator) that is a critical transcription factor controlling the expression of all HLA-II genes. In specific embodiments, the cell lacks the surface expression of one or more HLA-I and/or HLA-II molecules, or expresses reduced levels of such molecules by (or by at least) 50, 60, 70, 80, 90, 100% (or any range derivable therein). In some embodiments, the HLA-I or HLA-II are not expressed in the γδ T cell because the cell was manipulated by gene editing. In some embodiments, the gene editing involved is CRISPR-Cas9. Instead of Cas9, CasX or CasY may be involved. Zinc finger nuclease (ZFN) and TALEN are other gene editing technologies, as well as Cpf1, all of which may be employed. In other embodiments, the γδ T cell comprises one or more different siRNA or miRNA molecules targeted to reduce expression of HLA-I/II molecules, B2M, and/or CIITA.
In some embodiments, a γδ T cell of the invention comprises a recombinant vector or a nucleic acid sequence from a recombinant vector that was introduced into the cells. In certain embodiments the recombinant vector is or was a viral vector. In further embodiments, the viral vector is or was a lentivirus, a retrovirus, an adeno-associated virus (AAV), a herpesvirus, or adenovirus. It is understood that the nucleic acid of certain viral vectors integrate into the host genome sequence.
In some embodiments, a γδ T cell of the invention is disposed in selected media conditions during growth and differentiation (e.g., not disposed in media comprising animal serum). In further embodiments, a γδ T cell is or was frozen. In some embodiments, the γδ T cell has previously been frozen and the previously frozen cell is stable at room temperature for at least one hour. In some embodiments, the γδ T cell has previously been frozen and the previously frozen cell is stable at room temperature for at least 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, 24, 30, or 48 hours (or any derivable range therein). In certain embodiments, a γδ T cell or a population of γδ T cells in a solution comprises dextrose, one or more electrolytes, albumin, dextran, and/or DMSO. In a further embodiment, the cell is in a solution that is sterile, nonpyogenic, and isotonic.
In embodiments involving multiple cells, a γδ T cell population may comprise, comprise at least, or comprise at most about 102, 103, 104′, 105, 106, 107, 108, 109, 1010, 1011, 1012, 1013, 1014, 1015 cells or more (or any range derivable therein), which are engineered γδ T cells in some embodiments. In some cases, a cell population comprises at least about 106-1012 engineered γδ T cells. It is contemplated that in some embodiments, that a population of cells with these numbers is produced from a single batch of cells and are not the result of pooling batches of cells separately produced.
In specific embodiments, there is an T cell population comprising: clonal γδ T cells comprising one or more exogenous nucleic acids encoding an γδ T-cell receptor and a thymidine kinase suicide gene product, wherein the clonal γδ T cells have been engineered not to express functional beta-2-microglobulin (B2M), and/or class II, major histocompatibility complex, or transactivator (CIITA) and wherein the cell population is at least about 106-1012 total cells and comprises at least about 102-106 engineered γδ T cells. In certain instances, the cells are frozen in a solution.
A number of embodiments concern methods of preparing an γδ T cell or a population of cells, particularly a population in which some are all the cells are clonal. In certain embodiments, a cell population comprises cells in which at least or at most 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% (or any range derivable therein) of the cells are clonal, i.e., the percentage of cells that have been derived from the same ancestor cell as another cell in the population. In other embodiments, a cell population comprises a cell population that is comprised of cells arising from, from at least, or from at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 7, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 (or any range derivable therein) different parental cells.
Methods for preparing, making, manufacturing, and using engineered γδ T cells and γδ T cell populations are provided. Methods include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more of the following steps in embodiments: obtaining pluripotent cells; obtaining hematopoietic progenitor cells; obtaining progenitor cells capable of becoming one or more hematopoietic cells; obtaining progenitor cells capable of becoming γδ T cells; selecting cells from a population of mixed cells using one or more cell surface markers; selecting CD34+ cells from a population of cells; isolating CD34+ cells from a population of cells; separating CD34+ and CD34− cells from each other; selecting cells based on a cell surface marker other than or in addition to CD34; introducing into cells one or more nucleic acids encoding an γδ T-cell receptor (TCR); infecting cells with a viral vector encoding an γδ T-cell receptor (TCR); transfecting cells with one or more nucleic acids encoding an γδ T-cell receptor (TCR); transfecting cells with an expression construct encoding an γδ T-cell receptor (TCR); integrating an exogenous nucleic acid encoding an γδ T-cell receptor (TCR) into the genome of a cell; introducing into cells one or more nucleic acids encoding a suicide gene product; infecting cells with a viral vector encoding a suicide gene product; transfecting cells with one or more nucleic acids encoding a suicide gene product; transfecting cells with an expression construct encoding a suicide gene product; integrating an exogenous nucleic acid encoding a suicide gene product into the genome of a cell; introducing into cells one or more nucleic acids encoding one or more polypeptides and/or nucleic acid molecules for gene editing; infecting cells with a viral vector encoding one or more polypeptides and/or nucleic acid molecules for gene editing; transfecting cells with one or more nucleic acids encoding one or more polypeptides and/or nucleic acid molecules for gene editing; transfecting cells with an expression construct encoding one or more polypeptides and/or nucleic acid molecules for gene editing; integrating an exogenous nucleic acid encoding one or more polypeptides and/or nucleic acid molecules for gene editing; editing the genome of a cell; editing the promoter region of a cell; editing the promoter and/or enhancer region for an γδ TCR gene; eliminating the expression one or more genes; eliminating expression of one or more HLA-I/II genes in the isolated human CD34+ cells; transfecting into a cell one or more nucleic acids for gene editing; culturing isolated or selected cells; expanding isolated or selected cells; culturing cells selected for one or more cell surface markers; culturing isolated CD34+ cells expressing γδ TCR; expanding isolated CD34+ cells; culturing cells under conditions to produce or expand γδ T cells; culturing cells in an artificial thymic organoid (ATO) system to produce γδ T cells; culturing cells in serum-free medium; culturing cells in an ATO system, wherein the ATO system comprises a 3D cell aggregate comprising a selected population of stromal cells that express a Notch ligand and a serum-free medium. It is specifically contemplated that one or more steps may be excluded in an embodiment.
In some embodiments, there are methods of preparing a population of clonal γδ T cells comprising: a) selecting CD34+ cells from human peripheral blood cells (PBMCs); b) introducing one or more nucleic acids encoding a human γδ T-cell receptor (TCR); c) eliminating surface expression of one or more HLA-I/II genes in the isolated human CD34+ cells; and, d) culturing isolated CD34+ cells expressing γδ TCR (e.g. in an artificial thymic organoid system) to produce γδ T cells. Typically, the ATO system comprises a 3D cell aggregate comprising a selected population of stromal cells that express a Notch ligand and a serum-free medium.
