Primary NK car constructs and methods

Abstract

To provide technologies and methods that are able to modify NK cells for specific targeting of cancer cells.SOLUTION: Disclosed herein are recombinant nucleic acids, comprising a 5' untranslated (5'-UTR) sequence portion having a specific sequence, a signal peptide sequence portion, a single chain antibody fragment sequence portion, a hinge region sequence portion, a transmembrane domain sequence portion, and one or more intracellular domain sequence portions. Also disclosed herein are modified natural killer (NK) cells comprising the recombinant nucleic acid described above. Further disclosed herein are methods of treating a tumor in a subject by administering the modified NK cells.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] Sequence Listing The contents of the ASCII text file of the Sequence Listing entitled 104077.00016Pro_ST25, which is 42KB in size, was created on November 6, 2019, and was submitted electronically via EFS-Web herewith, are incorporated by reference in their entirety.

[0002] The field of the invention is recombinant nucleic acids and cells containing same, particularly as it relates to the treatment of cancer. [Background technology]

[0003] The background description includes information that may be useful in understanding the present invention. None of the information provided herein is admitted to be prior art or relevant to the invention(s) claimed in this application, nor is any publication expressly or impliedly mentioned admitted to be prior art.

[0004] Natural killer (NK) cell-based cancer immunotherapy has made significant progress in recent years. NK cells are cytotoxic lymphocytes that are an important component of the innate immune system. In most cases, NK cells, which account for approximately 4-10% of circulating lymphocytes, bind to and kill targeted cells (including virus-infected cells and many malignant cells). Killing by NK cells is nonspecific to a particular antigen and can occur without prior immune sensitization. Targeted cell killing is typically mediated by cytolytic proteins, including perforin, granzymes, and granulysin.

[0005] NK cells have been used as therapeutic agents. To this end, NK cells are isolated from the peripheral blood lymphocyte fraction of whole blood, expanded in cell culture to obtain sufficient numbers of cells, and then reinfused into subjects.NK cells have shown moderate efficacy in both in vitro and in vivo treatment, at least in some cases.However, cancers use various strategies to slow, alter, or stop antitumor immunity to prevent tumor growth from being controlled.

[0006] The antitumor response of NK cells also faces many limitations. First, the low ability of NK cells to reach tumor tissues limits their application as a treatment for solid tumors. This is a common problem of cellular immunotherapy approaches. Second, changes in NK cell activating receptors and their ligands in tumors may result in reduced therapeutic response and tumor progression. For example, high levels of NKG2D (natural killer group 2, member D) ligands are detected early in colon cancer, but their expression decreases as the disease progresses. Third, the tumor microenvironment (TME) remains a major obstacle to the efficacy of matched transplanted NK cells. For example, tumor-infiltrating immune cells such as dendritic cells (DCs), suppressive or tolerogenic macrophages and regulatory T (Treg) cells as well as cancer-associated fibroblasts embedded in the extracellular matrix may interfere with NK cell activation by secreting immunosuppressive cytokines or by interfering with receptor expression. Summary of the Invention

[0007] There remains a need in the art for techniques and methods to overcome the above challenges, which can modify NK cells for specific targeting of cancer cells.

[0008] The subject of the present invention relates to a recombinant nucleic acid comprising a T7 promoter sequence portion, a 5' untranslated (5'-UTR) sequence portion, a signal peptide sequence portion, a single-chain antibody fragment sequence portion, a hinge region sequence portion, a transmembrane domain sequence portion, and one or more intracellular domain sequence portions. The recombinant nucleic acid may further comprise a sequence encoding CD64. Furthermore, the 5'-UTR sequence portion may further comprise a Kozak sequence.

[0009] Preferably, the single-chain antibody fragment sequence portion comprises a sequence encoding a single-chain variable fragment adapted to bind to the PDL1 antigen or other tumor antigen. In certain embodiments, the recombinant nucleic acid may further comprise a sequence portion encoding CD16a and / or ER-IL2.

[0010] The hinge sequence portion provides a range of motion for the single chain antibody fragment sequence portion, while the transmembrane domain sequence portion allows insertion of the recombinant nucleic acid into the membrane.

