Antibody-loaded immune cells and methods of use in cancer therapy

Abstract

Antibody-loaded immune cells and methods of use in cancer treatment are provided. [Solution] Aspects of the present disclosure are directed to immune cells that contain a polynucleotide encoding an engineered receptor (e.g., chimeric antigen receptor, T cell receptor) and have an anti-CD20 antibody (or a fragment thereof) attached to their surface. Particular embodiments are directed to pre-activated and / or expanded natural killer cells that have an anti-CD20 antibody (or a fragment thereof) on their surface. Also disclosed are compositions that include such cells and methods of using such compositions to treat individuals with CD20-positive cancer. Further embodiments include cell preparation methods that include culturing natural killer cells with cytokines (e.g., IL-12, IL-15, and / or IL-18) and incubating the cells with an anti-CD20 antibody.

Description

[Technical field]

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 251,435, filed October 1, 2021, which is incorporated by reference herein in its entirety.

[0002] (Array Listing) This application contains a Sequence Listing submitted in XML format, which is incorporated herein by reference in its entirety. The XML copy created on Sep. 27, 2022 is named MDACP1317WO_Sequence_Listing_ST26.xml and is 33,741 bytes in size.

[0003] Embodiments of the present disclosure include the fields of medicine including at least cell biology, molecular biology, immunology, and cancer medicine. [Background technology]

[0004] Genetic reprogramming of natural killer (NK) cells for adoptive cancer immunotherapy has clinically relevant applications and advantages. However, certain challenges exist; for example, genetic modification of NK cells is expensive, poses technical challenges, and patients treated with modified (engineered) NK cells may relapse due to poor durability of treatment. There is a need for highly functional, long-lived NK cells and methods of using such cells in cancer therapy.

[0005] The present disclosure relates, in certain embodiments, to methods and compositions relating to the activation, expansion, and manipulation of NK cells for cancer-targeted cellular therapy. Summary of the Invention

[0006] The embodiments of the present disclosure encompass methods and compositions relating to modified cell receptors, including chimeric antigen receptors (CARs), and activated, expanded, and / or antibody-loaded immune cells. In certain embodiments, the modified receptors are in the form of polynucleotides, polypeptides, and / or are included on the surface of any type of cell, including any type of immune cell. In certain cases, the cells are immune cells, and in certain embodiments, the immune cells are NK cells, NKT cells, invariant NKT cells, gamma delta T cells, alpha beta T cells, regulatory T cells, B cells, macrophages, mesenchymal stromal cells (MSCs), dendritic cells, mixtures thereof, and the like, from any source. In some embodiments, the immune cells are NK cells. In certain cases, the NK cells of the present disclosure are loaded with one or more antibodies, in certain aspects anti-CD20 antibodies. In certain embodiments, umbilical cord blood-derived NK cells (CB-NK), particularly anti-CD20 antibody-loaded NK cells, are encompassed for targeting cancer. Such NK cells may be pre-activated (e.g., via incubation with IL-12, IL-15 and / or IL-18) and / or expanded (e.g., via expansion in cultures containing artificial antigen-presenting cells expressing the CD137 ligand).

[0007] Preactivation of NK cells with IL-12, IL-18, IL-2, IL-21, IL-15, or any combination thereof results in long-lived memory-like NK cells with enhanced cytokine production upon restimulation. The disclosed ex vivo preactivation and expansion strategy using umbilical cord blood (CB) as a source of NK cells generates a large number of highly functional NK cells, including those with a memory phenotype. As described herein, the specificity of these cells can be redirected to cancer antigens by combining them with one or more antibodies or fragments thereof (e.g., anti-CD20 antibodies such as obinutuzumab) either ex vivo and / or in vivo, resulting in highly potent NK cells with CAR-like properties. Thus, embodiments of the present disclosure include preactivated and expanded NK cells that are loaded with anti-CD20 antibodies (i.e., anti-CD20 antibodies are bound to the surface of the NK cells).

[0008] Certain embodiments of the present disclosure include polynucleotides encoding modified receptors (e.g., CARs). Immune cells (e.g., NK cells) comprising such polynucleotides are also disclosed. In certain cases, the immune cells are modified to express one or more cytokines that promote cell expansion and persistence. In certain cases, the one or more cytokines include interleukin (IL)-15, IL-2, IL-7, IL-12, IL-18, IL-21, and / or IL-23. In certain embodiments, the vector encoding the CAR also encodes one or more cytokines, each of which is ultimately produced as a separate polypeptide. In other embodiments, the CAR and the cytokine are encoded on separate vectors.

[0009] Certain embodiments of the present disclosure target cancer antigen (e.g., CD20) positive cells and allow for the use of pre-made immune cells, including at least NK cells, allogeneic to the recipient individual, which may or may not be transduced to express one or more cytokines, such as IL-15, IL-2, IL-21, IL-12, IL-23, IL-7 and / or IL-18.

[0010] In certain embodiments of the present disclosure, the expression of one or more endogenous genes in immune cells is altered, for example, expression may be partially or completely reduced. This alteration may occur by any means, but in certain embodiments, the expression of one or more genes is altered, for example, by reducing the expression level, which may occur by any suitable means, including at least CRISPR / Cas technology. By way of example only, the endogenous genes are selected from the group consisting of NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5, CD7, CTLA-4, TDAG8, CD38, CREM, and combinations thereof. The activity of the produced protein may also be partially or completely impaired, such as by antibodies and / or small molecules.

[0011] Certain aspects of the present disclosure are directed to modified immune cells, comprising (a) a polynucleotide encoding (i) a chimeric antigen receptor (CAR) or (ii) a T cell receptor (TCR), and (b) an anti-CD20 antibody or an antigen-binding fragment thereof bound to the surface of the immune cell. The CAR can be, for example, a CD19, CD70 or CD5 specific CAR, or a bispecific or trispecific CAR. The anti-CD20 antibody can be, for example, rituximab or obinutuaumab. In some embodiments, the anti-CD20 antibody is obinutuaumab.

[0012] In certain embodiments, the polynucleotide encoding the CAR of the present disclosure further encodes one or more additional polypeptides of interest. The sequence encoding the additional polypeptide of interest and the sequence encoding the CAR can be separated on the polynucleotide by any type of 2A element, such as an E2A element. In certain aspects, the polypeptide of interest is a therapeutic protein or a protein that enhances the activity, expansion and / or persistence of the cells. In some embodiments, the additional polypeptide of interest is a suicide gene, one or more cytokines, or one or more human or viral proteins that enhance the growth, expansion and / or metabolic fitness. In certain embodiments, the additional polypeptide of interest is a cytokine, such as IL-15, IL-2, IL-12, IL-18, IL-21, IL-23, or IL-7. In certain embodiments, the cytokine is IL-15. In one embodiment, the cytokine is IL-21.

[0013] The vector comprising the polynucleotide of the present disclosure is also provided herein.The vector contemplated herein includes viral vector (e.g., adenovirus vector, adeno-associated virus vector, lentivirus vector and retrovirus vector) and non-viral vector (e.g., plasmid).

[0014] The embodiments of the present disclosure include any type of immune cell comprising any polynucleotide and / or polypeptide encompassed herein. In certain embodiments, the immune cell is a NK cell, a T cell, a gamma delta T cell, an alpha beta T cell, an invariant NKT (iNKT) cell, a B cell, a macrophage, an MSC, a dendritic cell, or a mixture thereof. When the immune cell is a NK cell, the NK cell can be derived from umbilical cord blood (including pooled umbilical cord blood units), peripheral blood, induced pluripotent stem cells, bone marrow, hematopoietic stem cells, and / or can be derived from a cell line. In certain aspects, the NK cell line is the NK-92 cell line, or another NK cell line derived from tumor or healthy NK cells or progenitor cells.

[0015] In certain embodiments, the immune cells are NK cells, e.g., derived from umbilical cord blood, e.g., from umbilical cord blood mononuclear cells. NK cells, in certain cases, are CD56 + The immune cells may be NK cells. The NK cells may express one or more exogenously provided cytokines, such as IL-15, IL-2, IL-12, IL-18, IL-21, IL-23, IL-7, or a combination thereof. Certain embodiments include populations of any type of immune cells of the present disclosure, and the cells may be present in any type of suitable medium or suitable carrier.

[0016] Also disclosed is a population of NK cells bound to an anti-CD20 antibody or antigen-binding fragment thereof, the population of NK cells having been previously preactivated in a preactivation culture comprising an effective concentration of one or more of IL-12, IL-15 and IL-18. In some cases, the NK cells have been previously expanded in an expansion culture comprising artificial antigen presenting cells (aAPCs). In some embodiments, the aAPCs express CD137 ligand (also "4-1BB ligand" or "4-1BBL").

[0017] Methods of treating or preventing any type of cancer are encompassed herein, including administering antibody-loaded cells that can express a particular CAR and / or can be pre-activated and expanded in a therapeutically effective amount to ameliorate or prevent cancer, or reduce the risk of cancer, reduce the severity of one or more symptoms of cancer, reduce tumor burden, prevent metastasis or the risk thereof, or delay metastasis, or delay the onset of cancer. In some embodiments, methods of treating or preventing any type of cancer are encompassed herein, including administering the cells and antibodies encompassed herein in the same formulation or different formulations substantially simultaneously to an individual in need of treatment or prevention of any type of cancer. In certain embodiments, there are methods of treating or preventing any type of cancer by administering the cells and antibodies encompassed herein in different formulations at different times (including any order).

[0018] In some embodiments, a method of killing cancer cells in an individual is disclosed, comprising administering to the individual an effective amount of any of the antibody-loaded cells disclosed herein. In certain embodiments, the cells are NK cells, T cells, gamma delta T cells, alpha beta T cells, invariant NKT cells, B cells, macrophages, mesenchymal stromal cells (MSCs), dendritic cells, or mixtures thereof. In some embodiments, the cells are NK cells. The NK cells can be from cord blood, peripheral blood, induced pluripotent stem cells, hematopoietic stem cells, bone marrow, or cell lines. The NK cells can be from cord blood mononuclear cells. The cells can be allogeneic or autologous to the individual, which may be human or non-human. The cells can be administered to the individual by injection, intravenous, intraarterial, intraperitoneal, intratracheal, intratumoral, intramuscular, endoscopic, intralesional, intracranial, percutaneous, subcutaneous, topical, perfusion, tumor microenvironment, or combinations thereof.

[0019] In certain embodiments of the method, the cells may be administered to the individual one or more times. The period between administration of the cells to the individual may be 1-24 hours, 1-7 days, 1-4 weeks, 1-12 months, or more than a year. The method may further include providing the individual with an effective amount of an additional treatment, such as surgery, radiation, gene therapy, immunotherapy, and / or hormone therapy. The additional treatment may, in some cases, include one or more antibodies and / or antibody-based agents. In some aspects of the method, they may further include identifying any type of CD20 positive cells in the individual.

[0020] It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the invention, and vice versa. Further, compositions of the invention can be used to achieve methods of the invention.

[0021] The foregoing description has outlined rather broadly the characteristics and technical advantages of the present disclosure so that the following detailed description of the invention may be better understood. Additional characteristics and advantages will be described below which form the subject matter of the claims herein. As should be appreciated by those skilled in the art, the concepts and specific embodiments disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present design. As should also be appreciated by those skilled in the art, such equivalent constructions do not depart from the spirit and scope as set forth in the appended claims. The following description, when taken in conjunction with the accompanying drawings, will provide a better understanding of the novel features believed to be characteristic of the design disclosed herein, both as to its mechanism and method of operation, together with further objects and advantages. It should be expressly understood, however, that each of the figures is provided for the purpose of illustration and description only, and is not intended as a definition of the limits of the present disclosure. [Brief description of the drawings]

[0022] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0023] [Figure 1] FIG. 1 shows flow cytometry analysis of obinutuzumab-loaded NK cells.

[0024] [Figure 2A] FIG. 2A shows results from an Incucyte® killing assay against Raji CD19 knockout (KO) cells when co-cultured with obinutuzumab-loaded NK cells (NK; FIG. 2A). [Figure 2B] Figure 2B shows results from an Incucyte® killing assay on Raji CD19 knockout (KO) cells when co-cultured with obinutuzumab-loaded, cytokine (IL-12, IL-15 and IL-18) preactivated, expanded NK cells (NK P+E; Figure 2B). Raji CD19 KO cells were transduced with mKate2 so that the intensity of the red color corresponded to the tumor cell number.

[0025] [Diagram 3] FIG. 3 shows the percent cell killing of Raji cells at various effector to target cell ratios, as indicated, with or without obinutuzumab loading.

[0026] [Figure 4A] FIG. 4A shows results from an Incucyte® killing assay in Raji wild-type (WT) cells when co-cultured with obinutuzumab-loaded CD19 CAR NK cells (NK CD19 CAR; FIG. 4A). [Figure 4B]Figure 4B shows results from an Incucyte® killing assay in Raji wild type (WT) cells when loaded with obinutuzumab and co-cultured with cytokine (IL-12, IL-15 and IL-18) pre-activated and expanded CD19 CAR NK cells (NK P+E CD19 CAR; Figure 4B). Raji WT cells were transduced with mKate2 so that the intensity of the red color corresponded to the tumor cell number.

[0027] [Figure 5A] Figures 5A-5C show the results of in vivo analysis of CD19 CAR NK cells loaded with obinutuzumab. Figure 5A shows bioluminescence imaging of tumors from different treatment conditions. [Figure 5B] FIG. 5B shows quantification of the bioluminescence shown in FIG. 5A. [Figure 5C] FIG. 5C shows the survival curves for the different treatment conditions, as indicated.

[0028] [Figure 6A] FIG. 6A shows results from an Incucyte® killing assay in Raji CD19 knockout (KO) cells when co-cultured with obinutuzumab-loaded CD19 CAR NK cells (NK CD19 CAR; FIG. 6A). [Figure 6B] Figure 6B shows results from an Incucyte® killing assay in Raji CD19 knockout (KO) cells when co-cultured with obinutuzumab-loaded, cytokine (IL-12, IL-15 and IL-18) preactivated and expanded CD19 CAR NK cells (NK P+E CD19 CAR; Figure 6B). Raji cells were transduced with mKate2 so that color intensity corresponded to tumor cell number.

[0029] [Figure 7A]Figure 7A shows results from an Incucyte® killing assay in Raji CD19 knockout (KO) or Raji wild-type (WT) cells when co-cultured with obinutuzumab-loaded CD19 CAR NK cells (NK CD19 CAR; Figure 7A). [Figure 7B] Figure 7B shows results from an Incucyte® killing assay in Raji CD19 knockout (KO) or Raji wild type (WT) cells when co-cultured with obinutuzumab-loaded, cytokine (IL-12, IL-15 and IL-18) preactivated and expanded CD19 CAR NK cells (NK P+E CD19 CAR; Figure 7B). Raji cells were transduced with mKate2 so that the intensity of the red color corresponded to the tumor cell number.

[0030] While various embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes and substitutions may be made by those skilled in the art without departing from the present invention. It is understood that various alternatives to the embodiments of the present disclosure described herein may be used. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] I. Definition Examples In keeping with long-standing patent law conventions, the words "a" and "an" when used in conjunction with the word "comprising" herein, including the claims, refer to "one or more." Some embodiments of the present disclosure may consist of, or consist essentially of, one or more elements, method steps, and / or methods of the present disclosure. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein, and that different embodiments may be combined.

[0032] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises", and "comprising" are understood to mean the inclusion of the recited step or element, or group of steps or elements, but not the exclusion of any other step or element, or group of steps or elements. "Consisting of" means including and limited to whatever follows the word "consisting of". Thus, the word "consisting of" indicates that the recited elements are necessary or mandatory, and that no other elements may be present. "Consisting essentially of" means including any elements recited after the wording, limited to other elements that do not interfere with or contribute to the activity or action of the recited elements as specified in this disclosure. Thus, the word "consisting essentially of" indicates that the recited elements are necessary or mandatory, but that other elements are not optional and may or may not be present depending on whether they affect the activity or action of the recited elements.