Pluripotent cells that may be used to create engineered γδ T cells include CD34+ hematopoietic progenitor stem cells. Cells may be from peripheral blood mononuclear cells (PBMCs), bone marrow cells, fetal liver cells, embryonic stem cells, cord blood cells, induced pluripotent stem cells (iPS cells), or a combination thereof. In some embodiments, methods comprise isolating CD34− cells or separating CD34− and CD34+ cells. While embodiments involve manipulating the CD34+ cells further, CD34− cells may be used in the creation of γδ T cells. Therefore, in some embodiments, the CD34− cells are subsequently used, and may be saved for this purpose.
Certain methods involve culturing selected CD34+ cells in media prior to introducing one or more nucleic acids into the cells. Culturing the cells can include incubating the selected CD34+ cells with media comprising one or more growth factors. In some embodiments, one or more growth factors comprise c-kit ligand, flt-3 ligand, and/or human thrombopoietin (TPO). In further embodiments, the media includes c-kit ligand, flt-3 ligand, and TPO. In some embodiments, the concentration of the one or more growth factors is between about 5 ng/ml to about 500 ng/ml with respect to either each growth factor or the total of any and all of these particular growth factors. The concentration of a single growth factor or the combination of growth factors in media can be about, at least about, or at most about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 410, 420, 425, 430, 440, 441, 450, 460, 470, 475, 480, 490, 500 (or any range derivable) ng/ml or g/ml or more.
In typical embodiments, a nucleic acid may comprise a nucleotide sequence encoding an γ-TCR and/or a S-TCR, as discussed herein. In certain embodiments, one nucleic acid encodes both the gamma and delta chains of the TCR. In some embodiments, a further nucleic acid may comprise a nucleic acid sequence encoding an α-TCR and/or a β-TCR polypeptide, and/or one or more iNKT TCR polypeptides. In additional embodiments, a nucleic acid further comprises a nucleic acid sequence encoding a suicide gene product. In some embodiments, a nucleic acid molecule that is introduced into a selected CD34+ cell encodes the TCR, and the suicide gene product. In other embodiments, a method also involves introducing into the selected CD34+ cells a nucleic acid encoding a suicide gene product, in which case a different nucleic acid molecule encodes the suicide gene product than a nucleic acid encoding at least one of the TCR genes.
As discussed above, in some embodiments the γδ T cells do not express the HLA-I and/or HLA-II molecules on the cell surface, which may be achieved by disrupting the expression of genes encoding beta-2-microglobulin (B2M), transactivator (CIITA), or HLA-I and HLA-II molecules. In certain embodiments, methods involve eliminating surface expression of one or more HLA-I/II molecules in the isolated human CD34+ cells. In particular embodiments, eliminating expression may be accomplished through gene editing of the cell's genomic DNA. Some methods include introducing CRISPR and one or more guide RNAs (gRNAs) corresponding to B2M or CIITA into the cells. In particular embodiments, CRISPR or the one or more gRNAs are transfected into the cell by electroporation or lipid-mediated transfection. Consequently, methods may involve introducing CRISPR and one or more gRNAs into a cell by transfecting the cell with nucleic acid(s) encoding CRISPR and the one or more gRNAs. A different gene editing technology may be employed in some embodiments.
Similarly, in some embodiments, one or more nucleic acids encoding the TCR receptor are introduced into the cell. This can be done by transfecting or infecting the cell with a recombinant vector, which may or may not be a viral vector as discussed herein. The exogenous nucleic acid may incorporate into the cell's genome in some embodiments.
In some embodiments, cells are cultured in cell-free medium. In certain embodiments, the serum-free medium further comprises externally added ascorbic acid. In particular embodiments, methods involve adding ascorbic acid medium. In further embodiments, the serum-free medium further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or all 16 (or a range derivable therein) of the following externally added components: FLT3 ligand (FLT3L), interleukin 7 (IL-7), stem cell factor (SCF), thrombopoietin (TPO), stem cell factor (SCF), IL-2, IL-4, IL-6, IL-15, IL-21, TNF-alpha, TGF-beta, interferon-gamma, interferon-lambda, TSLP, thymopentin, pleotrophin, or midkine. In additional embodiments, the serum-free medium comprises one or more vitamins. In some cases, the serum-free medium includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the following vitamins (or any range derivable therein): comprise biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, vitamin B12, or a salt thereof. In certain embodiments, medium comprises or comprise at least biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, or combinations or salts thereof. In additional embodiments, serum-free medium comprises one or more proteins. In some embodiments, serum-free medium comprises 1, 2, 3, 4, 5, 6 or more (or any range derivable therein) of the following proteins: albumin or bovine serum albumin (BSA), a fraction of BSA, catalase, insulin, transferrin, superoxide dismutase, or combinations thereof. In other embodiments, serum-free medium comprises 1, 2, 3, 4, 5, 7, 8, 9, 10, or 11 of the following compounds: corticosterone, D-Galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triodo-I-thyronine, or combinations thereof. In further embodiments, serum-free medium comprises a B-27® supplement, xeno-free B-27® supplement, GS21TM supplement, or combinations thereof. In additional embodiments, serum-free medium comprises or further comprises amino acids, monosaccharides, and/or inorganic ions. In some aspects, serum-free medium comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of the following amino acids: arginine, cysteine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine, or combinations thereof. In other aspects, serum-free medium comprises 1, 2, 3, 4, 5, or 6 of the following inorganic ions: sodium, potassium, calcium, magnesium, nitrogen, or phosphorus, or combinations or salts thereof. In additional aspects, serum-free medium comprises 1, 2, 3, 4, 5, 6 or 7 of the following elements: molybdenum, vanadium, iron, zinc, selenium, copper, or manganese, or combinations thereof.
In some methods, cells are cultured in an artificial thymic organoid (ATO) system. The ATO system involves a three-dimensional (3D) cell aggregate, which is an aggregate of cells. In certain embodiments, the 3D cell aggregate comprises a selected population of stromal cells that express a Notch ligand. In some embodiments, a 3D cell aggregate is created by mixing CD34+ transduced cells with the selected population of stromal cells on a physical matrix or scaffold. In further embodiments, methods comprise centrifuging the CD34+ transduced cells and stromal cells to form a cell pellet that is placed on the physical matrix or scaffold. In certain embodiments, stromal cells express a Notch ligand that is an intact, partial, or modified DLL1, DLL4, JAG1, JAG2, or a combination thereof. In further embodiments, the Notch ligand is a human Notch ligand. In other embodiments, the Notch ligand is human DLL1.