[0011] It is envisaged that the intracellular domain sequence portion of the recombinant nucleic acid disclosed herein comprises a costimulatory or signal transduction sequence portion. In one embodiment, the intracellular domain sequence portion comprises CD28 and / or CD3ζ. In another embodiment, the intracellular domain sequence portion comprises CD28 and / or FcεRIγ. The recombinant nucleic acid according to any one of the preceding claims, further comprising the intracellular domain sequence portion capable of providing enhanced cytotoxic activity against tumor cells.

[0012] Preferably, the recombinant nucleic acid of the present disclosure comprises a 3'-untranslated region (3'-UTR) and a poly-A sequence portion. The 3'-UTR sequence portion provides RNA stability and translation initiation. The poly-A sequence portion preferably comprises at least 150 adenine nucleotides. The poly-A sequence portion provides RNA stability and translation initiation.

[0013] The recombinant nucleic acid vector of the present disclosure is preferably optimized to target tumor antigens. In some embodiments, the recombinant nucleic acid has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1. In other embodiments, the recombinant nucleic acid has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 2. In yet other embodiments, the recombinant nucleic acid has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 3. In yet other embodiments, the recombinant nucleic acid has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:4.

[0014] In another aspect of the present subject matter, the inventors disclose a modified NK cell comprising one or more nucleic acids encoding a 5' untranslated (5'-UTR) sequence portion, a signal peptide sequence portion, a single chain antibody fragment sequence portion, a hinge region sequence portion, a transmembrane domain sequence portion, and one or more intracellular domain sequence portions, the nucleic acid sequences being operably linked to each other as a single polynucleotide, the modified NK cell is envisioned to specifically target tumor cells.

[0015] In another aspect, the inventors disclose a method of generating modified NK cells or CAR-NK cells, comprising transfecting primary NK cells with a recombinant nucleic acid as disclosed above. Also disclosed is a composition comprising the modified NK cells and a pharma- ceutically acceptable excipient. Furthermore, the modified NK cells can be provided in a kit, e.g., the kit can include the NK cells disclosed herein and instructions for use.

[0016] In yet another aspect, a method of treating cancer or tumor in a subject is disclosed, comprising administering to the subject a therapeutically effective amount of modified NK cells or a composition comprising modified NK cells, wherein the administration treats the cancer or reduces the size of the tumor in the subject. A method of reducing cancer metastasis in a patient is also contemplated, wherein a therapeutically effective amount of modified NK cells or a composition comprising modified NK cells is administered to a subject with cancer metastasis. Preferably, the method is administered in a dose of 1 ml or more. 2 1x10 3 ~1×10 10 The NK cells are administered to the subject. Administration may be parenterally, intravenously, peritumorally, or by infusion. The method may also include further administration of an additional therapeutic agent to the subject.

[0017] In another embodiment, the inventors disclose a method of treating cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of any one of the genetically modified NK cells disclosed herein, thereby treating the cancer. The method may further comprise administering at least one additional therapeutic agent selected from the group consisting of viral cancer vaccines, bacterial cancer vaccines, yeast cancer vaccines, N-803, antibodies, stem cell transplants, and tumor-targeted cytokines. Cancers include, but are not limited to, leukemia, acute lymphocytic leukemia, acute myeloid leukemia, chronic leukemia, chronic myeloid (granular) leukemia, chronic lymphocytic leukemia, polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's disease, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, sarcomas and carcinomas, such as, but not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelioma, lymphangiosarcoma, lymphangioendothelioma, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma , colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, solid tumors including glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0018] Various objects, features, aspects and advantages of the present subject matter will become more apparent from the following detailed description of preferred embodiments, taken in conjunction with the accompanying drawings in which like numerals represent like elements. [Brief description of the drawings]