[0033] Throughout this specification, references to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "particular embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, mean that the particular features, structures, or characteristics described in connection with the embodiment are included in at least one embodiment of the invention. Thus, the appearances of such phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0034] As used herein, the terms "or" and "and / or" are utilized to describe multiple components in combination with each other or exclusively. For example, "x, y, and / or z" can refer to "x" alone, "y" alone, "z" alone, "x, y, and z," "(x and y) or z," "x or (y and z)," or "x or y or z." It is specifically contemplated that x, y, or z may be explicitly excluded from an embodiment.

[0035] Throughout this application, the term "about" is used according to its plain and ordinary meaning in the field of cell and molecular biology to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0036] The term "modified" as used herein refers to entities that are produced by the hand of man, including cells, nucleic acids, polypeptides, vectors, etc. In at least some cases, modified entities are synthetic and contain elements that are not found in nature or configured for use in the present disclosure.

[0037] The term "isolated" as used herein refers to a molecule or biologic or cellular material that is substantially free of other materials. In one embodiment, the term "isolated" refers to a nucleic acid, e.g., DNA or RNA, or a protein or polypeptide, or a cell or organelle, or a tissue or organ, respectively, that is separated from other DNA or RNA, or proteins or polypeptides, or cells or organelles, or tissues or organs, e.g., that are present in a natural source. The term "isolated" also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or medium when produced by recombinant DNA technology, or chemical precursors or other chemicals when chemically synthesized. Furthermore, "isolated nucleic acid" is meant to include nucleic acid fragments that are not naturally occurring as fragments and would not be found in the natural state. The term "isolated" is also used herein to refer to a polypeptide that is isolated from other cellular proteins, and is meant to include both purified and recombinant polypeptides. Also, the term "isolated" is used herein to refer to cells or tissues that are isolated from other cells or tissues, and is meant to include both cultured cells or tissues and modified (engineered) cells or tissues.

[0038] As used herein, "prevent" and similar terms such as "prevented," "prevention," and the like refer to an approach that prevents, inhibits, or reduces the likelihood of the onset or recurrence of a disease or condition, such as cancer. It also refers to delaying the onset or recurrence of a disease or condition, or delaying the onset or recurrence of symptoms of a disease or condition. As used herein, "prevention" and similar terms also include reducing the intensity, effect, symptoms, and / or burden of a disease or condition prior to the onset or recurrence of the disease or condition.

[0039] The term "sample" as used herein generally refers to a biological sample. A sample may be taken from tissue or cells from an individual. In some cases, a sample may include or be derived from tissue biopsy, blood (e.g., whole blood), plasma, extracellular fluid, dried blood spot, cultured cells, discarded tissue. A sample may be isolated from a source before collection. Non-limiting examples include blood, cerebrospinal fluid, pleural fluid, amniotic fluid, lymphatic fluid, saliva, urine, stool, tears, sweat, or mucosal secretions, and other bodily fluids isolated from a primary source before collection. In some cases, a sample is isolated from its primary source (cells, tissues, bodily fluids, e.g., blood, environmental samples, etc.) during sample preparation. A sample may or may not be purified or otherwise enriched from its primary source. In some cases, the primary source is homogenized before further processing. The sample may be filtered or centrifuged to remove buffy coat, lipids, or particulate matter. The sample may also be purified or enriched for nucleic acids, or may be treated with RNase. The sample may contain intact, fragmented, or partially degraded tissues or cells.

[0040] The term "subject" as used herein generally refers to an individual having a biological sample undergoing processing or analysis, and in certain cases, having or suspected of having cancer. A subject may be any living organism or animal subject that is the subject of a method or material, including mammals, such as humans, experimental animals (e.g., primates, rats, mice, rabbits), livestock (e.g., cows, sheep, goats, pigs, turkeys, and chickens), household pets (e.g., dogs, cats, and rodents), horses, and transgenic non-human animals. A subject may be a patient, and may have or be suspected of having a disease (which may be referred to as a medical condition), such as, for example, a benign or malignant neoplasm or cancer. A subject may be undergoing or have been undergoing treatment. A subject may be asymptomatic. A subject may be a healthy individual who desires to prevent cancer. The term "individual" may be used interchangeably, at least in some cases. As used herein, a "subject" or "individual" may or may not be housed in a medical facility, and may be treated as an outpatient in a medical facility. An individual may receive one or more pharmaceutical compositions via the Internet. An individual may include humans or non-human animals of any age, and thus includes both adults and juveniles (i.e., children), as well as infants, and includes intrauterine individuals. The term is not intended to imply a need for medical treatment, and thus an individual may be part of an experiment, whether voluntarily or involuntarily, whether clinical or in support of basic science research.

[0041] As used herein, "treatment" or "treating" includes any beneficial or desired effect on the symptoms or pathology of a disease or pathological condition, and may even include a minimal reduction in one or more measurable markers of the disease or condition being treated, such as cancer. Treatment may, in some cases, include either a reduction or amelioration of the symptoms of the disease or condition, or a delay in the progression of the disease or condition. "Treatment" does not necessarily indicate a complete eradication or cure of the disease or condition, or its associated symptoms.

[0042] Also, any method in the context of a therapeutic, diagnostic, or physiological purpose or effect may be described in "use" claim language, such as the "use of" any compound, composition, or agent discussed herein to achieve or carry out the described therapeutic, diagnostic, or physiological purpose or effect.

[0043] The present disclosure relates in particular to methods and compositions directed to treatments for various types of cancers utilizing adoptive cell therapy that targets cancer cells via antibodies (e.g., anti-CD20 antibodies) on the surface of the cells, and in some cases also via expression of genetically engineered receptors (e.g., chimeric antigen receptors (CARs)). In certain embodiments, compositions are provided that include antibody-loaded NK cells, including CAR NK cells and pre-activated, expanded NK cells. Methods for using such cells in the treatment of cancer are also disclosed.

[0044] II. Genetically engineered receptors The immune cells of the present disclosure may, in some cases, be genetically modified to express one or more antigen-binding receptors that target a cancer antigen, such as a modified CAR or alternatively a modified TCR. For example, the immune cells may be immune cells that are modified to express a CAR and / or TCR with antigen specificity for a cancer antigen. In some aspects, the immune cells are modified to express a cancer antigen-specific CAR, antigen-specific TCR or other modified protein by protein knock-in using CRISPR / Cas technology. A variety of cancer antigens are recognized in the art and contemplated herein for targeting by genetically modified receptors. Such cancer antigens include, for example, BCMA, CD5, CD19, CD20, CD22, CD33, CD38, CD70, CD138, FAP, HER2, MUC1 and NKG2D. In some embodiments, the CAR of the present disclosure is a CD19-specific CAR. In some embodiments, the CAR of the present disclosure is a CD70-specific CAR. In some embodiments, the CAR of the present disclosure is a CD5-specific CAR. In certain embodiments, it is specifically contemplated that the immune cells of the present disclosure are not genetically modified.

[0045] Suitable methods for modifying cells are known in the art.See, for example, Sambrook et al., 2001 and Ausubel et al., 1996.For example, cells can be transduced to express one or more CARs, TCRs and / or other modified proteins, or any heterologous protein, with antigen specificity for cancer antigens, using the transduction techniques described in Heemskerk et al., 2008 and Johnson et al., 2009.

[0046] In some embodiments, the cell contains one or more nucleic acids introduced via genetic modification that encode receptors that target one or more antigens, and the genetically modified products of such nucleic acids. In some embodiments, the nucleic acid is heterologous, i.e., not normally present in the cell or sample obtained from the cell, such as obtained from another organism or cell that is not normally found in the cell being modified and / or the organism from which such cell is derived. In some embodiments, the nucleic acid is not naturally occurring, such as a nucleic acid that is not found in nature (e.g., a chimera).

[0047] Exemplary antigen receptors, including CARs and recombinant TCRs, and methods for modifying and introducing receptors into cells are described, for example, in WO 200014257, WO 2013126726, WO 2012 / 129514, WO 2014031687, WO 2013 / 166321, WO 2013 / 071154, WO 2013 / 123061, U.S. Patent Application Publication Nos. 2002131960, 2013287748, and 20130149337. , U.S. Pat. Nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353 and 8,479,118 and EP 2 537 416 A1, and / or Sadelain et al., 2013; Davila et al., 2013; Turtle et al., 2012; Wu et al., 2012. In some embodiments, the genetically modified antigen receptor includes CARs as described in U.S. Pat. No. 7,446,190 and those described in WO2014055668A1.

[0048] A. Chimeric Antigen Receptor In some embodiments, modified (engineered) antigen receptors include CARs, including activating or stimulatory CARs, or costimulatory CARs (see WO2014 / 055668). CARs generally comprise an extracellular antigen (or ligand) binding domain that is linked to one or more intracellular signaling components, in some embodiments, via a linker and / or a transmembrane domain. Such molecules typically mimic or approximate the signaling through a natural antigen receptor, the signaling through such receptor in combination with a costimulatory receptor, and / or the signaling through a costimulatory receptor alone.

[0049] It is believed that the chimeric construct can be introduced into immune cells as naked DNA or in a suitable vector.Methods for stable transfection of cells by electroporation with naked DNA are known in the art.See, for example, U.S. Patent No. 6,410,319.Naked DNA generally refers to the DNA encoding the chimeric receptor contained in a plasmid expression vector in a suitable orientation for expression.

[0050] Alternatively, chimeric CAR constructs can be introduced into immune cells using viral vectors (e.g., retroviral vectors, adenoviral vectors, adeno-associated viral vectors, or lentiviral vectors).Vector suitable for use according to the method of the present disclosure is non-replicative in immune cells.Many virus-based vectors are known, such as HIV, SV40, EBV, HSV, or BPV-based vectors, whose virus copy number maintained in cells is low enough to maintain cell viability.

[0051] Certain embodiments of the present disclosure relate to the use of nucleic acids, including nucleic acids encoding cancer antigen-specific CAR polypeptides, including humanized CARs (hCARs) in some cases to reduce immunogenicity, that include at least one intracellular signaling domain, a transmembrane domain, and an extracellular domain that includes one or more signaling motifs. In certain embodiments, the cancer antigen-specific CARs can recognize epitopes that include a shared space between one or more antigens. In certain embodiments, the binding region can include a complementarity determining region of a monoclonal antibody, a variable region of a monoclonal antibody, and / or an antigen-binding fragment thereof. In another embodiment, the specificity is derived from a peptide (e.g., a cytokine) that binds to a receptor.

[0052] It is contemplated that the human CAR nucleic acid may be a human gene used to enhance cellular immunotherapy for human patients. In certain embodiments, the present disclosure includes a full-length CAR cDNA or coding region. The antigen-binding region or domain is the V of a single-chain variable fragment (scFv) derived from a particular human monoclonal antibody. H Chain and V L The fragments may include those described in the above paragraphs of the "fragments of the chains." Also, the fragments may be any number of different antigen-binding domains of a human antigen-specific antibody. In a more specific embodiment, the fragments are cancer antigen-specific scFvs encoded by sequences optimized for human codon usage for expression in human cells.

[0053] The configuration can be a multimer, such as a diabody or a multimer. The multimer is most likely formed by cross-pairing of the variable parts of the light and heavy chains into a diabody. The hinge part of the construct can have many options, from being completely deleted, to maintaining the first cysteine, to a proline instead of a serine substitution, to being truncated to the first cysteine. The Fc part can be deleted. Any protein that is stable and / or dimerizes can serve this purpose. Only one of the Fc domains can be used, for example, either the CH2 or CH3 domain from a human immunoglobulin. The hinge, CH2 and CH3 regions of a human immunoglobulin that have been modified to improve dimerization can also be used. Only the hinge part of an immunoglobulin can be used. Parts of CD28 and / or CD8 alpha can also be used.

[0054] The sequence of the open reading frame encoding the chimeric receptor can be obtained from genomic DNA sources, cDNA sources, or can be synthesized (e.g., via PCR), or a combination thereof. Depending on the size of the genomic DNA and the number of introns, it may be desirable to use cDNA or a combination thereof, since introns have been shown to stabilize mRNA. It may also be more advantageous to use endogenous or exogenous non-coding regions to stabilize the mRNA.

[0055] In some embodiments, the antigen-specific binding or recognition component is linked to one or more transmembrane domains and intracellular signaling domains. In some embodiments, the CAR comprises a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain that naturally binds to one of the domains in the CAR is used. In some examples, the transmembrane domain is selected or modified by amino acid substitution to avoid such domain binding to the transmembrane domain of the same or different surface membrane protein, and to minimize interaction with other members of the receptor complex. The transmembrane domain in some embodiments is derived from either natural or synthetic sources. When the source is natural, the domain in some aspects is derived from any membrane-bound or transmembrane protein. The transmembrane region may be derived from (i.e., at least includes) the alpha, beta, or zeta chain of the T cell receptor, CD28, DAP12, DAP10, NKG2D, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS / CD278, KIR such as KIR2DL4, GITR / CD357, and the like. Additionally, the transmembrane domain in some embodiments is synthetic. In some aspects, the synthetic transmembrane domain comprises primarily hydrophobic residues such as leucine and valine. In some aspects, triplets of phenylalanine, tryptophan, and valine are found at each end of the synthetic transmembrane domain.

[0056] In some embodiments, the CAR nucleic acid comprises a sequence encoding other costimulatory receptors, such as a transmembrane domain and one or more intracellular signaling domains. In addition to the primary T cell activation signal, such as that initiated by CD3ζ and / or FcεRIγ, additional stimulatory signals for immune effector cell proliferation and effector function after the chimeric receptor binds to the target antigen can be utilized. For example, some or all of the human costimulatory receptors can be utilized for enhanced cell activation, which can help improve in vivo persistence and improve the therapeutic success of adoptive immunotherapy. Examples include costimulatory domains from molecules such as DAP12, DAP10, NKG2D, CD2, CD28, CD27, 4-1BB, (CD137), OX40, ICOS, (CD278), CD30, HVEM, CD40, LFA-1 (CD11a / CD18), and / or ICAM-1, and / or portions of the KIR2DL4 cytoplasmic domain that can induce an activating signal, although in alternative specific embodiments any one of these listed may be excluded from use in a CAR.

[0057] In certain embodiments, the platform technology disclosed herein for genetically modifying immune cells, such as NK cells, includes (i) non-viral gene transfer using an electroporation device (e.g., nucleofector), (ii) CARs that signal through an endodomain (e.g., CD28 / CD3-ζ, CD137 / CD3-ζ, or other combinations), (iii) CARs with variable length extracellular domains linking the antigen recognition domain to the cell surface, and in some cases, (iv) CARs + These include K562-derived artificial antigen-presenting cells (aAPCs) that enable robust and numerical expansion of immune cells ( Singh et al., 2008 ; Singh et al., 2011 ).

[0058] B. Examples of Specific CAR Embodiments In certain embodiments, specific CAR molecules are encompassed herein. In some cases, the antigen-binding domain of the CAR is an scFv, and any scFv that binds to a cancer antigen can be utilized herein. When an scFv is utilized in the extracellular domain of the CAR, the variable heavy and variable light chains for the scFv can be in any order from N-terminus to C-terminus. For example, the variable heavy chain can be N-terminal to the variable light chain, or vice versa. The scFv and / or ligand that binds to the antigen in the CAR can be codon-optimized or not codon-optimized. In certain embodiments, the vector encodes a cancer antigen-specific CAR and also encodes one or more other molecules. For example, the vector can encode a CAR and also encode another protein of interest, such as another modified antigen receptor, a suicide gene, and / or a specific cytokine.

[0059] On the same molecule, cancer antigen-specific CAR can comprise one or more antigen-specific extracellular domains, specific hinges, specific transmembrane domains, one or more specific costimulatory domains and one or more specific activation signals.When multiple antigen-specific extracellular domains are utilized, such as for targeting two different antigens, there can be a linker between two antigen-specific extracellular domains.The examples of CAR contemplated herein include but are not limited to CD19-specific CAR, CD70-specific CAR and CD5-specific CAR.

[0060] In certain embodiments of specific CAR molecules, CARs may utilize DAP10, DAP12, 4-1BB, NKG2D or other costimulatory domains (herein referred to as intracellular or cytoplasmic domains). In some cases, CD3 zeta is utilized without a costimulatory domain. In certain embodiments of specific CAR molecules, CARs may utilize any suitable transmembrane domain from DAP12, DAP10, 4-1BB, 2B4, OX40, CD27, NKG2D, CD8 or CD28, etc.

[0061] Examples of specific sequence embodiments are provided below.