The methods of the disclosure may produce a population of cells (e.g. via a differentiation and/or expansion step) comprising at least 1×102, 1×103, 1×104, 1×105, 1×106, 1×107, 1×108, 1×109, 1×1010, 1×1011, 1×1012, 1×1013, 1×1014, 1×1015, 1×1016, 1×1017, 1×1018, 1×1019, 1×1020, or 1×1021 (or any derivable range therein) cells that may express a marker or have a high or low level of a certain marker. The cell population number may be one that is achieved without cell sorting based on marker expression or without cell sorting based on γδ T cell marker expression or without cell sorting based on T-cell marker expression. In some embodiments, the cell population size may be one that is achieved without cell sorting based on the binding of an antigen to a heterologous targeting element, such as a CAR, TCR, BiTE, or other heterologous tumor-targeting agent. Furthermore, the population of cells achieved may be one that comprises at least 1×102, 1×103, 1×104, 1×105, 1×106, 1×107, 1×108, 1×109, 1×1010, 1×1011, 1×1012, 1×1013, 1×1014, 1×1015, 1×1016, 1×1017, 1×1018, 1×1019, 1×1020, or 1×1021 (or any derivable range therein) cells that is made within a certain time period such as a time period that is at least, at most, or exactly 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 days or 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 weeks (or any derivable range therein).
In some embodiments, feeder cells used in methods comprise CD34− cells. These CD34− cells may be from the same population of cells selected for CD34+ cells. In additional embodiments, cells may be activated. In certain embodiments, methods comprise activating γδ T cells. In specific embodiments, γδ T cells have been activated and expanded with ZOL. Cells may be incubated or cultured with ZOL so as to activate and expand them. In some embodiments, feeder cells have been pulsed with ZOL.
Cells may be used immediately, or they may be stored for future use. In certain embodiments, cells that are used to create γδ T cells are frozen, while produced γδ T cells may be frozen in some embodiments. In some aspects, cells are in a solution comprising dextrose, one or more electrolytes, albumin, dextran, and DMSO. In other embodiments, cells are in a solution that is sterile, nonpyrogenic, and isotonic. In some embodiments, the engineered γδ T cell is derived from a hematopoietic stem cell. In some embodiments, the engineered γδ T cell is derived from a G-CSF mobilized CD34+ cells. In some embodiments, the cell is derived from a cell from a human patient that doesn't have cancer. In some embodiments, the cell doesn't express an endogenous TCR.
The number of cells produced by a production cycle may be about, at least about, or at most about 102, 103, 104, 105, 106, 107, 108, 109, 1010, 1011, 1012, 1013, 1014, 1015 cells or more (or any range derivable therein), which are engineered γδ T cells in some embodiments. In some cases, a cell population comprises at least about 106-1012 engineered γδ T cells. It is contemplated that in some embodiments, that a population of cells with these numbers is produced from a single batch of cells and are not the result of pooling batches of cells separately produced—i.e., from a single production cycle. In some embodiments, a cell population is frozen and then thawed. The cell population may be used to create engineered γδ T cells, or they may comprise engineered γδ T cells.
In some embodiments, methods include introducing one or more additional nucleic acids into the cell population, which may or may not have been previously frozen and thawed. This use provides one of the advantages of creating an off-the-shelf γδ T cell. In particular embodiments, the one or more additional nucleic acids encode one or more therapeutic gene products. Examples of therapeutic gene products include at least the following: 1. Antigen recognition molecules, e.g. CAR (chimeric antigen receptor) and/or TCR (T cell receptor); 2. Co-stimulatory molecules, e.g. CD28, 4-1BB, 4-1BBL, CD40, CD40L, ICOS; and/or 3. Cytokines, e.g. IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-7, IL-9, IL-15, IL-12, IL-17, IL-21, IL-23, IFN-γ, TNF-α, TGF-β, G-CSF, GM-CSF; 4. Transcription factors, e.g. T-bet, GATA-3, RORγt, FOXP3, and Bcl-6. Therapeutic antibodies are included, as are chimeric antigen receptors, single chain antibodies, monobodies, humanized, antibodies, bi-specific antibodies, single chain FV antibodies or combinations thereof.
In some embodiments, there are engineered γδ T cells produced by a method comprising: a) selecting CD34+ cells from human peripheral blood cells (PBMCs); b) culturing the CD34+ cells with medium comprising growth factors such as c-kit ligand, flt-3 ligand, and human thrombopoietin (TPO) or the like; c) transducing the selected CD34+ cells with a lentiviral vector comprising a nucleic acid sequence encoding γ-TCR, δ-TCR, thymidine kinase, and a reporter gene product; d) introducing into the selected CD34+ cells Cas9 and gRNA for beta 2 microglobulin (B2M) and/or CTIIA to eliminate expression of B2M or CTIIA; e) culturing the transduced cells for 2-10 weeks with an irradiated stromal cell line expressing an exogenous Notch ligand to expand γδ T cells in a 3D aggregate cell culture; f) selecting γδ T cells lacking expression of B2M and/or CTIIA; and, g) culturing the selected γδ T cells with irradiated feeder cells.
In particular embodiments, γδ T cells produced from transduced cells (e.g. HSPCs) are further modified to have one or more characteristics, including to render the cells suitable for allogeneic use or more suitable for allogeneic use than if the cells were not further modified to have one or more characteristics. The present disclosure encompasses UHSC-γδ T cells that are suitable for allogeneic use, if desired. In some embodiments, the HSC-γδ T cells are non-alloreactive and express an exogenous gamma delta TCR. These cells are useful for “off the shelf” cell therapies and do not require the use of the patient's own γδ T or other cells. Therefore, the current methods provide for a more cost-effective, less labor-intensive cell immunotherapy.