[0019] [Figure 1] FIG. 1 shows one embodiment of the generation of a chimeric antigen targeted to the PDL1 protein: pNBS-XL53. [Diagram 2] 1 shows one embodiment of a time course of XL53 PDL1 CAR expression after electroporation. [Diagram 3] One embodiment of cytotoxic activity in fLuc-expressing U251 and MS1 target cells is shown. [Figure 4] 1 shows one embodiment of a vector map of the XL53 construct. [Diagram 5] FIG. 1 shows one embodiment of the generation of a chimeric antigen targeted to the PDL1 protein, pNBS-XL53-150A. [Figure 6] 1 shows one embodiment of a time course of XL53-150A PDL1 CAR expression after electroporation. [Figure 7] 1 shows one embodiment of the cytotoxic activity of NK cells transfected with XL53-150A. [Figure 8] 1 shows one embodiment of a vector map for the XL53-150A construct. [Figure 9] FIG. 1 shows one embodiment of the generation of a chimeric antigen targeted at the PDL1 protein, NKW29-150A. [Figure 10] 1 shows one embodiment of a time course of NKW29-150A PDL1 CAR expression in NK cells. [Figure 11] 1 shows one embodiment of a vector map for the NKW29-150A construct. [Figure 12] FIG. 1 shows one embodiment of the generation of a chimeric antigen targeted at the PDL1 protein, tricistronic XL35. [Figure 13] 1 shows one embodiment of XL53-tricistronic PDL1 CAR expression 24 hours after electroporation. [Figure 14] 1 shows one embodiment of the cytotoxic activity of CAR-infected cells in MS1 ​​fLuc target cells. [Figure 15] 1 shows one embodiment of a vector map for the XL53-tricistronic construct. Description of the Invention

[0020] All publications and patent applications in this specification are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. If the definition or use of a term in an incorporated reference contradicts or is inconsistent with the definition of that term set forth herein, the definition of that term set forth herein shall apply and the definition of that term in the reference shall not apply. Unless otherwise defined, all technical and scientific terms used herein shall have the same meaning as commonly understood by those skilled in the art to which this invention belongs.

[0021] The present inventors disclose herein various engineered NK cells as a basis for improving immunotherapy against cancer and tumors. From a different perspective, the present inventors disclose nucleic acid constructs that target tumor antigens. Preferably, in one embodiment, the tumor antigen is PDL1. In one embodiment, these nucleic acid constructs can be chimeric antigen receptor constructs for transfecting primary NK cells to generate CAR-NK cells.

[0022] In one aspect of the inventive concept, the disclosure herein includes the generation of chimeric antigen RNA molecules (CARs) against PDL1 and potentially other tumor antigen targets. It is envisioned that the RNA generated from these types of DNA constructs will be delivered to natural killer cells for specific targeting of tumor cells.

[0023] In one embodiment, a recombinant nucleic acid is disclosed herein that includes a T7 promoter sequence portion, a 5' untranslated (5'-UTR) sequence portion, a signal peptide sequence portion, a single-chain antibody fragment sequence portion, a hinge region sequence portion, a transmembrane domain sequence portion, and one or more intracellular domain sequence portions. In one embodiment, the recombinant nucleic acid comprises, consists of, or consists essentially of an amino acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology to the nucleotide sequences of SEQ ID NOs: 1-4.

[0024] The recombinant nucleic acid construct of SEQ ID NO:1, also referred to herein as XL53, is targeted against the PD1 ligand, called PDL1. This molecule is designed for in vitro synthesis of an RNA molecule that will be delivered to natural killer cells for immunotherapy in cancer patients. In vitro transcription can be initiated at the T7 promoter using bacteriophage T7 RNA polymerase. The T7 promoter is flanked by a 42-bp untranslated sequence (5'UTR: 5' untranslated) that also contains a Kozak sequence upstream of the CAR gene. The secondary structure of the 5'UTR with the Kozak sequence promotes translation initiation. A short signal peptide (15-amino acids) from the CD64 protein marks the N-terminus of the CAR protein. The signal peptide is recognized by a signal recognition peptide (SRP) in the cytosol, which delivers the nascent polypeptide chain from the cytosol to the endoplasmic reticulum. The PDL1 binding site is a heterodimer of variable light and heavy chain domains. The two domains are connected to each other via a 20-aa (amino acid) linker. The hinge and transmembrane domains of the molecule are derived from the CD28 protein. The hinge region provides a range of movement and flexibility for the binding domain, while the transmembrane region allows for precise intramembrane insertion. The cytoplasmic domains of CD28 and CD3ζ are costimulatory domains involved in intracellular signaling pathways that promote the cytotoxic activity of transfected cells. At the 3' untranslated end of the construct, a 94-bp sequence from the 3'UTR of the Mus musculus hemoglobin α gene further stabilizes the construct. This 3'UTR is followed by a 22-bp polyA stretch. The combination of the 3'UTR and polyA confers stability to the RNA molecule. The main features of the construct of SEQ ID NO:1 are that (a) it has high binding affinity to the PDL1 protein, (b) it uses a combination of the intracellular domains of CD28 and CD3ζ for enhanced cytotoxic activity against target cells, and (c) it is RNA-based, so there is no problem with the integration of the construct into the host genome.