[0062] 1. Transmembrane domain Any suitable transmembrane domain can be used in the CAR of the present disclosure.Examples include at least the transmembrane domain from DAP10, DAP12, CD28, NKG2D, CD3 epsilon, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137 or CD154, from T cell receptor a or b chain, from CD3 zeta chain, from ICOS, their functional derivatives, and combinations thereof.In certain cases, the transmembrane domain from DAP10, DAP12, CD28, CD8 or NKG2D is used.Examples of specific transmembrane domain sequences can be used as follows:

[0063] CD28 transmembrane domain nucleotide sequence: TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG (SEQ ID NO: 1)

[0064] CD28 transmembrane domain amino acid sequence: FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 2)

[0065] CD8 transmembrane domain nucleotide sequence: ACCACAACACCAGCACCTAGACCTCCAACTCCAGCTCCTACAATCGCCAGCCAGCCTCTGTCTCTGAGGCCTGAAGCTTGTAGACCTGCTGGCGGAGCCGTGCATACCAGAGGACTGGATTTCGCCTGCGATATCTACATCTGGGCCCCTCTGGCTGGAACATGTGGCGTGCTGCTGCTGAGCCTCGTGATCACA (SEQ ID NO: 3)

[0066] CD8 transmembrane domain amino acid sequence: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 4)

[0067] 4-1BB transmembrane domain nucleotide sequence: ATCATCTCCTTCTTTCTTGCGCTGACGTCGACTGCGTTGCTCTTCCTGCTGTTCTTCCTCACGCTCCGTTTCTCTGTTGTT (SEQ ID NO: 5)

[0068] 4-1BB transmembrane domain amino acid sequence: IISFFLALTSTALLFLLFFLTLRFSVV (SEQ ID NO: 6)

[0069] DAP10 transmembrane domain nucleotide sequence: CTCCTGGCAGGCCTCGTGGCTGCTGATGCGGTGGCATCGCTGCTCATCGTGGGGGCGGTGTTC (SEQ ID NO: 7)

[0070] DAP10 transmembrane domain amino acid sequence: LLAGLVAADAVASLLIVGAVF (SEQ ID NO: 8)

[0071] DAP12 transmembrane domain nucleotide sequence: GGCGTGCTGGCAGGGATCGTGATGGGAGACCTGGTGCTGACAGTGCTCATTGCCCTGGCCGTG (SEQ ID NO: 9)

[0072] DAP12 transmembrane domain amino acid sequence: GVLAGIVMGDLVLTVLIALAV (SEQ ID NO: 10)

[0073] NKG2D transmembrane domain nucleotide sequence: GCGGTGATGATTATTTTTCGCATTGGCATGGCGGTGGCGATTTTTTGCTGCTTTTTTTTTCCG (SEQ ID NO: 11)

[0074] NKG2D transmembrane domain amino acid sequence: AVMIIFRIGMAVAIFCCFFFP (SEQ ID NO: 12)

[0075] Any polynucleotide encompassed by this disclosure may comprise SEQ ID NO: 15, 17, 19, 21, 23 or 25, or a sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more identical to SEQ ID NO: 15, 17, 19, 21, 23 or 25. Any polypeptide encompassed by this disclosure may comprise SEQ ID NO: 16, 18, 20, 22, 24 or 26, or a sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more identical to SEQ ID NO: 16, 18, 20, 22, 24 or 26.

[0076] 2. Intracellular domain One or more intracellular domains (which may also be referred to herein as signal activation domains or costimulatory domains, as appropriate) may or may not be utilized in a specific CAR of the present disclosure. Specific examples include intracellular domains derived from CD3 zeta, 4-1BB, NKG2D, OX-40, CD27, DAP10, DAP12, B7-1 / CD80, CD28, 2B4, 4-1BBL, B7-2 / CD86, CTLA-4, B7-H1 / PD-L1, ICOS, B7-H2, PD-1, B7-H3, PD-L2, B7-H4, PDCD6, BTLA, or combinations thereof.

[0077] Examples of particular intracellular domains that can be used in the CARs of the present disclosure are as follows:

[0078] 4-1BB intracellular domain nucleotide sequence: AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG (SEQ ID NO: 13)

[0079] 4-1BB intracellular domain amino acid sequence: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 14)

[0080] DAP10 intracellular domain nucleotide sequence: CTTTGCGCACGCCCACGCCGCAGCCCCGCCCAAGAAGATGGCAAAGTCTACATCAACATGCCAGGCAGGGGC (SEQ ID NO: 15)

[0081] DAP10 intracellular domain amino acid sequence: LCARPRRSPAQEDGKVYINMPGRG (SEQ ID NO: 16)

[0082] DAP12 intracellular domain nucleotide sequence: TACTTCCTGGGCCGCTGGTCCCTCGGGGGCGAGGGGCTGCGGAGGCAGCGACCCGGAAACAGCGTATCACTGAGACCGAGTCGCCTTATCAGGAGCTCCAGGGTCAGAGGTCGGATGTCTACAGCGACCTCAACACACAGAGGCCGTATTACAAA (SEQ ID NO: 17)

[0083] DAP12 intracellular domain amino acid sequence: YFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK (SEQ ID NO: 18)

[0084] NKG2D intracellular domain nucleotide sequence: AGCGCGAACGAACGCTGCAAAAGCAAAGTGGTGCCGTGCCGCCAGAAACAGTGGCGCACCAGCTTTGATAGCAAAAAACTGGATCTGAACTATAACCATTTTGAAAGCATGGAATGGAGCCATCGCAGCCGCCGCGGCCGCATTTGGGGCATG (SEQ ID NO: 19)

[0085] NKG2D intracellular domain amino acid sequence: SANERCKSKVVPCRQKQWRTSFDSKKLDLNYNHFESMEWSHRSRRGRIWGM (SEQ ID NO: 20)

[0086] Any polynucleotide encompassed by this disclosure may comprise SEQ ID NO:27, 29, 31 or 33, or a sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more identical to SEQ ID NO:27, 29, 31 or 33. Any polypeptide encompassed by this disclosure may comprise SEQ ID NO:28, 30, 32 or 34, or a sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more identical to SEQ ID NO:28, 30, 32 or 34.

[0087] 3. Hinge In some embodiments of the CAR, there is a hinge region between one or more extracellular antigen binding domains and the transmembrane domain. In certain embodiments, the hinge is a particular length, for example, 10-20, 10-15, 11-20, 11-15, 12-20, 12-15, or 15-20 amino acids in length. The hinge can be any suitable hinge, and in some cases includes a hinge from IgG or CD28. In certain embodiments, the hinge is a small flexible polypeptide that connects the CH2-CH3 domain and the CH1 domain of IgG Fc. For example, a CH2-CH3 hinge (part or all) from various IgG subclasses (IgG1-4, either modified or unmodified) can be utilized. However, in some cases, the entire CH2-CH3 hinge is not utilized, but instead a portion of the hinge is used (such as CH3 alone or a portion of CH3 alone). In certain embodiments, a CH2-CH3 hinge from IgG1 is utilized, and in some cases, either the entire CH2-CH3 hinge is used (all 229 amino acids), only the CH3 hinge (119 amino acids), or a short hinge (12 amino acids) is used.

[0088] In certain cases, the identity or length of the spacer and / or hinge may be altered to optimize the efficiency of the CAR. See, for example, Hudecek et al. (2014) and Jonnalagadda et al. (2015).

[0089] Thus, in certain embodiments, the IgG hinge region utilized is typically IgG1 or IgG4, and in some cases, the CAR comprises the CH2-CH3 domain of IgG Fc. The use of IgG Fc domains can provide flexibility to the CAR, making it less immunogenic, easier to detect CAR expression using anti-Fc reagents, and allowing one or more CH2 or CH3 modules to be removed to accommodate different spacer lengths. However, in one embodiment, mutations in certain spacers to avoid FcγR binding can improve the engraftment and anti-tumor efficacy of CAR+T cells to avoid soluble and cell surface Fc gamma receptor binding, while maintaining activity, for example, in mediating antigen-specific lysis. For example, an IgG4-Fc spacer modified in the CH2 region can be used. For example, the CH2 region can be mutated, including point mutations and / or deletions. Specific modifications have been demonstrated at two sites within the CH2 region (L235E; N297Q) and / or incorporating CH2 deletions (Jonnalagadda et al, 2015). In certain embodiments, the IgG4 hinge-CH2-CH3 domain (229 aa long) or the hinge domain alone (12 aa long) may be used (Hudececk et al., 2015).

[0090] In specific embodiments, the hinge is derived from IgG, CD28, CD8-alpha (CD8α), 4-1BB, 0X40, CD3-zeta (CD3ζ), T cell receptor a or b chain, CD3 zeta chain, CD28, CD3e, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, or CD154.

[0091] Examples of specific hinge arrangements that may be utilized include at least the following:

[0092] IgG hinge nucleotide sequence: GTACGGTCACTGTCTCTTCACAGGATCCCGCCGAGCCCAAATCTCCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAACCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAAAAGATCCCAAATT(SEQ ID NO: 21)

[0093] IgG hinge amino acid sequence TVTVSSQDPAEPKSPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL (SEQ ID NO: 22)

[0094] CD28 hinge nucleotide sequence: ATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCC (SEQ ID NO: 23)

[0095] CD28 hinge amino acid sequence IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 24)

[0096] CD8α hinge nucleotide sequence: AAGCCCACCACCACCCCTGCCCCTAGACCTCCAACCCCAGCCCCTACAATCGCCAGCCAGCCCCTGAGCCTGAGGCCCGAAGCCTGTAGACCTGCCGCTGGCGGAGCCGTGCACACCAGAGGCCTGGATTTCGCCTGCGACATCTACATCTGGGCCCCTCTGGCCGGCACCTGTGGCGTGCTGCTGCTGAGCCTGGTCATCACCCTGTACTGCAACCACCGGAAT (SEQ ID NO: 32)

[0097] CD8α hinge amino acid sequence KPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRN (SEQ ID NO: 33)

[0098] Any polynucleotide encompassed by this disclosure may comprise SEQ ID NO:21, 23 or 32, or a sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more identical to SEQ ID NO:21, 23 or 32. Any polypeptide encompassed by this disclosure may comprise SEQ ID NO:22, 24 or 33, or a sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more identical to SEQ ID NO:22, 24 or 33.

[0099] 4. Other proteins In some embodiments, one or more other proteins are utilized with the CAR of the present disclosure. One or more other proteins can be utilized for any reason, including promoting the effectiveness of the CAR itself and / or any type of cell expressing the CAR. In some cases, the other protein facilitates the treatment of an individual who receives the cell expressing the CAR as a therapy, regardless of whether the other protein directly or indirectly affects the activity of the CAR or the cell. In some cases, the other protein is a suicide gene, one or more cytokines, or both. In certain embodiments, one or more other proteins are produced from a vector, and are ultimately produced as two separate polypeptides. For example, the CAR and the other protein can be separated, for example, by a 2A sequence or by an IRES.

[0100] In certain embodiments, a cytokine such as IL-15 is utilized in conjunction with a CAR.

[0101] An example of an IL-15 nucleotide sequence is as follows:

[0102] IL-15 nucleotide sequence: GCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGACT GCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGGTGCACCCCAGCTGCAAGG TGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGG CAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGCTGACAATT (SEQ ID NO: 25)

[0103] IL-15 amino acid sequence: ISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 26)

[0104] In certain embodiments, a cytokine such as IL-21 is utilized in conjunction with a CAR.

[0105] An example of an IL-21 amino acid sequence is as follows: MRSSPGNMERIVICLMVIFLGTLVHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFCQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 27)

[0106] If it is intended that the CAR and another protein in the same vector be produced as two distinct polypeptides, a specific 2A sequence can be utilized.

[0107] The E2A amino acid sequence may be utilized as follows: QCTNYALLKLAGDVESNPGP (SEQ ID NO:28)

[0108] Other 2A examples may be used, such as:

[0109] T2A: EGRGSLLTCGDVEENPGP (SEQ ID NO: 29)

[0110] P2A: ATNFSLLKQAGDVEENPGP (SEQ ID NO: 30)

[0111] F2A: VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 31)

[0112] The present disclosure also encompasses specific CAR molecules, including those that are expressed in any type of immune effector cell.

[0113] CT cell receptor (TCR) In some embodiments, the genetically modified antigen receptor targeted to cancer antigen comprises a recombinant TCR and / or a TCR cloned from a natural T cell, or one or more parts thereof. "T cell receptor" or "TCR" refers to a molecule that contains variable a and β chains (also known as TCRα and TCRβ, respectively) or variable γ and δ chains (also known as TCRγ and TCRδ, respectively) and can specifically bind to an antigenic peptide bound to an MHC receptor. In some embodiments, the TCR is an αβ form.

[0114] Typically existing in αβ and γδ forms, TCRs are generally structurally similar, although T cells expressing them may have different anatomical locations or functions. TCRs may be found on the surface of cells or in soluble forms. Generally, TCRs are found on the surface of T cells (or T lymphocytes), where they are generally involved in the recognition of antigens bound to major histocompatibility complex (MHC) molecules. In some embodiments, TCRs may also contain constant domains, transmembrane domains and / or short cytoplasmic tails (see, e.g., Janeway et al, 1997). For example, in some aspects, each chain of a TCR may have one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region and a short cytoplasmic tail at the C-terminus. In some embodiments, TCRs are associated with invariant proteins of the CD3 complex, which are involved in mediating signal transduction. Unless otherwise stated, the term "TCR" should be understood to encompass functional TCR fragments thereof. The term also encompasses intact or full-length TCRs, including the αβ or γδ forms of the TCR.

[0115] Thus, for purposes herein, reference to a TCR includes any TCR or functional fragment, such as an antigen-binding portion of a TCR that binds to a specific antigenic peptide bound in an MHC molecule, i.e., an MHC-peptide complex. An "antigen-binding portion" or antigen-binding fragment of a TCR, which may be used interchangeably, refers to a molecule that contains a portion of the structural domain of the TCR but binds to the antigen (e.g., an MHC-peptide complex) that the complete TCR binds. In some cases, the antigen-binding portion contains sufficient variable domains of the TCR, such as the variable a chain and variable β chain of the TCR, to form a binding site for binding to a specific MHC-peptide complex, such as where each chain typically contains three complementarity determining regions.

[0116] In some embodiments, the variable domains of the TCR chains associate to form loops, or complementarity determining regions (CDRs) similar to immunoglobulins, which confer antigen recognition and determine peptide specificity by forming the binding site of the TCR molecule. Typically, like immunoglobulins, the CDRs are separated by framework regions (FRs) (see, for example, Jores et al., 1990; Chothia et al., 1988; Lefranc et al., 2003). In some embodiments, CDR3 is the main CDR involved in the recognition of processed antigens, although CDR1 of the alpha chain has also been shown to interact with the N-terminal portion of antigenic peptides, while CDR1 of the beta chain interacts with the C-terminal portion of peptides. CDR2 is believed to recognize MHC molecules. In some embodiments, the variable region of the beta chain may contain an additional hypervariable (HV4) region.

[0117] In some embodiments, the TCR chain contains a constant domain. For example, like an immunoglobulin, the extracellular portion of the TCR chain (e.g., a chain, β chain) comprises two immunoglobulin domains, an N-terminal variable domain (e.g., Va or Vp; typically, Kabat numbering Kabat et al., "Sequences of Proteins of Immunological Interest", US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5 th ed.), and one constant domain adjacent to the cell membrane (e.g., the a-chain constant domain or C a , typically amino acids 117-259 based on Kabat, and the β chain constant domain or Cp, typically amino acids 117-295 based on Kabat). For example, in some cases, the extracellular portion of the TCR formed by the two chains contains two membrane proximal constant domains and two membrane distal variable domains containing the CDRs. The constant domain of the TCR domain contains a short linking sequence in which cysteine ​​residues form disulfide bonds to link between the two chains. In some embodiments, the TCR may have an additional cysteine ​​residue in each of the α and β chains such that the TCR contains two disulfide bonds in the constant domain.

[0118] In some embodiments, the TCR chain may contain a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chain contains a cytoplasmic tail. In some cases, this structure allows the TCR to associate with other molecules, such as CD3. For example, the TCR contains a constant domain with a transmembrane region, which can anchor the protein to the cell membrane and associate with the invariant subunit of the CD3 signaling apparatus or complex.