In specific embodiments, HSC-γδ T cells are engineered to be HLA-negative to achieve safe and successful allogeneic engraftment without causing graft-versus-host disease (GvHD) and being rejected by host immune cells (HvG rejection). In specific embodiments, allogeneic HSC-γδ T cells do not express endogenous TCRs and do not cause GvHD, because the expression of the transgenic γδ TCR gene blocks the recombination of endogenous TCRs through allelic exclusion. In particular embodiments, allogeneic UHSC-γδ T cells do not express HLA-I and/or HLA-II molecules on cell surface and resist host CD8+ and CD4+ T cell-mediated allograft depletion and sr39TK immunogen-targeting depletion. Thus, in certain embodiments the engineered γδ T cells do not express surface HLA-I or -II molecules, achieved through disruption of genes encoding proteins relevant to HLA-I/II expression, including but not limited to beta-2− microglobulin (B2M), major histocompatibility complex II transactivator (CIITA), or HLA-I/II molecules. In some cases, the HLA-I or HLA-II are not expressed on the surface of γδ T cells because the cells were manipulated by gene editing, which may or may not involve CRISPR-Cas9.
In cases wherein the γδ T cells have been modified to exhibit one or more characteristics of any kind, the γδ T cells may comprise nucleic acid sequences from a recombinant vector that was introduced into the cells. The vector may be a non-viral vector, such as a plasmid, or a viral vector, such as a lentivirus, a retrovirus, an adeno-associated virus (AAV), a herpesvirus, or adenovirus.
The γδ T cells of the invention may or may not have been exposed to one or more certain conditions before, during, or after their production. In specific cases, the cells are not or were not exposed to media that comprises animal serum. The cells may be frozen. The cells may be present in a solution comprising dextrose, one or more electrolytes, albumin, dextran, and/or DMSO. Any solution in which the cells are present may be a solution that is sterile, nonpyogenic, and isotonic. The cells may have been activated and expanded by any suitable manner, such as activated with ZOL, for example.
Aspects of the disclosure relate to a human cell comprising: i) an exogenous expression or activity inhibitor of, or ii) a genomic mutation of: one or more of β2 microglobin (B2M), CIITA, TRAC, TRBC1, or TRBC2. In some embodiments, the cell comprises a genomic mutation. In some embodiments, the genomic mutation comprises a mutation of one or more endogenous genes in the cell's genome, wherein the one or more endogenous genes comprise the B2M, CIITA, TRAC, TRBC1, or TRBC2 gene. In some embodiments, the mutation comprises a loss of function mutation. In some embodiments, the inhibitor is an expression inhibitor. In some embodiments, the inhibitor comprises an inhibitory nucleic acid. In some embodiments, the inhibitory nucleic acid comprises one or more of a siRNA, shRNA, miRNA, or an antisense molecule. In some embodiments, the cells comprise an activity inhibitor. In some embodiments, following modification the cell is deficient in any detectable expression of one or more of B2M, CIITA, TRAC, TRBC1, or TRBC2 proteins. In some embodiments, the cell comprises an inhibitor or genomic mutation of B2M. In some embodiments, the cell comprises an inhibitor or genomic mutation of CIITA. In some embodiments, the cell comprises an inhibitor or genomic mutation of TRAC. In some embodiments, the cell comprises an inhibitor or genomic mutation of TRBC1. In some embodiments, the cell comprises an inhibitor or genomic mutation of TRBC2. In some embodiments, at least 90% of the genomic DNA encoding B2M, CIITA, TRAC, TRBC1, and/or TRBC2 is deleted. In some embodiments, at least or at most 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, or 100% (or any range derivable therein) of the genomic DNA encoding B2M, CIITA, TRAC, TRBC1, and/or TRBC2 is deleted. In other embodiments, a deletion, insertion, and/or substitution is made in the genomic DNA. In some embodiments, the cell is a progeny of the human stem or progenitor cell.
The UHSC-γδ T cells that are modified to be HLA-negative may be genetically modified by any suitable manner. The genetic mutations of the disclosure, such as those in the CIITA and/or B2M genes can be introduced by methods known in the art. In certain embodiments, engineered nucleases may be used to introduce exogenous nucleic acid sequences for genetic modification of any cells referred to herein. Genome editing, or genome editing with engineered nucleases (GEEN) is a type of genetic engineering in which DNA is inserted, replaced, or removed from a genome using artificially engineered nucleases, or “molecular scissors.” The nucleases create specific double-stranded break (DSBs) at desired locations in the genome and harness the cell's endogenous mechanisms to repair the induced break by natural processes of homologous recombination (HR) and nonhomologous end-joining (NHEJ). Non-limiting engineered nucleases include Zinc finger nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), the CRISPR/Cas9 system, and engineered meganuclease re-engineered homing endonucleases. Any of the engineered nucleases known in the art can be used in certain aspects of the methods and compositions.
In cases wherein the engineered γδ T cells comprise one or more suicide genes for subsequent depletion upon need, the suicide gene may be of any suitable kind. The γδ T cells of the disclosure may express a suicide gene product that may be enzyme-based, for example. Examples of suicide gene products include herpes simplex virus thymidine kinase (HSV-TK), purine nucleoside phosphorylase (PNP), cytosine deaminase (CD), carboxypetidase G2, cytochrome P450, linamarase, beta-lactamase, nitroreductase (NTR), carboxypeptidase A, or inducible caspase 9. Thus, in specific cases, the suicide gene may encode thymidine kinase (TK). In specific cases, the TK gene is a viral TK gene, such as a herpes simplex virus TK gene. In particular embodiments, the suicide gene product is activated by a substrate, such as ganciclovir, penciclovir, or a derivative thereof.
In some embodiments, the engineered γδ T cells are able to be imaged or otherwise detected. In particular cases, the cells comprise an exogenous nucleic acid encoding a polypeptide that has a substrate that may be labeled for imaging, and the imaging may be fluorescent, radioactive, colorimetric, and so forth. In specific cases, the cells are detected by positron emission tomography. The cells in at least some cases express sr39TK gene that is a positron emission tomography (PET) reporter/thymidine kinase gene that allows for tracking of these genetically modified cells with PET imaging and elimination of these cells through the sr39TK suicide gene function.
Encompassed by the disclosure are populations of engineered γδ T cells. In particular aspects, γδ T clonal cells comprise an exogenous nucleic acid encoding an γδ T-cell receptor and lack surface expression of one or more HLA-I or HLA-II molecules. The γδ T cells may comprise an exogenous nucleic acid encoding a suicide gene, including an enzyme-based suicide gene such as thymidine kinase (TK). The TK gene may be a viral TK gene, such as a herpes simplex virus TK gene. In the cells of the population the suicide gene may be activated by a substrate, such as ganciclovir, penciclovir, or a derivative thereof, for example. The cells may comprise an exogenous nucleic acid encoding a polypeptide that has a substrate that may be labeled for imaging, and in some cases a suicide gene product is the polypeptide that has a substrate that may be labeled for imaging. In specific aspects, the suicide gene is sr39TK. In particular cases for the γδ T cell population, the γδ T cells comprise nucleic acid sequences from a recombinant vector that was introduced into the cells, such as a viral vector (including at least a lentivirus, a retrovirus, an adeno-associated virus (AAV), a herpesvirus, or adenovirus).