[0025] FIG. 1 shows the generation of a chimeric antigen targeted at the PDL1 protein. Transcription of the RNA is initiated by the T7 promoter. The 5'-UTR / Kozak region of nucleotides significantly aids in translation initiation. A nucleic acid sequence encoding a CD64 signal peptide is present at the 3' end of the UTR / Kozak region, which directs the nascent protein to the ER. This is followed by an scFv region that binds to the PDL1 antigen. A hinge region is present adjacent to the scFv region, which provides a range of movement for the scFv. The hinge region is followed by a transmembrane domain that allows the insertion of the nucleotide construct into the membrane. This is followed by one or more intracellular domains that contain costimulatory and / or signaling elements. Finally, the 3'-UTR and poly-A region are present to provide stability and translation initiation to the RNA.

[0026] Figure 2 shows the time course of XL53 PDL1 CAR expression after electroporation, while cytotoxic activity in fLuc-expressing U251 and MS1 target cells is shown in Figure 3. Cytotoxicity assays shown in Figure 3 were set up up to 2 hours after transfection and after overnight incubation. The vector map of the XL53 construct is shown in Figure 4. Finally, Table 1 below shows the various sequence portions of the XL53 construct.

[0027] [Table 1]

[0028] In another embodiment, the inventors disclose a molecule XL53-150A comprising a recombinant nucleic acid construct of SEQ ID NO:2. This molecule is similar to the XL53 molecule, except for the following modifications: a 150-polyA stretch is added to the 3' untranslated end of the construct to further stabilize the RNA molecule. Also, an internal SapI restriction site is removed from the construct, while the same site is added to the end of the polyA tail. After linearization of the DNA template with SapI, only A nucleotides remain. This further facilitates translation of the RNA molecule. The main features of this construct are that it has a longer polyA tail and that it has a longer half-life compared to XL53.

[0029] FIG. 5 shows the generation of a chimeric antigen targeted to the PDL1 protein, pNBS-XL53-150A. As described in FIG. 1, transcription of the RNA is initiated by the T7 promoter. The 5'-UTR / Kozak region of nucleotides significantly aids in translation initiation. A nucleic acid sequence encoding a CD64 signal peptide is present at the 3' end of the UTR / Kozak region, which directs the nascent protein to the ER. This is followed by the scFv region that binds to the PDL1 antigen. A hinge region is present adjacent to the scFv region, which provides a range of movement for the scFv. After the hinge region, a transmembrane domain is followed, which allows the insertion of the nucleotide construct into the membrane. This is followed by one or more intracellular domains that contain costimulatory and / or signaling elements. Finally, the 3'-UTR and poly-A region are present to provide stability and translation initiation to the RNA. The longer poly-A region in this construct provides an RNA construct with higher stability and longer half-life.

[0030] Figure 6 shows the time course of XL53-150A PDL1 CAR expression after electroporation, while the cytotoxic activity of NK cells transfected with XL53-150A is shown in Figure 7. The vector map of the XL53-150A construct is shown in Figure 8. Finally, Table 2 below shows the various sequence portions of the XL53-150A construct.

[0031] [Table 2]

[0032] In another embodiment, the inventors disclose a molecule NKW29 comprising a recombinant nucleic acid construct of SEQ ID NO: 3. This molecule is very similar to the XL53-150A construct, except for the following modification: the CD3ζ intracellular domain of XL53-150A is replaced with the intracellular domain of FcεRIγ. The main features of this construct are that (i) it uses a combination of the intracellular domains of CD28 and FcεRIγ for enhanced cytotoxic activity against target cells, and (ii) it is relatively stable due to the long polyA tail.