[0119] Generally, CD3 is a complex of multiple proteins that may have three different chains (γ, δ, ε) and ζ chains in mammals. For example, in mammals, the complex may contain a homodimer of CD3γ, CD3δ, two CD3ε, and CD3ζ chains. CD3γ, CD3δ, and CD3ε chains are highly related cell surface proteins of the immunoglobulin superfamily that contain a single immunoglobulin domain. The transmembrane regions of CD3γ, CD3δ, and CD3ε chains are negatively charged, a feature that allows these chains to associate with the positively charged T cell receptor chains. The intracellular tails of CD3γ, CD3δ, and CD3ε chains each contain a single conserved motif known as an immunoreceptor tyrosine-based activation motif or ITAM, although each CD3ζ chain has three. Generally, ITAMs are involved in the signaling capacity of the TCR complex. These auxiliary molecules have negatively charged transmembrane regions and are responsible for propagating signals from the TCR into the cell. The CD3 chain and the ζ chain, together with the TCR, form what is known as the T cell receptor complex.

[0120] In some embodiments, the TCR may be a heterodimer of two chains, α and β (or optionally γ and δ), or may be a single chain TCR construct. In some embodiments, the TCR is a heterodimer containing two separate chains (α and β or γ and δ chains) linked, such as by disulfide bond(s). In some embodiments, a TCR against a target antigen (e.g., a cancer antigen) is identified and introduced into a cell. In some embodiments, a nucleic acid encoding the TCR may be obtained from a variety of sources, such as by polymerase chain reaction (PCR) amplification of a published TCR DNA sequence. In some embodiments, the TCR is obtained from a biological source, for example, a cell such as a T cell (e.g., a cytotoxic T cell), a T cell hybridoma, or other published source. In some embodiments, the T cell may be obtained from an isolated cell in vivo. In some embodiments, a high affinity T cell clone may be isolated from a patient and the TCR isolated. In some embodiments, the T cell may be a cultured T cell hybridoma or clone. In some embodiments, TCR clones against target antigens have been made in transgenic mice modified with human immune system genes (e.g., human leukocyte antigen system or HLA). See, e.g., tumor antigens (see, e.g., Parkhurst et al., 2009 and Cohen et al., 2005). In some embodiments, phage display is used to isolate TCRs against target antigens (see, e.g., Varela-Rohena et al., 2008 and Li, 2005). In some embodiments, TCRs or antigen-binding portions thereof can be made synthetically from knowledge of the sequence of the TCR.

[0121] III. Anti-CD20 Antibodies and Immune Cell Loading Certain aspects of the present disclosure include methods and compositions comprising anti-CD20 antibodies and antigen-binding fragments thereof. Full-length anti-CD20 antibodies as well as any fragments of anti-CD20 antibodies capable of binding to CD20 ("antigen-binding fragments") are contemplated, including, for example, Fab fragments, scFvs, and the like. The anti-CD20 antibodies of the present disclosure may have at least, up to, or exactly 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.5, or 100% sequence identity (or any range or value derivable therein) with one or more regions (e.g., VH, VL) of rituximab. In some embodiments, the anti-CD20 antibody of the present disclosure is rituximab. The anti-CD20 antibody of the present disclosure may have at least, up to, or exactly 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.5, or 100% sequence identity (or any range or value derivable therein) with one or more regions (e.g., VH, VL) of obinutuzumab. In some embodiments, the anti-CD20 antibody of the present disclosure is obinutuzumab. Additional anti-CD20 antibodies are recognized in the art and are contemplated herein, including, for example, ocrelizumab and ofatumumab. The compositions of the present disclosure may include one or more anti-CD20 antibodies. The methods of the present disclosure may include, for example, administering an anti-CD20 antibody alone or in combination with one or more other therapeutic agents, including a therapeutic agent that is an engineered immune cell.

[0122] Anti-CD20 antibodies and fragments thereof can be used in combination with immune cells of the present disclosure, including NK cells, activated NK cells, CAR NK cells, and other natural or modified immune cells, for the treatment of cancer. In some embodiments, immune cells of the present disclosure (e.g., CAR NK cells) are administered to a subject in combination with anti-CD20 antibodies or fragments thereof. The immune cells and anti-CD20 antibodies can be administered to a subject in separate compositions, either simultaneously or sequentially, or in the same composition.

[0123] As disclosed herein, immune cells can be incubated in vitro with an antibody (e.g., an anti-CD20 antibody) such that the antibody attaches to the surface of the immune cell, thereby "loading" the immune cell with the anti-CD20 antibody. As used herein, "loading" an immune cell with an antibody (or a fragment thereof) refers to incubating an immune cell with the antibody under conditions sufficient to allow the antibody to attach to the surface of the immune cell. Such immune cells that contain the antibody (or fragment) bound to their surface are described herein as "loaded" (or "antibody-loaded") immune cells. For example, NK cells that contain the antibody bound to their surface are described as antibody-loaded NK cells.

[0124] Loading the immune cells with antibody, in some embodiments, comprises incubating the immune cells with the antibody for at least, up to, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 90, 120, or 240 minutes (or any range or value derivable therein). The antibody may be provided to the immune cells in an amount of at least, up to, or about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μg / ml, or any range or value derivable therein. In some embodiments, the immune cells are incubated with the antibody for at least 15, 30, 45, 60, 90, or 120 minutes. In some embodiments, the immune cells are incubated with the antibody for at least 30 minutes. In some embodiments, the immune cells are incubated with the antibody for at least 60 minutes.

[0125] Aspects of the present disclosure include compositions of immune cells (e.g., activated NK cells, modified NK cells, e.g., CAR NK cells, etc.) having anti-CD20 antibodies or antigen-binding fragments thereof attached to their surface. Methods of treating a subject with cancer comprising administering such compositions are also disclosed. In some embodiments, methods are disclosed comprising administering a population of immune cells, e.g., loaded NK cells, loaded with anti-CD20 antibodies (e.g., obinutuzumab) to an individual with cancer. In some aspects, such loaded NK cells are loaded CAR NK cells, e.g., loaded CAR NK cells specific for CD19, CD70, and / or CD5. In some aspects, such loaded NK cells are activated NK cells (e.g., NK cells activated with IL-12, IL-15, and IL-18).

[0126] As used herein, "activated" or "preactivated" immune cells (e.g., preactivated NK cells) describe immune cells that have been activated, for example, by culturing with one or more cytokines, such as one or more of IL-12, IL-15, and IL-18. Aspects of the present disclosure are directed to methods that include activating (also "preactivating") NK cells by culturing a population of NK cells with one, two, or all of IL-12, IL-15, and IL-18. In some embodiments, the population of NK cells is cultured with IL-12 and IL-18. In some embodiments, the population of NK cells is cultured with IL-12, IL-15, and IL-18. In some embodiments, the preactivated NK cells are NK cells activated with IL-12, IL-15, and IL-18.

[0127] Preactivation of NK cells may include culturing isolated NK cells in the presence of one or more cytokines. NK cells may be stimulated with IL-2 or other cytokines that bind to a common gamma chain (e.g., IL-7, IL-12, IL-15, IL-21, etc.). In certain embodiments, the preactivation cytokines may be one, two, or all of IL-12, IL-15, and IL-18. In some embodiments, the preactivation cytokines are IL-18, IL-12, and IL-15. One or more additional cytokines may be used for the preactivation step. Preactivation may be for a short period of time, such as 5-72 hours, 10-50 hours, 10-20 hours, or 12, 13, 14, 15, 16, 17, 18, 19, or 20 hours, in some cases, about 16 hours. The pre-activation culture may comprise IL-18 and / or IL-15 at a concentration of 10-100 ng / mL, such as 40-60 ng / mL, or 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 or 55 ng / mL, in some cases about 50 ng / mL. The pre-activation culture may comprise IL-12 at a concentration of 0.1-150 ng / mL, such as 0.5-50 ng / mL, 1-20 ng / mL, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 ng / mL, in some cases about 10 ng / mL. Pre-activation may be before, during or after the expansion stage. Preactivation of NK cells may include culturing with IL-12, IL-18 and / or IL-15 either one or multiple times during the culture and expansion process. NK cells may be preactivated with IL-12, IL-18 and / or IL-15 one, two, three, four, five or more times during the processes or methods of the present disclosure.

[0128] The preactivated NK cells may be expanded to generate expanded NK cells, in some cases in the presence of artificial antigen presenting cells (aAPCs). The preactivated NK cells may be washed, for example, 2, 3, 4 or 5 times, in some cases 3 times, before expansion. The aAPCs may be modified to express CD137 ligand and / or membrane-bound cytokines. The membrane-bound cytokines may be membrane-bound IL-21 (mIL-21) or membrane-bound IL-15 (mIL-15). In certain embodiments, the aAPCs are modified to express CD137 ligand and mIL-21. The aAPCs may be derived from cancer cells, such as leukemia cells. The aAPCs may not express endogenous HLA class I, II or CD1d molecules. They may express ICAM-1 (CD54) and LFA-3 (CD58). In particular, the aAPCs can be K562 cells, such as K562 cells modified to express CD137 ligand and mIL-21. The aAPCs can be irradiated. The modification can be by any method known in the art, such as retroviral transduction. The aAPCs can be non-cellular; for example, the aAPCs can be by microparticles or other synthetic aAPCs. The cell expansion culture can be for about 2-30 days, such as 3-20 days, 12-16 days, or 12, 13, 14, 15, 16, 17, 18 or 19 days, and in some cases about 14 days. The preactivated NK cells and the aAPCs can be in a ratio of about 3:1 to 1:3, such as 2:1, 1:1 or 1:2, and in some cases about 1:2. The expansion culture can further include a cytokine to promote the expansion culture, such as IL-2. IL-2 may be present at a concentration of about 10-500 U / mL, e.g., 100-300 U / mL, particularly about 200 U / mL. IL-2 may be replenished, e.g., every 2-3 days, during expansion. aAPC may be added to the culture at least a second time, e.g., on about day 7 of expansion.

[0129] Contemplated herein are pre-activated and expanded NK cells, as well as methods for producing such cells, such as those described in U.S. Patent Application Publication No. 2020 / 0390816A1 and Kerbauy et al., Clin Cancer Res. 2021;27(13):3744-3756, both of which are incorporated by reference in their entireties. IV. Cytokines

[0130] One or more cytokines may be utilized in some instances with one or more engineered antigen-targeting receptors, such as antigen-specific CARs. In some cases, one or more cytokines are present on the same vector molecule as the engineered receptor, while in other cases, they are present on separate vector molecules. In some cases, the immune cells of the present disclosure express cytokines and do not express antigen-specific CARs. In certain embodiments, one or more cytokines are co-expressed from the same vector as the engineered receptor. As an example, interleukin-15 (IL-15) is utilized. IL-15 may be used because, for example, it is tissue-restricted and is only observed in serum or systemically at some level under pathological conditions. IL-15 has several attributes that are desirable for adoptive therapy. IL-15 is a homeostatic cytokine that induces the development and cell proliferation of natural killer cells, promotes the eradication of established tumors through the relief of functional inhibition of tumor-resident cells, and inhibits activation-induced cell death. In addition to IL-15, other cytokines are envisioned. These include, but are not limited to, cytokines, chemokines, and other molecules that contribute to the activation and proliferation of cells used in human applications. As an example, the one or more cytokines are IL-15, IL-12, IL-2, IL-18, IL-21, IL-23, IL-7, or a combination thereof. NK cells that express IL-15 can be utilized and are capable of continued supportive cytokine signaling, which is useful for survival after infusion.

[0131] In certain embodiments, the NK cells express one or more exogenously provided cytokines. The cytokines can be exogenously provided to the NK cells because they are expressed from an expression vector in the cells and / or provided in the culture medium of the cells. Alternatively, the endogenous cytokines in the cells are upregulated by regulatory engineering of the expression of the endogenous cytokine, such as genetic engineering at the promoter site of the cytokine. When the cytokine is provided to the cells on an expression construct, the cytokine can be encoded from the same vector as the suicide gene. The cytokine can be expressed as a polypeptide molecule separate from the suicide gene and as a polypeptide separate from the engineered receptor of the cell. In some embodiments, the present disclosure relates to the co-use of CAR and / or TCR vectors with IL-15, particularly in NK cells. V. Suicide Genes

[0132] In certain embodiments, suicide genes are utilized with any type of cell therapy to control its use and allow for the termination of cell therapy at a desired event and / or time. Suicide genes are used in transduced cells to induce the death of the transduced cells as needed. The antigen-targeted cells of the present disclosure modified to have vectors encompassed by the present disclosure may contain one or more suicide genes. In some embodiments, the term "suicide gene" as used herein is defined as a gene that results in the transfer of the gene product to a compound that will kill the host cell upon administration of a prodrug or other agent. In other embodiments, the suicide gene encodes a gene product that, if desired, is targeted by an agent (such as an antibody) that targets the suicide gene product.

[0133] Examples of suicide gene / prodrug combinations that can be used include Herpes Simplex Virus-thymidine kinase (HSV-tk) and ganciclovir, acyclovir, or FIAU; oxidoreductase and cycloheximide; cytosine deaminase and 5-fluorocytosine; thymidine kinase thymidilate kinase (Tdk::Tmk) and AZT; and deoxycytidine kinase and cytosine arabinoside. E. coli purine nucleoside phosphorylase, a suicide gene that converts the prodrug 6-methylpurine deoxyriboside to the toxic purine 6-methylpurine, can be used. Other examples of suicide genes used in prodrug therapy include the E. coli cytosine deaminase gene and the HSV thymidine kinase gene.

[0134] Exemplary suicide genes include CD20, CD52, EGFRv3, or inducible caspase 9. In one embodiment, a truncated version of EGFR variant III (EGFRv3) can be used as a suicide antigen that can be removed by cetuximab. Additional suicide genes known in the art that can be used in the present disclosure include purine nucleoside phosphorylase (PNP), cytochrome p450 enzymes (CYP), carboxypeptidase (CP), carboxylesterase (CE), nitroreductase (NTR), guanine ribosyltransferase (XGRTP), glycosidase enzymes, methionine-alpha, gamma-lyase (MET), and thymidine phosphorylase (TP). In some embodiments, inducible caspase 9 (iC9) is used.

[0135] In certain embodiments, the vector encoding the antigen-targeted CAR, or any vector in the NK cells encompassed herein, comprises one or more suicide genes. The suicide genes may or may not be on the same vector as the antigen-targeted CAR. VI. Vector

[0136] The antigen-targeting CAR of the present disclosure can be delivered to recipient immune cells by any suitable vector, including viral vector or non-viral vector.Examples of viral vector include at least retrovirus, lentivirus, adenovirus, or adeno-associated virus vector.Examples of non-viral vector include at least plasmid, transposon, lipid, nanoparticle, etc.

[0137] When an immune cell is transduced with a vector encoding an antigen-targeting CAR and also requires the transduction of another gene, such as a suicide gene and / or a cytokine and / or an optional therapeutic gene product, into the cell, the antigen-targeting CAR, the suicide gene, the cytokine, and the optional therapeutic gene may or may not be included in the same vector. In some cases, the antigen-targeting CAR, the suicide gene, the cytokine, and the optional therapeutic gene are expressed from the same vector molecule, such as the same viral vector molecule. In such cases, the expression of the antigen-targeting CAR, the suicide gene, the cytokine, and the optional therapeutic gene may or may not be regulated by the same regulatory element. When the antigen-targeting CAR, the suicide gene, the cytokine, and the optional therapeutic gene are on the same vector, they may or may not be expressed as separate polypeptides. When they are expressed as separate polypeptides, they may be separated on the vector, for example, by a 2A element or an IRES element (or both types may be used on the same vector, one or more times). A. General embodiment

[0138] Those of skill in the art would be well-versed in constructing vectors by standard recombinant techniques (see, e.g., Sambrook et al., 2001 and Ausubel et al., 1996, both of which are incorporated herein by reference) for expression of the antigen receptors of the present disclosure. 1. Adjustment element

[0139] The expression cassette contained in the vector useful in the present disclosure contains, inter alia, a eukaryotic transcriptional promoter operably linked to a protein coding sequence, a splice signal with intervening sequences, and a transcription termination / polyadenylation sequence (5' to 3' direction). The promoters and enhancers that control the transcription of protein coding genes in eukaryotic cells can be composed of multiple genetic elements. The cellular machinery can collect and incorporate the regulatory information transmitted by each element, allowing different genes to evolve different, often complex patterns of transcriptional regulation. Promoters used in connection with the present disclosure include, for example, constitutive promoters, inducible promoters, and tissue-specific promoters. When the vector is utilized for the production of cancer treatments, the promoter can be effective under conditions of hypoxia. 2. Promoter / Enhancer

[0140] The expression constructs provided herein include promoters for driving the expression of antigen receptors and other cistron gene products. Promoters generally contain sequences that function to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters that lack a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late gene, separate elements that cover the start site itself help to fix the location of start. Additional promoter elements regulate the frequency of transcription initiation. Typically, this is located in the region upstream of the start site, but some promoters have been shown to contain functional elements downstream of the start site as well. To place a coding sequence "under the control" of a promoter, the 5' end of the transcription start site of the transcriptional reading frame is placed "downstream" (i.e., 3') of the selected promoter. The "upstream" promoter stimulates transcription of DNA to promote expression of the encoded RNA.