In certain embodiments, the cells of the γδ T cell population may or may not have been exposed to, or are exposed to, one or more certain conditions. In certain cases, for example, the cells of the population not exposed or were not exposed to media that comprises animal serum. The cells of the population may or may not be frozen. In some cases, the cells of the population are in a solution comprising dextrose, one or more electrolytes, albumin, dextran, and/or DMSO. The solution may comprise dextrose, one or more electrolytes, albumin, dextran, and DMSO. The cells may be in a solution that is sterile, nonpyogenic, and isotonic. In specific cases the γδ T cells have been activated, such as activated with ZOL. In specific aspects, the cell population comprises at least about 102-106 clonal cells. The cell population may comprise at least about 106-1012 total cells, in some cases.
In particular embodiments there is an gamma delta (γδ) T cell population comprising: clonal γδ T cells comprising one or more exogenous nucleic acids encoding an γδ T-cell receptor and a thymidine kinase suicide, wherein the clonal γδ T cells have been engineered not to express functional beta-2-microglobulin (B2M), major histocompatibility complex class II transactivator (CIITA), and/or HLA-I and HLA-II molecules and wherein the cell population is at least about 106-1012 total cells and comprises at least about 102-106 clonal cells. In some cases, the cells are frozen in a solution.
In particular embodiments, the UHSC-γδ T cells and/or precursors thereto may be specifically formulated and/or they may be cultured in a particular medium (whether or not they are present in an in vitro ATO culture system) at any stage of a process of generating the UHSC-γδ T cells. The cells may be formulated in such a manner as to be suitable for delivery to a recipient without deleterious effects.
The medium in certain aspects can be prepared using a medium used for culturing animal cells as their basal medium, such as any of AIM V, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL 1066, Glasgow MEM, Improved MEM Zinc Option, IMDM, Medium 199, Eagle MEM, αMEM, DMEM, Ham, RPMI-1640, and Fischer's media, as well as any combinations thereof, but the medium may not be particularly limited thereto as far as it can be used for culturing animal cells. Particularly, the medium may be xeno-free or chemically defined.
The medium can be a serum-containing or serum-free medium, or xeno-free medium. From the aspect of preventing contamination with heterogeneous animal-derived components, serum can be derived from the same animal as that of the stem cell(s). The serum-free medium refers to medium with no unprocessed or unpurified serum and accordingly, can include medium with purified blood-derived components or animal tissue-derived components (such as growth factors).
The medium may contain or may not contain any alternatives to serum. The alternatives to serum can include materials which appropriately contain albumin (such as lipid-rich albumin, bovine albumin, albumin substitutes such as recombinant albumin or a humanized albumin, plant starch, dextrans and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3′-thiolgiycerol, or equivalents thereto. The alternatives to serum can be prepared by the method disclosed in International Publication No. 98/30679, for example (incorporated herein in its entirety). Alternatively, any commercially available materials can be used for more convenience. The commercially available materials include knockout Serum Replacement (KSR), Chemically-defined Lipid concentrated (Gibco), and Glutamax (Gibco).
In further embodiments, the medium may be a serum-free medium that is suitable for cell development. For example, the medium may comprise B-27© supplement, xeno-free B-27© supplement (available at world wide web at thermofisher.com/us/en/home/technical-resources/media-formulation.250.html), NS21 supplement (Chen et al., J Neurosci Methods, 2008 Jun. 30; 171(2): 239-247, incorporated herein in its entirety), GS21™ supplement (available at world wide web at amsbio.com/B-27.aspx), or a combination thereof at a concentration effective for producing T cells from the 3D cell aggregate.
Cell expressing polypeptides comprising an amino acid sequence shown in Table 1 (SEQ ID NO: 1-SEQ ID NO: 52) and/or other γδ T cells may be produced by any suitable method(s). The method(s) may utilize one or more successive steps for one or more modifications to cells and/or utilize one or more simultaneous steps for one or more modifications to cells. In specific embodiments, a starting source of cells are modified to become functional as γδ T cells followed by one or more steps to add one or more additional characteristics to the cells, such as the ability to be imaged, and/or the ability to be selectively killed, and/or the ability to be able to be used allogeneically. In specific embodiments, at least part of the process for generating UHSC-γδ T cells occurs in a specific in vitro culture system. An example of a specific in vitro culture system is one that allows differentiation of certain cells at high efficiency and high yield. In specific embodiments the in vitro culture system is an artificial thymic organoid (ATO) system.
In specific cases, UHSC-γδ T cells may be generated by the following: 1) genetic modification of donor HSCs to express γδ TCRs (for example, via lentiviral vectors) and to eliminate expression of HLA-I/II molecules (for example, via CRISPR/Cas9-based gene editing); 2) in vitro differentiation into γδ T cells via an ATO culture, 3) in vitro γδ T cell purification and expansion, and 4) formulation and cryopreservation and/or use.
Particular embodiments of the disclosure provide methods of preparing a population of clonal gamma delta (T6) T cells comprising: a) selecting CD34+ cells from human peripheral blood cells (PBMCs); b) introducing one or more nucleic acids encoding a human γδ T-cell receptor (TCR); c) eliminating expression of one or more HLA-I/II genes in the isolated human CD34+ cells; and, d) culturing isolated CD34+ cells expressing γδ TCR in an artificial thymic organoid (ATO) system to produce γδ T cells, wherein the ATO system comprises a 3D cell aggregate comprising a selected population of stromal cells that express a Notch ligand and a serum-free medium. The method may further comprise isolating CD34− cells. In alternative embodiments, other culture systems than the ATO system is employed, such as a 2-D culture system or other forms of 3-D culture systems (e.g., FTOC-like culture, metrigel-aided culture).
Specific aspects of the disclosure relate to a novel three-dimensional cell culture system to produce γδ T cells from less differentiated cells such as embryonic stem cells, pluripotent stem cells, hematopoietic stem or progenitor cells, induced pluripotent stem (iPS) cells, or stem or progenitor cells. Stem cells of any type may be utilized from various resources, including at least fetal liver, cord blood, and peripheral blood CD34+ cells (either G-CSF-mobilized or non-G-CSF-mobilized), for example.