[0033] FIG. 9 shows the generation of a chimeric antigen targeted at the PDL1 protein, NKW29-150A. As described in FIG. 1 and 5, transcription of the RNA is initiated by the T7 promoter. The 5'-UTR / Kozak region of nucleotides significantly aids in translation initiation. A nucleic acid sequence encoding a CD64 signal peptide is present at the 3' end of the UTR / Kozak region, which directs the nascent protein to the ER. This is followed by the scFv region that binds to the PDL1 antigen. A hinge region is present adjacent to the scFv region, which provides a range of movement for the scFv. The hinge region is followed by a transmembrane domain that allows insertion of the nucleotide construct into the membrane. This is followed by one or more intracellular domains that contain costimulatory and / or signaling elements. The intracellular domain is shown in FIG. 9, where SEQ ID NO: 3 is the intracellular domain of FcεRIγ. Finally, the 3'-UTR and poly-A region are present to provide stability and translation initiation to the RNA. The longer poly-A region in this construct provides an RNA construct with greater stability and a longer half-life.

[0034] Figure 10 shows the time course of NKW29-150A PDL1 CAR expression in NK cells 24 and 48 hours after electroporation. In vitro transcription was performed using Sapl digested NKW29-150A DNA. NK cells were transfected with in vitro transcribed RNA (2ug / 1e6 cells). PDL1 expression was determined using flow cytometry and biotinylated PDL1 / streptavidin APC.

[0035] The vector map of the NKW29-150A construct is shown in Figure 11. Finally, Table 3 below shows the various sequence portions of the NKW29-150A construct.

[0036] [Table 3]

[0037] In another embodiment, the inventors disclose a XL53-tricistronic molecule, which shows the recombinant nucleic acid construct of SEQ ID NO: 4. This molecule is similar to XL53, except that it co-expresses three genes: PDL1 CAR, CD16a and ER-retained Il2. A P2A sequence and an EMCV IRES precede the CD16a and ER-IL2 genes, respectively, allowing independent translation of these genes. The main features of this construct are: (a) it expresses CD16a, which is involved in ADCC (antibody-dependent cellular cytotoxicity), further triggering NK cell lysis of target cells, and (b) it expresses IL-2, a cytokine that is an important growth factor for the growth and cytotoxic activity of NK cells.

[0038] FIG. 12 shows the generation of a chimeric antigen targeted to the PDL1 protein, a tricistronic XL53. As described in FIG. 1, transcription of the RNA is initiated by the T7 promoter. The 5'-UTR / Kozak region of nucleotides significantly aids in translation initiation. A nucleic acid sequence encoding a CD64 signal peptide is present at the 3' end of the UTR / Kozak region, which directs the nascent protein to the ER. This is followed by the scFv region that binds to the PDL1 antigen. A hinge region is present adjacent to the scFv region, which provides a range of movement for the scFv. The hinge region is followed by a transmembrane domain that allows insertion of the nucleotide construct into the membrane. This is followed by one or more intracellular domains that contain costimulatory and / or signaling elements. The costimulatory and / or signaling elements are followed by P2A for ribosome entry, CD16a important for ADCC, EMCV, which is a ribosome entry site, and ER-IL2. Finally, the 3'-UTR and poly-A region are present to provide stability and translation initiation to the RNA. The longer poly-A region in this construct provides an RNA construct with greater stability and a longer half-life.

[0039] Figure 13 shows XL53-tricistronic PDL1 CAR expression 24 hours after electroporation. The cytotoxic activity of CAR-infected cells in MS1 ​​fLuc target cells is shown in Figure 14. In this case, the transfected cells were mixed with the target cells 2 hours after electroporation for overnight incubation. The vector map of the XL53-tricistronic construct is shown in Figure 15. Finally, the following Table 4 shows the various sequence parts of the XL53-tricistronic construct.

[0040] [Table 4]

[0041] Most currently available CAR technologies use viral vectors as a means of delivery of DNA molecules to cells. Viral DNA can enter the nucleus and fuse with the host genome. The present inventors have developed a new approach using RNA molecules, since only RNA can enter the cytoplasm and be ready to be translated. The present inventors have overcome the problem of degradation of RNA molecules by introducing several elements, such as 5' and 3' UTR and long poly-A, to improve the stability of the molecules disclosed herein.

[0042] Some variations of the inventive concept envisaged by the inventors would be the introduction of various 5' or 3' UTR elements that may improve the stability of the RNA molecule. The construct may also be modified by the addition (or swapping) of additional costimulatory domains. The addition of other cytokine genes to the same construct (as a bicistronic or tricistronic) may also improve the activity of the molecule.