[0141] Spacing between promoter elements is frequently flexible enough that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, for example, spacing between promoter elements can be increased to 50 bp before activity begins to decline. Depending on the promoter, individual elements appear to be able to function cooperatively or independently to activate transcription. Promoters may or may not be used in conjunction with "enhancers," which refer to cis-acting regulatory sequences involved in the transcriptional activation of a nucleic acid sequence.

[0142] A promoter may be one that is naturally associated with a nucleic acid sequence, such as may be obtained by isolating 5' non-coding sequences located upstream of a coding segment and / or exon. Such a promoter may be referred to as "endogenous". Similarly, an enhancer may be one that is naturally associated with a nucleic acid sequence and located either downstream or upstream of said sequence. Certain advantages may also be obtained by placing a coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer also refers to an enhancer that is not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, as well as promoters or enhancers isolated from any other virus or prokaryotic or eukaryotic cell, as well as promoters or enhancers that are not "naturally occurring", i.e., that contain different elements of different transcriptional regulatory regions and / or mutations that alter expression. For example, promoters most commonly used in recombinant DNA construction include the β-lactamase (penicillinase), lactose, and tryptophan (trp-) promoter systems. In addition to producing promoter and enhancer nucleic acid sequences synthetically, sequences may be produced using recombinant cloning and / or nucleic acid amplification techniques, including PCR™, in conjunction with the compositions disclosed herein. Furthermore, it is contemplated that control sequences that direct transcription and / or expression of sequences in non-nuclear organelles, such as mitochondria, chloroplasts, etc., may be used as well.

[0143] Of course, it will be important to use a promoter and / or enhancer that effectively directs the expression of the DNA segment in the organelle, cell type, tissue, organ, or organism selected for expression. Those skilled in the art of molecular biology are generally aware of the use of promoter, enhancer, and cell type combinations for protein expression (see, for example, Sambrook et al. 1989, which is incorporated herein by reference). The promoter used may be constitutive, tissue-specific, inducible, and / or useful under appropriate conditions to direct high-level expression of the introduced DNA segment, such as being advantageous in large-scale production of recombinant proteins and / or peptides. The promoter may be heterologous or endogenous.

[0144] Additionally, expression can be driven using any promoter / enhancer combination (e.g., according to the Eukaryotic Promoter Data Base EPDB, via the World Wide Web at epd.isb-sibi.ch / ). Use of the T3, T7, or SP6 cytoplasmic expression systems is another possible embodiment. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided as part of the delivery complex or as an additional gene expression construct.

[0145] Non-limiting examples of promoters include early or late viral promoters, such as SV40 early or late promoters, cytomegalovirus (CMV) immediate early promoter, Rous sarcoma virus (RSV) early promoter, eukaryotic promoters, such as beta actin promoter, GADPH promoter, metallothionein promoter, and linked response element promoters, such as cyclic AMP response element promoter (cre), serum response element promoter (sre), phorbol ester promoter (TPA), and response element promoter near minimal TATA box (tre). It is also possible to use a human growth hormone promoter sequence (e.g., human growth hormone minimal promoter described in GenBank®, Accession No. X05244, nucleotides 283-341) or a mouse mammary tumor promoter (available from the ATCC, Cat No. ATCC 45007). In certain embodiments, the promoter is a CMV IE, Dectin-1, Dectin-2, human CD11c, F4 / 80, SM22, RSV, SV40, Ad MLP, beta-actin, MHC class I, or MHC class II promoter. However, any other promoter useful for driving expression of therapeutic genes can be used in the practice of the present disclosure.

[0146] In certain embodiments, the methods of the present disclosure also relate to enhancer sequences, i.e., nucleic acid sequences that have the potential to increase the activity of a promoter and act in cis and regardless of its orientation, even over relatively long distances (up to several kilobases away from the target promoter). However, enhancer function is not necessarily limited to such long distances, since it may function in close proximity to a given promoter. 3. Initiation Signals and Linked Expression

[0147] Also, specific initiation signals can be used in the expression constructs provided in this disclosure for efficient translation of the coding sequence. Such signals include the ATG initiation codon or adjacent sequences. It may be necessary to provide exogenous translation control signals including the ATG initiation codon. Those skilled in the art can easily determine this and provide the necessary signals. It is well known that to ensure translation of the entire insert, the initiation codon must be "in frame" with the reading frame of the desired coding sequence. Exogenous translation control signals and initiation codons can be either natural or synthetic. The efficiency of expression can be enhanced by including appropriate transcription enhancer elements.

[0148] In certain embodiments, an internal ribosome entry site (IRES) element is used to produce multigene or polycistronic messages. IRES elements can bypass the ribosome scanning model of 5' methylated Cap-dependent translation and initiate translation at an internal site. IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) as well as IRES from mammalian messages have been described. IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES resulting in a polycistronic message. Thanks to the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter / enhancer to transcribe a single message.

[0149] As detailed elsewhere herein, certain 2A sequence elements can be used to provide for linked or co-expression of genes in constructs provided in this disclosure. For example, a truncation sequence can be used to link open reading frames to form a single cistron, thereby allowing genes to be co-expressed. Exemplary truncation sequences include Equine Rhinitis A Virus (E2A) or F2A (Foot and Mouth Disease Virus 2A) or "2A-like" sequences (e.g., Thosea asigna Virus 2A; T2A) or Porcine Teschovirus-1 (P2A). In certain embodiments, in a single vector, multiple 2A sequences are not identical, but in alternative embodiments, the same vector utilizes two or more identical 2A sequences. Examples of 2A sequences are described in U.S. Patent Application Publication No. 2011 / 0065779, which is incorporated herein by reference in its entirety. 4. Origin of replication

[0150] To propagate the vector in a host cell, the vector may contain one or more origin of replication sites (often referred to as "ori"), such as a nucleic acid sequence corresponding to the EBV oriP described above, or a genetically engineered oriP with similar or enhanced function in programming, which is a specific nucleic acid sequence from which replication is initiated. Alternatively, the origin of replication of other extrachromosomally replicating viruses described above, or an autonomously replicating sequence (ARS), may be used. 5. Selectable and Screenable Markers

[0151] In some embodiments, NK cells containing the constructs of the present disclosure can be identified in vitro or in vivo by including a marker in the expression vector. Such a marker will confer an identifiable change to the cells that allows easy identification of cells containing the expression vector. In general, a selection marker is one that confers a property that allows for selection. A positive selection marker is one whose presence allows for selection, and a negative selection marker is one whose presence prevents selection. An example of a positive selection marker is a drug resistance marker.

[0152] In general, the inclusion of a drug selection marker aids in cloning and identification of transformants; for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and histidinol are useful selection markers. In addition to markers that confer a phenotype that allows the identification of transformants based on the implementation of conditions, other types of markers are also contemplated, including screenable markers such as GFP, whose basis is colorimetric analysis. Other screenable enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) can be utilized as negative selection markers. Also, those skilled in the art will likely know how to use immunological markers in combination with FACS analysis. The marker used is not believed to be important, so long as it can be expressed simultaneously with the nucleic acid encoding the gene product. Further examples of selection and screenable markers are well known to those skilled in the art. B. Multicistronic Vectors

[0153] In certain embodiments, the antigen-targeting CAR, optional suicide gene, optional cytokine, and / or optional therapeutic gene are expressed from a multicistronic vector (the term "cistron" as used herein refers to a nucleic acid sequence from which a gene product can be generated). In certain embodiments, the multicistronic vector encodes the antigen-targeting CAR, the suicide gene, and at least one cytokine and / or engineered receptor, such as a T cell receptor. Optionally, the multicistronic vector encodes at least one antigen-targeting CAR and at least one cytokine. The cytokine can be a specific type of cytokine, such as human or mouse, or any species. In certain cases, the cytokine is IL15, IL12, IL2, IL18, and / or IL21. In some embodiments, the cytokine is IL15. In some aspects, the cytokine is IL21.

[0154] In certain embodiments, the present disclosure provides a flexible modular system utilizing polycistronic vectors capable of expressing multiple cistrons at substantially the same level (the term "module" as used herein refers to a cistron or a component of a cistron, allowing its interchangeability, such as by removal and replacement of the entire cistron or each of the components of the cistron, for example, by using standard recombinant techniques). The system can be used for cell engineering to allow combinatorial expression (including overexpression) of multiple genes. In certain embodiments, one or more of the genes expressed by the vector includes one, two, or more antigen receptors. The multiple genes can include, but are not limited to, CARs, TCRs, cytokines, chemokines, homing receptors, CRISPR / Cas9-mediated gene mutations, decoy receptors, cytokine receptors, chimeric cytokine receptors, and the like. The vector can further include: (1) one or more reporters, such as fluorescent or enzymatic reporters for cell assays and animal imaging, and the like; (2) one or more cytokines or other signaling molecules; and / or (3) a suicide gene.

[0155] In certain cases, the vector may contain at least four cistrons separated by any kind of cleavage site, such as 2A cleavage sites. The vector may or may not be based on Moloney Murine Leukemia Virus (MoMLV or MMLV) containing 3' and 5' LTR with psi packaging sequence in pUC19 backbone. The vector may contain four or more cistrons with three or more 2A cleavage sites and multiple ORFs for gene exchange. The system allows for combinatorial overexpression of multiple genes (seven or more) in some embodiments flanked by restriction sites for rapid integration by subcloning, and also contains at least three 2A self-cleavage sites. Thus, the system allows for the expression of multiple CARs, TCRs, signaling molecules, cytokines, cytokine receptors, and / or homing receptors. The system may also be used for other viral and non-viral vectors, including but not limited to lentivirus, adenovirus AAV, and non-viral plasmids.

[0156] The modularity of the system also allows for efficient subcloning of genes into each of the four cistrons within the polycistronic expression vector, and swapping of genes for rapid testing, etc. Strategically positioned restriction sites within the polycistronic expression vector allow genes to be swapped efficiently.

[0157] Embodiments of the present disclosure encompass systems that utilize polycistronic vectors in which at least a portion of the vector is modular, for example, by allowing for the removal and replacement of one or more cistrons (or components of one or more cistrons), such as by utilizing one or more restriction enzyme sites whose identities and locations are specifically selected to facilitate modular use of the vector. Vectors also have embodiments that confer the advantage that the vector expresses separate gene products in substantially equimolar concentrations, with multiple cistrons being translated into a single polypeptide and processed into separate polypeptides.

[0158] The vectors of the present disclosure are configured such that modularity can be altered for one or more cistrons of the vector and / or one or more components of one or more particular cistrons. The vectors can be designed to take advantage of unique restriction enzyme sites flanking the ends of one or more cistrons and / or flanking the ends of one or more components of a particular cistron.

[0159] The embodiments of the present disclosure include polycistronic vectors that include at least two, at least three, or at least four cistrons, each flanked by one or more restriction enzyme sites, with at least one cistron encoding at least one antigen receptor. In some cases, two, three, four, or more cistrons are translated into a single polypeptide and cleaved into separate polypeptides, while in other cases, multiple cistrons are translated into a single polypeptide and cleaved into separate polypeptides. Adjacent cistrons on a vector can be separated by a self-cleaving site, such as a 2A self-cleaving site. In some cases, each cistron expresses a separate polypeptide from the vector. In certain cases, adjacent cistrons on a vector are separated by an IRES element.

[0160] In certain embodiments, the present disclosure provides a system for cell engineering that allows for combinatorial expression, including overexpression, of multiple cistrons, which may include, for example, one, two, or more antigen receptors. In certain embodiments, the use of polycistronic vectors described herein allows the vectors to generate equimolar levels of multigene products from the same mRNA. Multigenes may include, but are not limited to, CARs, TCRs, cytokines, chemokines, homing receptors, CRISPR / Cas9-mediated gene mutations, decoy receptors, cytokine receptors, chimeric cytokine receptors, and the like. The vectors may further include one or more fluorescent or enzymatic reporters for cell assays, animal imaging, and the like. The vectors may also include suicide gene products for the termination of cells carrying the vector when the cells are no longer needed or become harmful to the host to which it is provided.

[0161] In certain embodiments, the vector is a viral vector (e.g., a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector) or a non-viral vector. The vector may include Moloney Murine Leukemia Virus (MMLV) 5'LTR, 3'LTR, and / or psi packaging elements. In certain cases, the psi packaging is integrated between the 5'LTR and the antigen receptor coding sequence. The vector may or may not include a pUC19 sequence. In some embodiments of the vector, at least one cistron encodes a cytokine (e.g., IL-15, IL-7, IL-21, IL-23, IL-18, IL-12, or IL-2), a chemokine, a cytokine receptor, and / or a homing receptor.

[0162] If 2A cleavage sites are utilized in the vector, the 2A cleavage sites may include a P2A, T2A, E2A, and / or F2A site.

[0163] The restriction enzyme site may be of any type and may contain any number of bases in its recognition site, for example, 4-8 bases; the number of bases in the recognition site may be at least 4, 5, 6, 7, 8, or more. The site when cleaved may generate blunt cut ends or sticky ends. The restriction enzyme may be, for example, type I, type II, type III, or type IV. Restriction enzyme sites may be obtained from available databases, for example, the Integrated relational Enzyme database (IntEnz) or BRENDA (The Comprehensive Enzyme Information System).

[0164] An exemplary vector may be circular, and by convention, position 1 (the 12 o'clock position at the top of the circle, with the remaining sequences in a clockwise direction) is set at the start of the 5'LTR.

[0165] In embodiments in which a self-cleaving 2A peptide is utilized, the 2A peptide can be a viral oligopeptide 18-22 amino acids (aa) long that mediates the "cleavage" of the polypeptide during translation in eukaryotic cells. The designation "2A" refers to a specific region of the viral genome, and various viral 2As are commonly named after the viruses from which they are derived. The first 2A discovered was F2A (foot and mouth disease virus), and subsequently E2A (equine rhinitis A virus), P2A (porcine teschovirus-1 2A), and T2A (Thosea asigna virus 2A) were also identified. The mechanism of 2A-mediated "self-cleavage" was discovered to be ribosomal skipping, which skips the formation of a glycyl-prolyl peptide bond at the C-terminus of 2A.

[0166] In certain cases, the vector can be a gamma-retroviral transfer vector. Retroviral transfer vectors can include a backbone based on a plasmid, such as pUC19 plasmid (large fragment (2.63 kb) between the restriction enzyme sites of HindIII and EcoRI). The backbone can have viral components from Moloney Murine Leukemia Virus (MoMLV), including 5'LTR, psi packaging sequence, and 3'LTR. LTRs are long terminal repeat sequences found on both sides of retroviral proviruses, and in the case of transfer vectors, they surround the genetic cargo of interest. Also, the psi packaging sequence, which is the target site for packaging by nucleocapsid, is integrated in cis and sandwiched between the 5'LTR and the CAR coding sequence. Thus, the basic structure of an example transfer vector can be constructed as follows: pUC19 sequence-5'LTR-psi packaging sequence-genetic cargo of interest-3'LTR-pUC19 sequence. The system can also be used with other viral and non-viral vectors, including but not limited to lentiviruses, adenoviruses AAV, and non-viral plasmids. VII.Cells

[0167] The present disclosure encompasses any type of immune cell or stem cell with at least one vector encoding an antigen-targeting CAR and may also encode at least one cytokine and / or at least one suicide gene. In some cases, the various vectors encode the CAR versus encoding the suicide gene and / or cytokine. Immune cells, including NK cells, can be derived from umbilical cord blood (including pooled umbilical cord blood from multiple sources), peripheral blood, induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), hematopoietic stem cells (HSCs), bone marrow, or mixtures thereof. NK cells can be derived from cell lines, such as, for example, but not limited to, NK-92 cells. NK cells are characterized by CD56 + They may be cord blood mononuclear cells, such as NK cells.