In particular embodiments, the system involves using serum-free medium. In certain aspects, the system uses a serum-free medium that is suitable for cell development for culturing of a three-dimensional cell aggregate. Such a system produces sufficient amounts of UHSC-γδ T cells. In embodiments of the disclosure, the 3D cell aggregate is cultured in a serum-free medium comprising insulin for a time period sufficient for the in vitro differentiation of stem or progenitor cells to UHSC-γδ T cells or precursors to UHSC-γδ T cells.
Embodiments of a cell culture composition comprise an ATO 3D culture that uses highly-standardized, serum-free components and a stromal cell line to facilitate robust and highly reproducible T cell differentiation from human HSCs. In certain embodiments, cell differentiation in ATOs closely mimicked endogenous thymopoiesis and, in contrast to monolayer co-cultures, supported efficient positive selection of functional UHSC-γδ T. Certain aspects of the 3D culture compositions use serum-free conditions, avoid the use of human thymic tissue or proprietary scaffold materials, and facilitate positive selection and robust generation of fully functional, mature human UHSC-γδ T cells from source cells.
Cells produced by the preparation methods may be frozen. The produced cells may be in a solution comprising dextrose, one or more electrolytes, albumin, dextran, and DMSO. The solution may be sterile, nonpyogenic, and isotonic.
Genetic modification may also be introduced to certain components to generate antigen-specific T cells, and to model positive and negative selection. Examples of these modifications include transduction of HSCs with a lentiviral vector encoding an antigen-specific T cell receptor (TCR) or chimeric antigen receptor (CAR) for the generation of antigen-specific, allelically excluded naïve T cells; transduction of HSCs with gene/s to direct lineage commitment to specialized lymphoid cells. For example, transduction of HSCs with a gamma delta (76) associated TCR to generate functional γδ T cells in ATOs; transduction of the ATO stromal cell line (e.g., MS5-hDLL1) with human MHC genes (e.g. human CD1d gene) to enhance positive selection and maturation of both TCR engineered or non-engineered T cells in ATOs; and/or transduction of the ATO stromal cell line with an antigen plus costimulatory molecules or cytokines to enhance the positive selection of CAR T cells in ATOs.
In producing the engineered γδ T cells, CD34+ cells from human peripheral blood cells (PBMCs) may be modified by introducing certain exogenous gene(s) and by knocking out certain endogenous gene(s). The methods may further comprise culturing selected CD34+ cells in media prior to introducing one or more nucleic acids into the cells. The culturing may comprise incubating the selected CD34+ cells with medium comprising one or more growth factors, in some cases, and the one or more growth factors may comprise c-kit ligand, flt-3 ligand, and/or human thrombopoietin (TPO), for example. The growth factors may or may not be at a certain concentration, such as between about 5 ng/ml to about 500 ng/ml.
In particular methods the nucleic acid(s) to be introduced into cells are one or more nucleic acids that comprise a nucleic acid sequence encoding an γ-TCR and a S-TCR (e.g., SEQ ID NO: 1-SEQ ID NO: 52). The methods may comprise introducing into the selected cells a nucleic acid encoding a suicide gene. In specific aspects, one nucleic acid encodes both the γ-TCR and the S-TCR, or one nucleic acid encodes the γ-TCR, the S-TCR, and the suicide gene. The suicide gene may be enzyme-based, such as thymidine kinase (TK) including a viral TK gene such as one from herpes simplex virus TK gene. The suicide gene may be activated by a substrate, such as ganciclovir, penciclovir, or a derivative thereof. The cells may be engineered to comprise an exogenous nucleic acid encoding a polypeptide that has a substrate that may be labeled for imaging. In some cases, a suicide gene product is a polypeptide that has a substrate that may be labeled for imaging, such as sr39TK.
In manufacturing the engineered γδ T cells, the cells may be present in a particular serum-free medium, including one that comprises externally added ascorbic acid. In specific aspects, the serum-free medium further comprises externally added FLT3 ligand (FLT3L), interleukin 7 (IL-7), stem cell factor (SCF), thrombopoietin (TPO), stem cell factor (SCF), thrombopoietin (TPO), IL-2, IL-4, IL-6, IL-15, IL-21, TNF-alpha, TGF-beta, interferon-gamma, interferon-lambda, TSLP, thymopentin, pleotrophin, midkine, or combinations thereof. The serum-free medium may further comprise vitamins, including biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, vitamin B12, or combinations thereof or salts thereof. The serum-free medium may further comprise one or more externally added (or not) proteins, such as albumin or bovine serum albumin, a fraction of BSA, catalase, insulin, transferrin, superoxide dismutase, or combinations thereof. The serum-free medium may further comprise corticosterone, D-Galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triodo-I-thyronine, or combinations thereof. The serum-free medium may comprise a B-27© supplement, xeno-free B-27© supplement, GS21™ supplement, or combinations thereof. Amino acids (including arginine, cysteine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine, or combinations thereof), monosaccharides, and/or inorganic ions (including sodium, potassium, calcium, magnesium, nitrogen, or phosphorus, or combinations or salts thereof, for example) may be present in the serum-free medium. The serum-free medium may further comprise molybdenum, vanadium, iron, zinc, selenium, copper, or manganese, or combinations thereof.
Further aspects and embodiments of the invention are discussed in the following sections.
Human Vγ9Vδ2 TCRs (referred to as γδ TCRs herein) were cloned from healthy donor peripheral blood mononuclear cells (PBMCs)-derived γδ T (PBMC-γδT) cells. Illustrative working embodiments of the methods disclosed herein as well as γδ TCR sequences (e.g., amino acid sequences and/or gene coding sequences) and illustrative γδ TCR gene delivery vectors are discussed below.
Human γδ T cells can be generated through γδ TCR gene-engineering of stem and progenitor cells (e.g., CD34+ HSCs, ESCs, iPSCs), followed by differentiation (in vivo or ex vivo) into transgenic γδ T cells.
HSCs refer to human CD34+ hematopoietic progenitor and stem cells, that can be directly isolated from cord blood or G-CSF-mobilized peripheral blood (CB HSCs or PBSCs), or can be derived from embryonic or induced pluripotent stem cells (ES-HSCs or iPS-HSCs). HSCs can be gene engineered via vector-dependent or vector-independent gene delivery methods, or via other gene editing methods (e.g., CRISPR, TALEN, Zinc finger and the like.
In addition to the antigen-specificity endowed by the monoclonal transgenic γδ TCR, HSC-γδT can be further engineered to express additional targeting molecules to enhance their disease-targeting capacity. Such targeting molecules can be Chimeric Antigen Receptors (CARs), natural or synthetic receptors/ligands, or others. The resulting CAR-γδT cells can then be utilized for off-the-shelf disease-targeting cellular therapy.