[0043] In one embodiment, a recombinant nucleic acid is disclosed herein, comprising a T7 promoter sequence portion, a 5' untranslated (5'-UTR) sequence portion, a signal peptide sequence portion, a single-chain antibody fragment sequence portion, a hinge region sequence portion, a transmembrane domain sequence portion, and one or more intracellular domain sequence portions. The signal peptide sequence portion further comprises a sequence encoding CD64. The RNA formed from the recombinant DNA nucleic acid is stabilized by the 5'-UTR sequence portion and / or the Kozak sequence. The Kozak sequence (or Kozak consensus sequence) is a nucleic acid motif that functions as a translation initiation site in most mRNA transcripts. It is considered the optimal sequence for initiating translation in eukaryotes, and this sequence is an essential aspect of protein regulation. This sequence is generally defined as 5'-(gcc)gccRccAUGG-3', where R represents a purine (adenine or guanine). Of course, variations of the Kozak sequence are known to those skilled in the art and are contemplated herein by the inventors.

[0044] The single-chain antibody fragment sequence portion of the recombinant nucleic acid comprises a sequence encoding a single-chain variable fragment adapted to bind to the PDL1 antigen. The hinge portion serves to provide a range of movement for the single-chain antibody fragment sequence portion. The transmembrane domain sequence portion allows the recombinant nucleic acid to be inserted into a membrane. The intracellular domain sequence portion comprises a costimulatory or signal transduction sequence portion, such as CD28, CD3ζ and / or FcεRIγ. The intracellular domain sequence portion is selected to provide enhanced cytotoxic activity against tumor cells. The 3′-UTR region toward the 3′ end of the recombinant nucleic acid provides stability and translation initiation to the RNA. Additionally, a poly-A sequence portion may be present for additional stability. In some embodiments, the poly-A sequence portion comprises at least 150 adenine nucleotides. In some embodiments, the recombinant nucleic acid may be tricistronic, in other words, the nucleic acid may have sequences encoding PDL1-CAR, CD16a and ER-IL2.

[0045] In another aspect of the present disclosure, provided herein is a modified NK cell comprising one or more nucleic acids encoding a T7 promoter sequence portion, a 5' untranslated (5'-UTR) sequence portion, a signal peptide sequence portion, a single chain antibody fragment sequence portion, a hinge region sequence portion, a transmembrane domain sequence portion, and one or more intracellular domain sequence portions, wherein the nucleic acid sequences are operably linked to each other as a single polynucleotide.

[0046] Natural killer (NK) cells are cells of the immune system that kill target cells in the absence of specific antigenic stimulation and without restriction according to major histocompatibility complex (MHC) class. NK cells are characterized by the presence of CD56 and the absence of CD3 surface markers. Endogenous NK cells are generally a heterogeneous population of cells enriched for NK cells. Endogenous NK cells can be intended for the treatment of autologous or allogeneic transplants in patients.

[0047] As used herein, "immunotherapy" refers to the use of modified or unmodified NK cells, natural or modified NK cells, or T cells, whether alone or in combination, capable of inducing cytotoxicity upon contact with target cells.

[0048] Cancer Treatment Provided herein is a method of treating cancer or tumor in a subject, comprising administering to a patient in need thereof a therapeutically effective amount of the modified NK cells disclosed above or a composition comprising the modified NK cells disclosed above. It is envisioned that the administration treats the cancer, reduces the size of the tumor in the subject, or reduces cancer metastasis in the subject.

[0049] The term "cancer" refers to any type of cancer, neoplasm, or malignant tumor found in a mammal, including leukemia, carcinoma, and sarcoma. Exemplary cancers include brain, breast, cervical, colon, head and neck, liver, kidney, lung, non-small cell lung, melanoma, mesothelioma, ovarian, sarcoma, stomach, uterine cancer, and medulloblastoma. Additional examples include Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, primary brain tumors, cancer, malignant pancreatic insulinoma, malignant carcinoid, bladder cancer, precancerous skin lesions, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, endometrial cancer, adrenal cortical carcinoma, endocrine and exocrine pancreatic neoplasms, and prostate cancer.