[0168] The present disclosure encompasses any type of immune cell or other cell, including conventional T cells, gamma-delta T cells, NK T and invariant NKT cells, regulatory T cells, macrophages, B cells, dendritic cells, mesenchymal stromal cells (MSCs), or mixtures thereof.

[0169] In some cases, the cells are expanded in the presence of an effective amount of universal antigen presenting cells (UAPC), including any suitable ratio. The cells can be cultured with UAPC at a ratio of 10:1 to 1:10; 9:1 to 1:9; 8:1 to 1:8; 7:1 to 1:7; 6:1 to 1:6; 5:1 to 1:5; 4:1 to 1:4; 3:1 to 1:3; 2:1 to 1:2; or 1:1, including, for example, a ratio of 1:2. In some cases, the NK cells were expanded in the presence of IL-2 at a concentration of, for example, 10-500, 10-400, 10-300, 10-200, 10-100, 10-50, 100-500, 100-400, 100-300, 100-200, 200-500, 200-400, 200-300, 300-500, 300-400, or 400-500 U / mL.

[0170] After genetic modification with the vector, the NK cells may be infused immediately or stored. In certain embodiments, after genetic modification, the cells may be expanded ex vivo as a bulk population for days, weeks, or months within about 1, 2, 3, 4, 5 days or more after gene introduction into the cells. In further embodiments, the transfectants are cloned (clones showing the presence of a single integrated or episomally maintained expression cassette or plasmid) and the expression of the antigen-targeting CAR is expanded ex vivo. Clones selected for expansion demonstrate the ability to specifically recognize and lyse antigen-expressing target cells. The recombinant immune cells may be expanded by stimulation with IL-2 or other cytokines that bind to the common gamma chain (e.g., IL-7, IL-12, IL-15, IL-21, IL-23, etc.). The recombinant immune cells may be expanded by stimulation with artificial antigen-presenting cells. In further embodiments, the genetically modified cells may be cryopreserved.

[0171] Embodiments of the present disclosure include cells expressing one or more antigen-targeting CARs and one or more suicide genes encompassed herein.

[0172] The cells may be obtained directly from an individual or from a repository or other storage facility. Cells as a therapy may be autologous or allogeneic to the individual to whom the cells are provided as a therapy.

[0173] The cells may be derived from an individual in need of treatment for a medical condition and, after engineering to express the antigen-targeting CAR, optional suicide gene, optional cytokine, and optional therapeutic gene product (e.g., using standard techniques for transduction and expansion for adoptive cell therapy), may be returned to the individual from whom they were originally sourced. In some cases, the cells are stored for later use in the individual or another individual.

[0174] The immune cells may be included within a population of cells, the population being predominantly transduced with one or more antigen targeting receptors and / or one or more suicide genes and / or one or more cytokines. The cell population may include 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, or 100% of immune cells transduced with one or more antigen targeting receptors and / or one or more suicide genes and / or one or more cytokines. The one or more antigen targeting receptors and / or the one or more suicide genes and / or the one or more cytokines may be separate polypeptides.

[0175] Immune cells can be generated with one or more antigen-targeting receptors and / or one or more suicide genes and / or one or more cytokines, with the intention of being modular with respect to a particular purpose. For example, cells expressing an antigen-targeting CAR and / or one or more suicide genes and / or one or more cytokines (or having nucleic acids encoding mutants distributed for subsequent transduction) can be generated, for example, for commercial distribution, and the user can modify the cells to express one or more other genes of interest (including therapeutic genes) depending on the intended purpose.

[0176] In certain embodiments, NK cells can be utilized to modify the genome of transduced NK cells, which express one or more antigen targeting receptors and / or one or more suicide genes and / or one or more cytokines.Genome can be modified in any manner, but in certain embodiments, genome is modified by, for example, CRISPR gene editing.Genome of cell can be modified to enhance the effectiveness of cell for any purpose. VIII. Gene editing of cells

[0177] In certain embodiments, the cell of the present invention is genetically edited to modify the expression of one or more endogenous genes in the cell.In certain cases, the cell is modified to have the expression level of one or more endogenous genes reduced, including the expression inhibition (can be called knockout) of one or more endogenous genes.Such cells can be expanded or not.

[0178] In certain cases, one or more endogenous genes of a cell are modified, for example, expression is disrupted, expression is partially or completely reduced. In certain cases, one or more genes are knocked down or knocked out using the process of the present disclosure. In certain cases, multiple genes are knocked down or knocked out, which may or may not occur at the same step in their production. The gene edited in a cell can be of any kind, but in certain embodiments, the gene is a gene whose gene product inhibits the activity and / or proliferation of a cell (an example is an antigen-specific CAR NK cell, such as one derived from umbilical cord blood). In certain cases, the gene edited in a cell allows the cell to act more effectively in the tumor microenvironment. In certain cases, the genes are one or more of NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5, CD7, and CD38. In certain embodiments, the TGFBR2, CISH, and / or CD38 genes are knocked out or knocked down in the cell.

[0179] In some embodiments, gene editing is performed using one or more DNA-binding nucleic acids, such as modification via RNA-guided endonucleases (RGENs). For example, modification can be performed using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins. In some embodiments, CpF1 is utilized instead of Cas9. In general, "CRISPR system" refers to the transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated ("Cas") genes, including sequences encoding Cas genes, tracr (transactivating CRISPR) sequences (e.g., tracrRNA or active portion tracrRNA), tracr-mate sequences ("direct repeats", encompassing tracrRNA-processed portion direct repeats in the context of endogenous CRISPR systems), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR systems), and / or other sequences and transcripts from CRISPR loci.

[0180] A CRISPR / Cas nuclease or CRISPR / Cas nuclease system can include a non-coding RNA molecule (guide) RNA that binds to DNA in a sequence-specific manner, and a Cas protein (e.g., Cas9) that has nuclease functionality (e.g., two nuclease domains). One or more elements of the CRISPR system can be derived from a type I, type II, or type III CRISPR system, e.g., derived from a particular organism that contains an endogenous CRISPR system, such as Streptococcus pyogenes.

[0181] In some embodiments, Cas nuclease and gRNA (including a fusion of a crRNA specific for a target sequence and a fixed tracrRNA) are introduced into cells. In general, a target site at the 5' end of the gRNA targets the Cas nuclease to a target site, e.g., a gene, using complementary base pairing. The target site can be selected based on its location immediately 5' of a protospacer adjacent motif (PAM) sequence, typically NGG or NAG. In this regard, the gRNA is targeted to a desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 14, 12, 11, or 10 nucleotides of the guide RNA to correspond to the target DNA sequence. In general, CRISPR systems feature elements that promote the formation of a CRISPR complex at the site of the target sequence. Typically, a "target sequence" generally refers to a sequence that the guide sequence is designed to have complementarity with, and hybridization between the target sequence and the guide sequence promotes the formation of a CRISPR complex. Absolute complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex.

[0182] The CRISPR system can induce a double-strand break (DSB) at the target site, followed by destruction or modification, as discussed herein. In other embodiments, a Cas9 variant considered a "nickase" is used to nick a single strand at the target site. Paired nickases can be used, for example, to improve specificity, each guided by a pair of different gRNA targeting sequences, such that a 5' overhang is introduced when a nick is introduced simultaneously. In other embodiments, catalytically inactive Cas9 is fused to a heterologous effector domain, such as a transcriptional repressor or activator, to affect gene expression.

[0183] The target sequence may comprise any polynucleotide, for example DNA or RNA polynucleotide. The target sequence may be located in the nucleus or cytoplasm of a cell, for example in an organelle of a cell. In general, the sequence or template that can be used for recombination into the target locus that comprises the target sequence is referred to as "editing template" or "editing polynucleotide" or "editing sequence". In some embodiments, the exogenous template polynucleotide may be referred to as editing template. In some embodiments, the recombination is homologous recombination.

[0184] Typically, in the context of an endogenous CRISPR system, the formation of a CRISPR complex (including a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in the cleavage of one or both strands within or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from the target sequence). Also, the tracr sequence may comprise or consist of all or a portion of a wild-type tracr sequence (e.g., about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of the wild-type tracr sequence), but may form part of a CRISPR complex, for example, by hybridization with all or a portion of a tracr mate sequence operably linked to the guide sequence along at least a portion of the tracr sequence. The tracr sequence has sufficient complementarity to the tracr mate sequence (such as at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence complementarity along the length of the tracr mate sequence when optimally aligned) to hybridize and participate in the formation of a CRISPR complex.

[0185] One or more vectors driving the expression of one or more elements of the CRISPR system can be introduced into a cell, such that the expression of the elements of the CRISPR system directs the formation of a CRISPR complex at one or more target sites. Also, the components can be delivered to the cell as proteins and / or RNA. For example, the Cas enzyme, the guide sequence linked to the tracr-mate sequence, and the tracr sequence can each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more of the elements expressed from the same or different regulatory elements can be combined in a single vector with one or more additional vectors that provide any components of the CRISPR system that are not included in the first vector. The vector can include one or more insertion sites (also referred to as "cloning sites"), such as restriction endonuclease recognition sequences. In some embodiments, the one or more insertion sites are located upstream and / or downstream of one or more sequence elements of one or more vectors. When multiple different guide sequences are used, a single expression construct can be used to target CRISPR activity to multiple different corresponding target sequences in a cell.

[0186] The vector may include a regulatory element operably linked to an enzyme coding sequence encoding a CRISPR enzyme, such as a Cas protein, non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, and the like. , Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csfl, Csf2, Csf3, Csf4, Cpf1 (Cas12a), their homologs, or modified versions thereof. These enzymes are known. For example, the amino acid sequence of the S. pyogenes Cas9 protein can be found in the SwissProt database under the accession number Q99ZW2.

[0187] The CRISPR enzyme may be Cas9 (e.g., from S. pyogenes or S. pneumonia). In some cases, Cpf1 (Cas12a) may be used as an endonuclease instead of Cas9. The CRISPR enzyme may direct the cleavage of one or both strands at the location of the target sequence, such as within the target sequence and / or within the complement of the target sequence. The vector may encode a CRISPR enzyme that is mutated relative to the corresponding wild-type enzyme, such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing the target sequence. For example, an aspartic acid to alanine substitution (D10A) in the RuvCI catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (that cleaves a single strand). In some embodiments, Cas9 nickases can be used in combination with guide sequences, such as two guide sequences, that target the sense and antisense strands of a DNA target, respectively, allowing both strands to be nicked and used to induce NHEJ or HDR.

[0188] In some embodiments, the enzyme coding sequence encoding the CRISPR enzyme is codon-optimized for expression in a particular cell, such as a eukaryotic cell. The eukaryotic cell may be of or derived from a particular organism, such as a mammal, including but not limited to a human, mouse, rat, rabbit, dog, or non-human primate. In general, codon optimization refers to the process of modifying a nucleic acid sequence to enhance expression in a host cell of interest by replacing at least one codon of the native sequence with a codon that is more frequently or most frequently used in the genes of the host cell, while maintaining the native amino acid sequence. Different species show a particular bias for a particular codon of a particular amino acid. Codon bias (the difference in codon usage between organisms) often correlates with the translation efficiency of messenger RNA (mRNA), which is believed to depend, among other things, on the properties of the codon to be translated and the availability of a particular transfer RNA (tRNA) molecule. The dominance of a selected tRNA in a cell is generally a reflection of the codon that is most frequently used in peptide synthesis. Thus, based on codon optimization, genes can be tailored for optimal gene expression in a given organism.

[0189] In general, a guide sequence is any polynucleotide sequence that has sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of CRISPR complex to the target sequence.In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence is about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or more when optimally aligned using a suitable alignment algorithm.

[0190] Optimal alignment can be determined by use of any algorithm suitable for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., Burrows Wheeler Aligner), Clustal W, Clustal X, BLAT, Novoalign (Novocraft Technologies), ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).

[0191] CRISPR enzyme can be part of a fusion protein that contains one or more heterologous protein domains. CRISPR enzyme fusion protein can contain any additional protein sequence, and optionally a linker sequence between any two domains. Examples of protein domains that can be fused to CRISPR enzyme include, but are not limited to, epitope tags, reporter gene sequences, and protein domains that have one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tag, V5 tag, FLAG tag, influenza hemagglutinin (HA) tag, Myc tag, VSV-G tag, and thioredoxin (Trx) tag. Examples of reporter genes include, but are not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), autofluorescent proteins including HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and blue fluorescent protein (BFP). CRISPR enzymes can be fused to gene sequences that code for proteins or fragments of proteins that bind to DNA molecules or other cellular molecules, including, but not limited to, maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4A DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. Additional domains that can form part of fusion proteins that contain CRISPR enzymes are described in US Patent Publication No. 2011 / 0059502, which is incorporated herein by reference. IX. Treatment Method

[0192] In various embodiments, diseased or other cells expressing endogenous antigens (e.g., CD20) on their surface are targeted to improve a medical condition in an individual with the medical condition, or to reduce the risk or delay the severity and / or onset of a medical condition in an individual. In certain cases, cancer cells expressing endogenous antigens are targeted in order to kill the cancer cells.

[0193] The antigen-targeting CAR constructs, nucleic acid sequences, vectors, immune cells, etc. contemplated herein, and / or pharmaceutical compositions comprising same, are used for the prevention, treatment, or amelioration of cancerous diseases, such as neoplastic diseases.

[0194] The immune cells in which the antigen targeting receptor is utilized may be, in certain embodiments, NK cells, T cells, gamma delta T cells, alpha beta T cells, or NKT or invariant NKT (iNKT), or invariant NKT cells engineered for cell therapy for mammals. When the cells are NK cells, the NK cell therapy may be of any type, and the NK cells may be of any type. In some embodiments, the NK cells of the present disclosure are engineered to express one or more suicide genes and / or one or more cytokines, and do not express an antigen targeting CAR. In certain embodiments, the NK cells of the present disclosure are not engineered to express any exogenous genes.

[0195] In certain embodiments, the present disclosure contemplates, in part, antigen CAR expressing cells, antigen targeted CAR constructs, antigen targeted CAR nucleic acid molecules, and antigen targeted CAR vectors that can be administered alone or in any combination using standard vectors and / or gene delivery systems, and in at least some aspects, with a pharma- ceutically acceptable carrier or excipient. In certain embodiments, after administration, the nucleic acid molecule or vector can be stably integrated into the subject's genome.

[0196] In certain embodiments, viral vectors can be used that are specific to certain cells or tissues and persist in NK cells.Suitable pharmaceutical carriers and excipients are well known in the art.The composition prepared according to the present disclosure can be used for the prevention or treatment or delay of the above-mentioned specified diseases.

[0197] Furthermore, the present disclosure relates to a method for the prevention, treatment, or amelioration of a neoplastic disease, comprising administering to a subject in need thereof an effective amount of cells expressing an antigen-targeting CAR, nucleic acid sequence, vector as contemplated herein and / or produced by a process as contemplated herein.

[0198] The possible indications for administration of the exemplary antigen-targeting cell composition are cancerous diseases, including, for example, neoplastic diseases, including, for example, B-cell malignancies, multiple myeloma, leukemia, breast cancer, glioblastoma, renal cancer, pancreatic cancer, or lung cancer. Administration of the disclosed composition is useful for all stages (I, II, III, or IV) and types of cancer, including, for example, minimal residual disease, early cancer, advanced cancer, and / or metastatic and / or refractory cancer.

[0199] The present disclosure further includes co-administration protocols with other compounds, such as bispecific antibody constructs, targeted toxins, or other compounds that act through immune cells.The clinical regimen for co-administration of the compounds of the present invention may include co-administration at the same time, before, or after administration of other components.Specific combination therapies include chemotherapy, radiation, surgery, hormone therapy, or other types of immunotherapy.