In addition to the antigen-specificity endowed by the monoclonal transgenic TCR, HSC-γδT can be further engineered to express additional targeting molecules to enhance their disease-targeting capacity. Such targeting molecules can be Chimeric Antigen Receptors (CARs), natural or synthetic receptors/ligands, or others. The resulting CAR-γδT cells can then be utilized for off-the-shelf disease-targeting cellular therapy.
The HSC-γδT cells and derivatives can also be further engineered to overexpress genes encoding T cell stimulatory factors, or to disrupt genes encoding T cell inhibitory factors, resulting in functionally enhanced HSC-γδT cells and derivatives.
A γδ TCR gene-engineered HSC adoptive transfer method is disclosed that can generate HSC-γδT cells in vivo, cells that can potentially provide patients with a life-long supply of engineered HSC-γδT cells targeting diseases.
The procedure includes 1) genetic modification of human CD34+ hematopoietic stem cells (HSCs) to express a selected γδ TCR gene; 2) adoptive transfer γδ TCR gene engineered HSCs into a patient; 3) in vivo generation of HSC-γδT cells; 4) due to longevity of self-renewal of HSCs, this method can potentially protect patient with life-long supplies of HSC-γδT cells.
Ex vivo differentiation culture methods are disclosed to generate AlloHSC-γδT cells for off-the-shelf cell therapy applications.
The procedure includes 1) genetic modification of human CD34+ hematopoietic stem cells (HSCs) to express a selected γδ TCR gene; 3) ex vivo generation of AlloHSC-γδT cells with feeder cells (e.g., artificial thymic organoid culture; 3) ex vivo expansion of differentiated AlloHSC-γδT cells.
The production procedure includes 1) genetic modification of human CD34+ hematopoietic stem cells (HSCs) to express a selected TCR gene; 2) ex vivo differentiation AlloHSC-γδT cells without feeder cells; and 3) ex vivo expansion of differentiated AlloHSC-γδT cells.
Engineered γδ T cells can be used to target multiple diseases including cancer and infectious diseases.
Proof of principle data are provided for treating a large collection of human cancers, including blood cancer (e.g., multiple myeloma) and solid tumor (e.g., ovarian, melanoma, prostate, breast, and lung cancer).
Proof of principle data are provided for targeting COVID-19.
Human CD34+ HSCs were cultured for no more than 48 hours in X-VIVO 15 serum-free hematopoietic cell medium containing recombinant human Flt3 ligand, SCF, TPO, and IL-3 in no-tissue culture-treated plates coated with Retronectin. Viral transduction was performed at 24 hours by adding concentrated lentivector directly to the culture medium. At around 48 hours CD34+ cells were collected and intravenously (i.v.) injected in NOD.Cg-Prkdscid Il2rgtm1/Wji/SzJ (NSG) mice that had received 270 rads of total body irradiation. 1-2 fragments of human fetal or postnatal thymus were implanted under the kidney capsule of each recipient NSG mice.
Fresh or frozen/thawed CD34+ HSCs are cultured in stem cell culture media (base medium supplemented with cytokine cocktails including IL-3, IL-7, IL-6, SCF, EPO, TPO, FLT3L, and others) for 12-72 hours in flasks coated with retronectin, followed by addition of the TCR gene-delivery vector, and culturing for an additional 12-48 hours. TCR gene-modified HSCs are then differentiated into AlloHSC-γδT cells in a feeder-dependent culture (e.g., artificial thymic organoid culture) over 4-10 weeks. Artificial thymic organoid (ATO) was generated following a previously established protocol (Seet et al., Cell Stem Cell. 2019 Mar. 7; 24(3):376-389).
At Stage 2, differentiated AlloHSC-γδT cells are stimulated with TCR cognate antigens (proteins, peptides, lipids, phosphor-antigens, small molecules, and others) or non-specific TCR stimulatory reagents (anti-CD3/anti-CD28 antibodies or antibody-coated beads, Concanavalin A, PMA/Ionomycin, and others), and expanded for up to 1 month in T cell culture media. The culture can be supplemented with T cell supporting cytokines (IL-2, IL-7, IL-15, and others). AlloHSC-γδT Cell Derivatives
In some embodiments, AlloHSC-γδT cells can be further engineered to express additional transgenes. In one embodiment, such transgenes encode disease targeting molecules such as chimeric antigen receptors (CARs), T-cell receptors (TCRs), and other native or synthetic receptor/ligands. In another embodiment, such transgenes can encode T cell regulatory proteins such as IL-2, IL-7, IL-15, IFN-γ, TNF-α, CD28, 4-1BB, OX40, ICOS, FOXP3, and others. Transgenes can be introduced into post-expansion AlloHSC-γδT cells or their progenitor cells (HSCs, newly differentiated AlloHSC-γδT cells, in-expansion AlloHSC-γδT cells) at various culture stages.
In some embodiments, AlloHSC-γδT cells can be further engineered to disrupt selected genes using gene editing tools (CRISPR, TALEN, Zinc-Finger, and others). In one embodiment, disrupted genes encode T cell immune checkpoint inhibitors (PD-1, CTLA-4, TIM-3, LAG-3, and others). Deficiency of these negative regulatory genes may enhance the disease fighting capacity of AlloHSC-γδT cells, making them resistance to disease-induced anergy and tolerance.
Fresh or frozen/thawed CD34+ HSCs are cultured in stem cell culture media (base medium supplemented with cytokine cocktails including IL-3, IL-7, IL-6, SCF, EPO, TPO, FLT3L, and others) for 12-72 hours in flasks coated with retronectin, followed by addition of the TCR gene-delivery vector, and culturing for an additional 12-48 hours.
TCR gene-modified HSCs are then differentiated into AlloHSC-γδT cells in a differentiation medium over a period of 4-10 weeks without feeders. Non-tissue culture-treated plates are coated with a AlloHSC-γδT Culture Coating Material (DLL-1/4, VCAM-1/5, retronectin, and others). CD34+ HSCs are suspended in an Expansion Medium (base medium containing serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, SCF, TPO, IL-3, IL-6, Flt3 ligand, human LDL, UM171, and additives), seeded into the coated wells of a plate, and cultured for 3-7 days. Expansion Medium is refreshed every 3-4 days. Cells are then collected and suspended in a Maturation Medium (base medium containing serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, SCF, IL-3, IL-6, IL-7, IL-15, Flt3 ligand, ascorbic acid, and additives). Maturation Medium is refreshed 1-2 times per week.
Differentiated AlloHSC-γδT cells are stimulated with TCR cognate antigens (proteins, peptides, lipids, phosphor-antigens, small molecules, and others) or non-specific TCR stimulatory reagents (anti-CD3/anti-CD28 antibodies or antibody-coated beads, Concanavalin A, PMA/Ionomycin, and artificial APCs), and expanded for up to 1 month in T cell culture media. The culture can be supplemented with T cell supporting cytokines (IL-2, IL-7, IL-15, and others).
In some embodiments, AlloHSC-γδT cells can be further engineered to express additional transgenes. In one embodiment, such transgenes encode disease targeting molecules such as chimeric antigen receptors (CARs), T-cell receptors (TCRs), and other native or synthetic receptor/ligands. In another embodiment, such transgenes can encode T cell regulatory proteins such as IL-2, IL-7, IL-15, IFN-γ, TNF-α, CD28, 4-1BB, OX40, ICOS, FOXP3, and others. Transgenes can be introduced into post-expansion AlloHSC-γδT cells or their progenitor cells (HSCs, newly differentiated AlloHSC-γδT cells, in-expansion AlloHSC-γδT cells) at various culture stages.
In some embodiments, AlloHSC-γδT cells can be further engineered to disrupt selected genes using gene editing tools (CRISPR, TALEN, Zinc-Finger, and others). In one embodiment, disrupted genes encode T cell immune checkpoint inhibitors (PD-1, CTLA-4, TIM-3, LAG-3, and others). Deficiency of these negative regulatory genes may enhance the disease fighting capacity of AlloHSC-γδT cells, making them resistance to disease-induced anergy and tolerance.
In some embodiments, AlloHSC-γδT cells or enhanced AlloHSC-γδT cells can be further engineered to make them suitable for allogeneic adoptive transfer, thereby suitable for serving as off-the-shelf cellular products. In one embodiment, genes encoding MHC molecules or MHC expression/display regulatory molecules [MHC molecules, B2M, CIITA (Class II transcription activator control induction of MHC class II mRNA expression), and others]. Lack of MHC molecule expression on AlloHSC-γδT cells makes them resistant to allogeneic host T cell-mediated depletion. In another embodiment, MHC class-I deficient AlloHSC-γδT cells will be further engineered to overexpress an HLA-E gene that will endow them resistant to host NK cell-mediated depletion.
AlloHSC-γδT cells and derivatives can be used freshly or cryopreserved for further usage. Moreover, various intermediate cellular products generated during AlloHSC-γδT cell culture can be paused for cryopreservation, stored and recovered for continued production.
Compared to the method of generating AlloHSC-γδT cells using a feeder-dependent culture (e.g., ATO culture), this invention offers an in vitro differentiation method that does not require feeder cells. This new method greatly improves the process for the scale-up production and GMP-compatible manufacturing of therapeutic cells for human applications.
The cell products, AlloHSC-γδT cells, display phenotypes/functionalities distinct from that of their native counterpart T cells as well as their counterpart T cells generated using other ex vivo culture methods (e.g., ATO culture method), making AlloHSC-γδT cells unique cellular products.
Unique features of the AlloHSC-γδT cell differentiation culture include:
1) It is Ex Vivo and Feeder-Free.
2) It does not support TCR V/D/J recombination, so no randomly rearranged endogenous TCRs, thereby no GvHD risk.
3) It supports the synchronized differentiation of transgenic AlloHSC-γδT cells, thereby eliminating the presence of un-differentiated progenitor cells and other lineages of bystander immune cells.
4) As a result, the AlloHSC-γδT cell product comprises a homogenous and pure population of monoclonal TCR engineered T cells. No escaped random T cells, no other lineages of immune cells, and no un-differentiated progenitor cells. Therefore, no need for a purification step.
5) High yield. About 1013 AlloHSC-γδT cells (10,000-100,000 doses) can be generated from PBSCs of a healthy donor, and about 1013 AlloHSC-γδT cells (10,000-100,000 doses) can be generated from CB HSCs of a healthy donor.
6) Unique phenotype of AlloHSC-γδT cells—transgenicTCR+endogenousTCR−CD3+.
(Note: These unique features of the AlloHSC-γδT cell differentiation culture distinct it from other methods to generate off-the-shelf T cell products, including the healthy donor PBMC-based T cell culture, the ATO culture, and the others.)
Proof-of-principle studies have been performed, showing the successful generation of AlloHSC-γδT cells. Further engineering of AlloCAR-γδT cells to additionally express a BCMA CAR (AlloBCAR-γδT cell product) and together with Interleukin-15 (IL-15) (Allo15BCAR-γδT cell product) were also proved successful. Pilot CMC, pharmacology, efficacy, and safety studies were performed analyzing these cell products.
AAATAAAAGTTTTTGGACCAGGAACAAAACTGATAATTACGGATAAACAGCTTGATGCAG
CGTGTGACACGCTCGGAATGGGAGGGGAATACACTGACAAACTCATCTTCGGAAAGGGTA
CCAGAGTGACAGTAGAGCCAAGGAGCCAACCGCATACAAAACCTTCTGTTTTTGTGAT
GTGACACGGTAGGGGGTGCAACTGACAAACTCATCTTCGGAAAGGGTACCAGAGTGACA
GTAGAGCCAAGGAGCCAACCGCATACAAAACCTTCTGTTTTTGTGATGAAGAATGGA
All publications mentioned herein (e.g., PCT Published International Application Nos. PCT/US19/36786 and. PCT/US2020/037486; U.S. patent application Ser. No. 15/320,037; as well as Zarin et al., Cell Immunol. 2015 July; 296(1):70-5. doi: 10.1016/j.cellimm.2015.03.007. Epub 2015, those listed above etc.) are incorporated by reference to disclose and describe aspects, methods and/or materials in connection with the cited publications. Many of the techniques and procedures described or referenced herein are well understood and commonly employed by those skilled in the art.
Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.
This application claims the benefit under 35 U.S.C. Section 119(e) of co-pending and commonly-assigned U.S. Provisional Patent Application Ser. No. 63/131,170, filed on Dec. 28, 2020, and entitled “ENGINEERED GAMMA DELTA (γδ) T CELLS AND METHODS OF MAKING AND USING THEREOF” which application is incorporated by reference herein.
| Number | Date | Country | |
|---|---|---|---|
| 63131170 | Dec 2020 | US |