[0200] Embodiments relate to kits comprising a construct as defined herein, a nucleic acid sequence as defined herein, a vector as defined herein, and / or a host cell (such as an immune cell) as defined herein. It is also contemplated that the kits of the present disclosure comprise a pharmaceutical composition as described herein above, alone or in combination with additional agents to be administered to an individual in need of medical treatment or intervention. A. Pharmaceutical Compositions

[0201] Also provided herein are pharmaceutical compositions and formulations comprising NK cells (including transduced NK cells, preactivated and expanded NK cells, loaded NK cells) and a pharma- ceutically acceptable carrier. The transduced cells may be contained in a medium suitable for transfer into an individual and / or a medium suitable for storage, such as cryopreservation, including prior to transfer into an individual.

[0202] The pharmaceutical compositions and formulations described herein comprise an active ingredient (such as cells) having a desired purity in the form of a lyophilized formulation or an aqueous solution, optionally in one or more pharma- ceutical acceptable carriers (see Remington's Pharmaceutical Sciences 22). ndPharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations used, and include, but are not limited to, buffers such as phosphate, citric acid, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (about 10 residues) soluble ... (less than 1000) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmacologic carriers herein further include interstitial drug dispersion agents, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), such as human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one embodiment, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases. B. Combination Therapy

[0203] In certain embodiments, the compositions and methods of the present embodiment include immune cell populations (including NK cell populations) combined with at least one additional therapy.Additional therapy can be radiation therapy, surgery (e.g., lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, virus therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, hormone therapy, oncolytic virus, or combinations of the above.Additional therapy can be in the form of adjuvant therapy or neoadjuvant therapy.

[0204] In some embodiments, the additional therapy is administration of a small molecule enzyme inhibitor or an anti-metastatic agent. In some embodiments, the additional therapy is administration of a side effect limiting agent (e.g., an agent intended to reduce the occurrence and / or severity of side effects of treatment, such as an antiemetic agent, etc.). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy is a therapy targeting the PBK / AKT / mTOR pathway, an HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and / or a chemopreventive agent. The additional therapy can be one or more chemotherapeutic agents known in the art.

[0205] In certain embodiments, in addition to the inventive cell therapy of the present disclosure, the individual may have been, is being, and / or will be receiving certain additional therapies for cancer, including one or more of surgery, radiation, immunotherapy (other than the cell therapy of the present disclosure), hormone therapy, gene therapy, chemotherapy, and the like.

[0206] Immune cell therapy may be administered before, during, after, or in various combinations with additional cancer therapy. Administration may be at intervals ranging from simultaneous to minutes to days to weeks. In embodiments where immune cell therapy is administered to a patient separately from additional therapeutic agents, one skilled in the art would generally ensure that a significant period does not lapse between each delivery time so that the two compounds can still exert their beneficially combined effect on the patient. In such cases, it is believed that the antibody therapy and the anti-cancer therapy may be administered to a patient within about 12 to 24 or 72 hours of each other, more specifically within about 6 to 12 hours of each other. In some situations, it may be desirable to extend the treatment period significantly when several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) pass between the respective administrations.

[0207] Various combinations can be used. In the following examples, the immune cell therapy is "A" and the anti-cancer therapy is "B". A / B / AB / A / BB / B / AA / A / BA / B / BB / A / AA / B / B / BB / A / B / B B / B / B / AB / B / A / BA / A / B / BA / B / A / BA / B / B / AB / B / A / A B / A / B / AB / A / A / BA / A / A / BB / A / A / AA / B / A / AA / A / B / A

[0208] Administration of any compound or cell therapy of the present embodiments to a patient will follow standard protocols for administration of such compounds, taking into account the toxicity, if any, of the agent. Thus, in some embodiments, there is a step of monitoring for toxicity resulting from combination therapy. 1.Chemotherapy

[0209] A wide variety of chemotherapeutic agents may be used in accordance with this embodiment. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to mean a compound or composition administered in the treatment of cancer. The agents or drugs are classified according to their mode of activity within the cell, for example, whether and at what stage they affect the cell cycle. Alternatively, agents may be characterized based on their ability to directly crosslink DNA, intercalate into DNA, or induce chromosomal and mitotic abnormalities by affecting nucleic acid synthesis.

[0210] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylameramines (including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylmelamine); acetogenins (particularly bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 2). liptophysin 8; dolastatins; duocarmycins (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictin; spongistatins; nitrogen mustards, such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembitine, phenesterine, prednimustine, trophosphamide, and uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicins, especially calicheamicin gamma II (calicheamicin gammaII and calicheamicin omegaII); dynemicins (including dynemicin A); bisphosphonates, such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, autarmicin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxo rubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins, such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycin, peplomycin, potfilomycin, puromycin, queramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as denopterin, promycin, and folic acid analogs. teropterin, and trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; anti-adrenals such as mitotane and trilo Stans; folic acid supplements, such as floric acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformitin; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol;Nitraerine; pentostatin; phenameth; pirarubicin; rosoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; schizophyllan; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, veraculin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; Arabinosides ("Ara-C"); cyclophosphamide; taxoids, such as paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g. CPT-11); topoisomerase inhibitors RFS 2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, farnesyl-protein transferase inhibitors, transplatinum, as well as pharma- ceutically acceptable salts, acids, or derivatives of any of the foregoing. 2. Radiation therapy

[0211] Other agents that cause DNA damage and have been widely used include gamma radiation, X-rays, and / or what is commonly known as the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging agents are also contemplated, such as microwaves, proton beam irradiation (US Pat. Nos. 5,760,395 and 4,870,287), and UV irradiation. All of these agents most likely affect a wide range of damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. X-ray dose ranges from daily doses of 50-200 roentgens for prolonged periods (3-4 weeks) to single doses of 2000-6000 roentgens. Dose ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and uptake by neoplastic cells. 3. Immunotherapy

[0212] Those skilled in the art will understand that immunotherapy can be used in conjunction or in combination with the methods of the embodiments. In the context of cancer treatment, immunotherapy generally relies on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab (RITUXAN®) is such an example. The immune effector can be, for example, an antibody specific to some marker on the surface of tumor cells. The antibody can function alone as an effector of therapy or can recruit other cells to actually affect cell death. The antibody can also be conjugated to a drug or toxin (chemotherapeutic agent, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) to function as a targeting agent. Alternatively, the effector can be a lymphocyte carrying a surface molecule that directly or indirectly interacts with the tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[0213] Antibody-drug conjugates have emerged as a revolutionary approach to the development of cancer therapeutics. Cancer is one of the leading causes of death in the world. Antibody-drug conjugates (ADCs) contain a monoclonal antibody (MAb) covalently linked to a cell-killing drug. This approach combines the high specificity of MAbs for antigen targets with highly potent cytotoxic drugs, resulting in "armed" MAbs that deliver the payload (drug) to tumor cells enriched with antigen levels. Targeted delivery of the drug also minimizes exposure in normal tissues, resulting in reduced toxicity and improved therapeutic index. The FDA's approval of two ADC drugs, ADCETRIS® (brentuximab vedotin) in 2011 and KADCYLA® (trastuzumab emtansine or T-DM1) in 2013, has validated the approach. Currently, there are more than 30 ADC drug candidates in various stages of clinical trials for cancer treatment (Leal et al., 2014). As antibody engineering and linker-payload optimization become more and more mature, the discovery and development of new ADCs increasingly depends on the identification and validation of new targets suitable for this approach and the generation of targeted MAbs. Two criteria for ADC targets are up-regulation / high levels of expression in tumor cells and robust internalization.

[0214] In one aspect of immunotherapy, the tumor cells must have some marker suitable for targeting, i.e., not present on the majority of other cells. There are many tumor markers, any of which may be suitable for targeting in the context of this embodiment. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B, and p155. An alternative aspect of immunotherapy is to combine anti-cancer effects with immune stimulatory effects. There are also immune stimulatory molecules, including cytokines such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines such as MIP-1, MCP-1, IL-8, and growth factors such as FLT3 ligand.

[0215] Examples of immunotherapies currently under investigation or in use include immune adjuvants, such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Pat. Nos. 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998); cytokine therapies, such as interferon alpha, beta, and gamma, IL-1, GM-CSF, and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998); gene therapies, such as TNF, IL-1, IL-2, and p53 (Qin et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Patent Nos. 5,830,880 and 5,846,945; and monoclonal antibodies, such as anti-CD20, anti-ganglioside GM2, and anti-p185 (Hollander, 2012; Hanibuchi et al., 1998; U.S. Patent No. 5,824,311). It is contemplated that one or more anti-cancer therapies may be used in conjunction with the antibody therapies described herein.

[0216] In some embodiments, the immunotherapy can be an immune checkpoint inhibitor. Immune checkpoints make signals (e.g., costimulatory molecules) stronger or weaker. Inhibitory immune checkpoints that can be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAG3), programmed death 1 (PD-1), T cell immunoglobulin domain and mucin domain 3 (TIM-3), and V domain Ig suppressor of T cell activation (VISTA). In particular, immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4.

[0217] The immune checkpoint inhibitor may be a drug, such as a small molecule, a recombinant form of a ligand or receptor, or an antibody, such as a human antibody (e.g., WO 2015 / 016718; Pardoll, Nat Rev Cancer, 12(4):252-64, 2012; both of which are incorporated herein by reference). Known inhibitors of immune checkpoint proteins or analogs thereof may be used, particularly chimeric, humanized, or human forms of antibodies. Alternative and / or equivalent names known to those skilled in the art may be used for the specific antibodies referred to in this disclosure. Such alternative and / or equivalent names are interchangeable in the context of this disclosure. For example, it is known that lambrolizumab is also known by the alternative and equivalent names MK-3475 and pembrolizumab.

[0218] In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In certain embodiments, the PD-1 ligand binding partner is PDL1 and / or PDL2. In another embodiment, the PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partner. In certain embodiments, the PDL1 binding partner is PD-1 and / or B7-1. In another embodiment, the PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partner. In certain embodiments, the PDL2 binding partner is PD-1. The antagonist can be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Exemplary antibodies are described in U.S. Pat. Nos. 8,735,553, 8,354,509, and 8,008,449, all of which are incorporated herein by reference. Other PD-1 axis antagonists for use in the methods provided herein are known in the art, such as those described in U.S. Patent Application Publication No. 2014 / 0294898, U.S. Patent Application Publication No. 2014 / 022021, and U.S. Patent Application Publication No. 2011 / 0008369, all of which are incorporated herein by reference.

[0219] In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 binding antagonist is AMP-224. Nivolumab is also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, and is an anti-PD-1 antibody described in WO 2006 / 121168. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in WO 2009 / 114335. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in WO 2009 / 101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in WO 2010 / 027827 and WO 2011 / 066342.

[0220] Another immune checkpoint that can be targeted in the methods provided herein is cytotoxic T lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an "off" switch when bound to CD80 or CD86 on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of helper T cells and transmits inhibitory signals to T cells. CTLA4 is similar to the T cell costimulatory protein CD28, and both molecules bind to CD80 and CD86, also called B7-1 and B7-2, respectively, on antigen-presenting cells. CTLA4 transmits inhibitory signals to T cells, while CD28 transmits stimulatory signals. Intracellular CTLA4 is also found in regulatory T cells and may be important for their function. T cell activation via the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for the B7 molecule.

[0221] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

[0222] Anti-human CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, any art-recognized anti-CTLA-4 antibody can be used. For example, anti-CTLA-4 antibodies disclosed in U.S. Pat. No. 8,119,129, WO 01 / 14424, WO 98 / 42752, WO 00 / 37504 (CP675,206, also known as tremelimumab; formerly ticilimumab), U.S. Pat. No. 6,207,156; Hurwit Zeta l. (1998) Proc Natl Acad Sci USA 95(17):10067-10071; Camacho et al. (2004) J Clin Oncology 22(145):Abstract No. 2505 (antibody CP-675206); and Mokyr et al. (1998) Cancer Res 58:5301-5304 may be used in the methods disclosed herein. The teachings of each of the above publications are incorporated herein by reference.Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 can also be used.For example, humanized CTLA-4 antibodies are described in WO 2001 / 014424, WO 2000 / 037504, and U.S. Patent No. 8,017,114 (all of which are incorporated herein by reference).

[0223] An exemplary anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or antigen-binding fragments and variants thereof (see, e.g., WO 01 / 14424). In other embodiments, the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab, and the CDR1, CDR2, and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for binding to and / or binds to the same epitope on CTLA-4 as the above-mentioned antibodies. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the above-mentioned antibodies (e.g., at least about 90%, 95%, or 99% variable region identity with ipilimumab).

[0224] Other molecules for modulating CTLA-4 include CTLA-4 ligands and receptors such as those described in U.S. Pat. No. 5,844,905, U.S. Pat. No. 5,885,796, and WO 1995 / 001994 and WO 1998 / 042752 (all of which are incorporated by reference herein), and immunoadhesins such as those described in U.S. Pat. No. 8,329,867 (which is incorporated by reference herein). 4.Surgery

[0225] Approximately 60% of people with cancer will undergo some kind of surgery, including preventive, diagnostic, or staging, curative surgery, and palliative surgery. Curative surgery includes resection, in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed, and may be used in combination with other therapies, such as the treatment of the present embodiment, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapy. Tumor resection refers to the physical removal of at least a part of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microsurgery (Mohs surgery).

[0226] When part or all of the cancerous cells, tissue, or tumor is removed, a cavity may be formed in the body. Treatment may be achieved by perfusion, direct injection, or local application of the area with additional anticancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may also vary in dosage. 5. Other drugs

[0227] It is believed that other agents can be used in combination with certain aspects of the present embodiment to improve the therapeutic efficacy of the treatment. These additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of hyperproliferative cells to apoptosis inducers, or other biological agents. Increasing intercellular signaling by increasing the number of GAP junctions will increase the anti-hyperproliferative effect on adjacent hyperproliferative cell populations. In other embodiments, cytostatic or differentiation agents can be used in combination with certain aspects of the present embodiment to improve the anti-hyperproliferative effect of the treatment. Inhibitors of cell adhesion are believed to improve the efficacy of the present embodiment. Examples of cell adhesion inhibitors include focal adhesion kinase (FAK) inhibitors and lovastatin. In addition, it is believed that other agents that increase the sensitivity of hyperproliferative cells to apoptosis, such as antibody c225, can be used in combination with certain aspects of the present embodiment to improve the efficacy of the treatment. X. Protein

[0228] As used herein, a "protein" or "polypeptide" refers to a molecule that comprises at least three amino acid residues. As used herein, the term "wild type" refers to the endogenous version of a molecule that occurs naturally in an organism. In some embodiments, a wild type version of a protein or polypeptide is used. However, in many embodiments of the present disclosure, modified proteins or polypeptides are used to generate an immune response. The above terms may be used interchangeably. "Modified protein" or "modified polypeptide" or "variant" refers to a protein or polypeptide whose chemical structure, particularly its amino acid sequence, has been altered relative to the wild type protein or polypeptide. In some embodiments, a modified / variant protein or polypeptide has at least one modified activity or function (recognizing that a protein or polypeptide may have multiple activities or functions). It is specifically contemplated that a modified / variant protein or polypeptide may be altered with respect to one activity or function, but may retain the wild type activity or function in other respects, such as immunogenicity.

[0229] When a protein is specifically mentioned herein, it generally refers to a natural (wild type) or recombinant (modified) protein, or a protein that, optionally, has any signal sequence removed. A protein may be directly isolated from a natural organism, produced by recombinant DNA / exogenous expression methods, or produced by solid phase peptide synthesis (SPPS) or other in vitro methods. In certain embodiments, there are isolated nucleic acid segments and recombinant vectors that incorporate a nucleic acid sequence that encodes a polypeptide (e.g., an antibody or fragment thereof). The term "recombinant" may be used with a polypeptide, or with the name of a particular polypeptide, generally referring to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or is a product of replication of such a molecule.

[0230] In certain embodiments, the size of a protein or polypeptide (wild-type or modified) may include, but is not limited to, the following: 5, 6, 7, 8, 9, 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, 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, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500, 1750, 2000, 2250, 2500 amino acid residues or more, and any range derivable therein, or derivatives of the corresponding amino acid sequences described or referenced herein. It is contemplated that polypeptides may be mutated by truncation, shortened from their wild-type counterparts, and modified by fusing or attaching heterologous protein or polypeptide sequences having specific functions (e.g., for targeting or localization, to enhance immunogenicity, for purification purposes, etc.) As used herein, the term "domain" refers to any discrete functional or structural unit of a protein or polypeptide, generally a sequence of amino acids having a structure or function recognizable to one of skill in the art.

[0231] A polypeptide, protein, or a polynucleotide encoding such a polypeptide or protein of the present disclosure may include: or 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, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (or an inducible range thereof) or more mutated amino acid or nucleic acid substitutions relative to SEQ ID NOs: 1-31; or is at least 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%, or 100% (or a range derivable therein) similar, identical, or homologous to Up to 3, 4, 5, 6, 7, 8, 9, 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, 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, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222,223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 400, 500, 550, 1000 or more consecutive amino acids or nucleic acids or derived ranges thereof.

[0232] In some embodiments, the protein, polypeptide, or polynucleotide may include: 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, 27, 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, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218,219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418,419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618,619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818,819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000 (or a derivable range) consecutive amino acids or nucleotides.

[0233] In some embodiments, a polypeptide, protein, or polynucleotide may include at least, at most, or exactly the following: Any one of SEQ ID NOs: 1 to 31 in the sequence listing 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, 27, 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, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222,223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422,423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622,623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822,823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000 (or a derivable range) contiguous amino acids that are at least, at most, or exactly 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%, or 100% (or derivable range) similar, identical, or homologous to any one of SEQ ID NOs:1-31.

[0234] In one embodiment, a nucleic acid molecule or polypeptide starting at the following position in any one of SEQ ID NOs: 1 to 31: 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, 27, 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, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222,223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422,423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622,623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822,823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000, and at least, at most, or exactly 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, 27, 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, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214,215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414,415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614,615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814,815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000 (or a derivable range) consecutive amino acids or nucleotides.

[0235] Nucleotide and protein, polypeptide and peptide sequences of various genes have been disclosed previously and can be found in known computerized databases.Two commonly used databases are the Genbank and GenPept databases of the National Center for Biotechnology Information (ncbi.nlm.nih.gov / on the World Wide Web) and The Universal Protein Resource (UniProt; uniprot.org on the World Wide Web).The coding regions of these genes can be amplified and / or expressed using the techniques disclosed herein or known to those skilled in the art.

[0236] It is contemplated that in the compositions of the present disclosure, there is about 0.001 mg to about 10 mg of total polypeptide, peptide, and / or protein per ml. The concentration of protein in the composition is about, at least about, or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg / ml or more (or any range derivable therein). XI. Kits of the Disclosure

[0237] Any of the compositions described herein may be included in the kit. In a non-limiting example, cells, reagents for generating cells, vectors, and reagents for generating vectors and / or components thereof may be included in the kit. In certain embodiments, NK cells may be included in the kit, which may or may not express antigen targeting receptors, optional cytokines, or optional suicide genes. Such kits may or may not have one or more reagents for manipulating cells. Such reagents include, for example, small molecules, proteins, nucleic acids, antibodies, buffers, primers, nucleotides, salts, and / or combinations thereof. Nucleotides encoding one or more antigen targeting CARs, suicide gene products, and / or cytokines may be included in the kit. Proteins such as cytokines or antibodies, including monoclonal antibodies, may be included in the kit. Nucleotides encoding components of modified (engineered) CAR receptors may be included in the kit, including reagents for generating the components.

[0238] In certain embodiments, the kit also includes the NK cell therapy of the present disclosure and another cancer treatment. In some cases, the kit also includes a second cancer treatment, such as, for example, chemotherapy, hormone therapy, and / or immunotherapy, in addition to the cell therapy embodiment. The kit can be tailored to the specific cancer of the individual and can include the respective second cancer treatment for the individual.

[0239] The kit may include a properly aliquoted composition of the present disclosure. The components of the kit may be packaged in either aqueous media or lyophilized form. The container means of the kit generally includes at least one vial, test tube, flask, bottle, syringe, or other container means into which the components may be placed and preferably properly aliquoted. If there are multiple components in the kit, the kit may also generally contain a second, third, or other additional container into which the additional components may be placed separately. However, various combinations of components may be included in a vial. The kit of the present invention also typically includes a means for containing the composition and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are held. EXAMPLES

[0240] The following examples are included to illustrate specific embodiments of the present invention. Those skilled in the art should understand that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to work well in the practice of the present invention, and therefore can be considered to constitute specific modes for its practice. However, those skilled in the art should understand in light of this disclosure that many changes can be made in the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the present invention.

[0241] Example 1 - Validation of Obinutuzumab Binding to NK Cells NK cells were derived from umbilical cord blood and expanded in complete serum-free stem cell growth medium (SCGM) with irradiated (100 Gy) UAPC feeder cells (2:1 feeder:NK ratio) and recombinant human IL-2 (200 U / ml). NK cells were either non-transduced (NT) or transduced with CD19 chimeric antigen receptor (CD19 CAR). NK cells were loaded with obinutuzumab (10 μg) for 1 h at 37°C in SCGM medium and after washing, obinutuzumab binding was verified by flow cytometry. Alexa-Fluor 647 affinity purified F(ab') 2 NK cells were stained with a fragmented goat anti-human IgG (H+L) antibody and analyzed by flow cytometry. Figure 1 shows the results of the staining, demonstrating that obinutuzumab was able to bind to NK cells and was expressed on the surface of NK cells to the same extent as CD19 CAR.

[0242] Example 2 - Obinutuzumab-loaded NK cells showed enhanced killing of Raji cells compared to unloaded or rituximab-loaded NK cells NK cells were derived from umbilical cord blood and either expanded (NK) or preactivated with cytokines (IL-12, IL-15, and IL-18) and expanded (NK P+E). NK cells were loaded with either obinutuzumab (10 μg) or rituximab (10 μg) for 1 h in SCGM medium at 37°C, washed, and then cocultured with Raji CD19 KO cells at a 1:1 ratio (100,000 cells for each condition). As shown in Figure 2A, obinutuzumab-loaded NK cells showed enhanced killing of Raji CD19 KO cells when compared to unloaded or rituximab-loaded NK cells. NK P+E cells were loaded with obinutuzumab (10 μg) or rituximab (10 μg) for 1 h at 37°C in SCGM medium, washed, and then cocultured with Raji CD19 KO cells at a 1:1 ratio (100,000 cells for each condition). As shown in Figure 2B, NK P+E cells loaded with obinutuzumab showed enhanced killing of Raji CD19 KO cells when compared to unloaded or rituximab-loaded NK cells.

[0243] Example 3 - Obinutuzumab-loaded NK cells exhibited enhanced cytotoxicity against Raji cells compared to unloaded NK cells NK cells were derived from umbilical cord blood and transduced with CD19 CAR to generate NK CD19 CAR cells. Both non-transduced (NT) and CD19 CAR-transduced NK cells (CD19) were loaded with obinutuzumab (10 μg) for 1 hour at 37° C. in SCGM medium, washed, and then co-cultured with Raji cells at various effector-to-target ratios as described in the figures. As shown in FIG. 3, compared to unloaded NK cells, obinutuzumab-loaded NK cells showed improved cytotoxicity of Raji cells for both NT NK cells and CD19 CAR NK cells as shown by chromium release assay, indicating that obinutuzumab loading improves the cytotoxic activity of NK cells. Maximum toxicity was observed for obinutuzumab-loaded CD19 CAR NK cells.

[0244] Example 4 - Obinutuzumab-loaded CD19 CAR NK cells showed better killing of Raji cells (Raji WT) compared to non-loaded CD19 CAR NK cells NK cells were derived from umbilical cord blood and either expanded (NK) or preactivated with cytokines (IL-12, IL-15 and IL-18) and expanded (NK P+E). CD19 CAR NK cells were loaded with obinutuzumab (10 μg) for 1 h at 37°C in SCGM medium, washed and then co-cultured with Raji WT cells at a 1:1 ratio (100,000 cells for each condition). As shown in Figure 4B, CD19 CAR NK cells loaded with obinutuzumab showed better killing of Raji WT cells when compared to non-loaded CD19 CAR NK cells.

[0245] CD19 CAR NK P+E cells were loaded with obinutuzumab (10 μg) for 1 hour at 37° C. in SCGM medium, washed, and then co-cultured with Raji WT cells at a 1:1 ratio (100,000 cells for each condition). As shown in Figure 4B, obinutuzumab-loaded CD19 CAR NK P+E cells showed better killing of Raji CD19 KO cells when compared to unloaded CD19 CAR NK cells.

[0246] Example 5 - Obinutuzumab-loaded CD19 CAR NK cells were better at controlling Raji cell tumor growth in mice compared to unloaded CD19 CAR NK cells 20,000 luciferase-labeled Raji cells (Raji-FFluc) with high expression of CD19 were transplanted into NSG mice. NK cells were derived from umbilical cord blood, preactivated with cytokines (IL-12, IL-15 and IL-18) and expanded (NK P+E). These NK P+E cells were transduced with CD19 CAR to generate NK P+E CD19 CAR cells, loaded with obinutuzumab (10 μg) for 1 h at 37°C in SCGM medium, washed twice and then injected into mice. As shown in Figure 5A and 5B, obinutuzumab-loaded NK P+E CD19 CAR cells were better at controlling Raji cell tumors in mice compared to unloaded NK P+E CD19 CAR cells. As shown in Figure 5C, obinutuzumab-loaded NK P+E CD19 CAR cells improved survival of NSG mice injected with Raji cells compared with unloaded NK P+E CD19 CAR cells.

[0247] Example 6 - Obinutuzumab-loaded CD19 CAR NK cells showed enhanced killing of Raji cells with antigen loss (Raji CD19 KO) NK cells were derived from umbilical cord blood and were either expanded (NK) or preactivated with cytokines and expanded (NK P+E). CD19 CAR NK cells were loaded with obinutuzumab (10 μg) for 1 hour at 37° C. in SCGM medium, washed, and then cocultured with Raji CD19 KO cells at a 1:1 ratio (100,000 cells for each condition). As shown in Figure 6A and Figure 7A, CD19 CAR NK cells loaded with obinutuzumab showed enhanced killing of Raji CD19 KO cells when compared to unloaded CD19 CAR NK cells. CD19 CAR NK P+E cells were loaded with obinutuzumab (10 μg) for 1 hour at 37° C. in SCGM medium, washed, and then cocultured with Raji CD19 KO cells at a 1:1 ratio (100,000 cells for each condition). As shown in Figure 6B and Figure 7B, CD19 CAR NK P+E cells loaded with obinutuzumab showed enhanced killing of Raji CD19 KO cells when compared to unloaded CD19 CAR NK cells.

[0248] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of this disclosure. Although the compositions and methods of the present invention have been described with reference to certain embodiments, it will be apparent to those skilled in the art that variations can be applied to the methods described herein and in the steps of the methods, or in the sequence of steps of the methods, without departing from the concept, spirit and scope of the present invention. More specifically, it will be apparent that certain agents that are chemically and physiologically related may be substituted for the agents described herein while still achieving the same or similar results. All such similar substitutes and modifications that are apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the present invention as defined by the appended claims.

Claims

1. 1. A modified immune cell comprising: (a) a polynucleotide encoding (i) one or more chimeric antigen receptors (CARs) and / or (ii) one or more T cell receptors (TCRs); (b) an anti-CD20 antibody or antigen-binding fragment thereof, optionally bound to the surface of said immune cells; Modified immune cells, including:

2. The modified immune cell of claim 1, wherein the polynucleotide encodes the CAR, and the CAR is a CD19-specific CAR, a CD70-specific CAR, or a CD5-specific CAR.

3. The modified immune cell of claim 1 or 2, wherein the polynucleotide further encodes an additional polypeptide of interest.

4. 4. The modified immune cell of claim 3, wherein the additional polypeptide of interest is a therapeutic protein or a protein that enhances cell activity, expansion and / or persistence, or the additional polypeptide of interest is a suicide gene, a cytokine, or a human or viral protein that enhances growth, expansion and / or metabolic fitness.

5. 5. The modified immune cell of claim 4, wherein the cytokine is IL-15, IL-2, IL-12, IL-18, IL-21, IL-23, or IL-7.

6. 6. The modified immune cell of any one of claims 1 to 5, wherein the immune cell is a natural killer (NK) cell, a T cell, a gamma delta T cell, an alpha beta T cell, an invariant NKT (iNKT) cell, a B cell, a macrophage, a mesenchymal stromal cell, a dendritic cell, or a mixture thereof.

7. 10. A composition for treating an individual having CD20-positive cancer cells, comprising an effective amount of a population of immune cells comprising the modified immune cells of any one of claims 1 to 6.

8. 8. The composition of claim 7, wherein the individual has B-cell non-Hodgkin's lymphoma, chronic lymphocytic leukemia, or acute lymphoblastic leukemia.

9. A population of cells comprising the modified immune cells of any one of claims 1 to 8.

10. A population of NK cells bound to an anti-CD20 antibody or antigen-binding fragment thereof, the population of NK cells having been previously preactivated in a preactivation culture containing effective concentrations of one or more of IL-12, IL-15, and IL-18.

11. The population of NK cells according to claim 10, which has been previously pre-activated in a pre-activation culture comprising effective concentrations of IL-12, IL-15 and IL-18.

12. The population of NK cells according to claim 10 or 11, which has been pre-activated by multiple treatments with one or more of IL-12, IL-15 and IL-18.

13. A composition for treating an individual with a CD20-positive cancer, comprising: (a) a population of NK cells that have been pre-activated in a pre-activation culture containing effective concentrations of one or more of IL-12, IL-15, and IL-18; and (b) an effective amount of an anti-CD20 antibody or antigen-binding fragment thereof.

14. 14. The composition of claim 13, wherein the population of NK cells has been previously preactivated in a preactivation culture comprising effective concentrations of IL-12, IL-15 and IL-18.

15. The composition of claim 13 or 14, wherein the population of NK cells and the anti-CD20 antibody or antigen-binding fragment thereof are administered in the same composition.

16. The composition of claim 15, wherein the anti-CD20 antibody or antigen-binding fragment thereof is bound to the surface of the NK cell.

17. The composition of claim 13 or 14, wherein the population of NK cells and the anti-CD20 antibody or antigen-binding fragment thereof are administered in different compositions.

18. 1. A composition for treating an individual with a CD20-positive cancer, comprising: (a) an engineered immune cell comprising a polynucleotide encoding (i) one or more CARs and / or (ii) one or more TCRs; and (b) a composition comprising an effective amount of an anti-CD20 antibody or antigen-binding fragment thereof.

19. 1. A method for preparing a population of modified NK cells, comprising: (a) culturing a population of NK cells with one or more of IL-12, IL-15 and IL-18; (b) incubating the population of NK cells with an anti-CD20 antibody or antigen-binding fragment thereof; A method comprising:

20. 20. The method of claim 19, comprising: (a) culturing the population of NK cells with two or more of IL-12, IL-15, and IL-18.

21. 21. The method of claim 19 or 20, wherein (b) is carried out for at least 30 minutes, or at least 60 minutes.

22. 1. A modified immune cell comprising: (a) a polynucleotide encoding a CAR; and (b) obinutuzumab bound to the surface of the immune cell; Modified immune cells, including:

23. A population of NK cells bound to obinutuzumab, the population of NK cells having been previously preactivated in a preactivation culture comprising effective concentrations of one or more of IL-12, IL-15 and IL-18.

24. 24. The population of NK cells according to claim 23, which has been previously pre-activated in a pre-activation culture comprising an effective concentration of one or more of IL-12, IL-15 and IL-18.

25. A composition for treating an individual with CD20-positive cancer, comprising: (a) a population of NK cells that have been pre-activated in a pre-activation culture containing effective concentrations of one or more of IL-12, IL-15, and IL-18; and (b) an effective amount of obinutuzumab.

26. 1. A composition for treating an individual having a CD20-positive cancer, comprising: (a) modified immune cells comprising a polynucleotide encoding (i) a CAR or (ii) a TCR; and (b) an effective amount of obinutuzumab.

27. A method for preparing a population of NK cells, comprising: (a) culturing a population of NK cells with one or more of IL-12, IL-15 and IL-18; and (b) incubating the population of NK cells with obinutuzumab; A method comprising:

Images