[0050] The terms "metastasis", "metastatic" and "metastatic cancer" can be used interchangeably and refer to the spread of a proliferative disease or disorder, such as cancer, from one organ or another non-adjacent organ or body part. Cancer begins at a site of origin, such as the breast, which is called a primary tumor, such as a primary breast cancer. Some cancer cells in the primary tumor or site of origin acquire the ability to penetrate and invade surrounding normal tissues locally and / or penetrate the walls of the lymphatic or vascular system and circulate through the system to other sites and tissues in the body. A second clinically detectable tumor formed from cancer cells of the primary tumor is called a metastatic or secondary tumor. When cancer cells metastasize, the metastatic tumor and its cells are presumed to be similar to those of the primary tumor. Thus, if lung cancer metastasizes to the breast, the secondary tumor at the site of the breast is composed of abnormal lung cells, not abnormal breast cells. A secondary tumor in the breast is called metastatic lung cancer. Thus, the phrase metastatic cancer refers to a disease in which a subject has or has had a primary tumor and has one or more secondary tumors. The phrase non-metastatic cancer or subject with non-metastatic cancer refers to a disease in which the subject has a primary tumor but does not have one or more secondary tumors. For example, metastatic lung cancer refers to a disease in a subject who has a primary lung tumor or has a history of a primary lung tumor and has one or more secondary tumors in a second location or multiple locations, such as the breast.

[0051] As used herein, "treating" a condition, disease or disorder or a symptom associated with a condition, disease or disorder or "treatment" thereof refers to an approach to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results may include, but are not limited to, alleviating or ameliorating one or more symptoms or conditions, whether partial or total, reducing the extent of a condition, disorder or disease, stabilizing the condition, disorder or disease state, preventing the onset of a condition, disorder or disease, preventing the spread of a condition, disorder or disease, delaying or slowing the progression of a condition, disorder or disease, delaying or slowing the onset of a condition, disorder or disease, improving or palliating and remission of the condition, disorder or disease state. "Treating" may also mean extending the survival of a subject beyond the expected survival in the absence of treatment. "Treating" may also mean inhibiting the progression of a condition, disorder or disease, slowing the progression of a condition, disorder or disease temporarily, although in some cases it includes permanently halting the progression of a condition, disorder or disease. As used herein, the terms treatment, treat or treating refer to a method of reducing the effects of one or more symptoms of a disease or condition characterized by expression of a protease or a symptom of a disease or condition characterized by expression of a protease. Thus, in the disclosed methods, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% reduction in the severity of the disease, condition or symptoms of the disease or condition suffered. For example, a method for treating a disease is considered to be therapeutic if there is a 10% reduction in one or more symptoms of the disease in a subject compared to a control. Thus, the reduction can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or any percentage reduction between 10% and 100% compared to untreated or control levels. It is understood that treatment does not necessarily refer to a cure or complete elimination of the disease, condition, or symptoms of a disease or condition.Furthermore, as used herein, references to reduction, decrease or inhibition include a change of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to a control level, and such terms can include, but do not necessarily include, complete elimination.

[0052] Terms such as subject, patient, individual, etc. are not intended to be limiting and are generally interchangeable. That is, an individual described as a patient does not necessarily have a given disease, but may simply be under the care of a physician. As used throughout, a subject may be a vertebrate, more specifically a mammal (e.g., human, horse, cat, dog, cow, pig, sheep, goat, mouse, rabbit, rat, and guinea pig), bird, reptile, amphibian, fish, and any other animal. The term does not indicate a particular age or sex. Thus, it is intended to include adult and newborn subjects, whether male or female. As used herein, patient, individual, and subject may be used interchangeably, and these terms are not intended to be limiting. That is, an individual described as a patient does not necessarily have a given disease, but may simply be under the care of a physician. The term patient or subject includes human and animal subjects.

[0053] "Administration" or "administering" as used herein refers to providing, contacting and / or delivering one or more compounds by any suitable route to achieve a desired effect. Administration may include, but is not limited to, oral, sublingual, parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular, intra-articular, intra-arterial, intrasynovial, intrasternal, intrathecal, intralesional or intracranial injection), transdermal, topical, buccal, rectal, intravaginal, intranasal, intraocular, inhalation and by implantation. Optionally, the NK cells are administered parenterally. Optionally, the NK cells are administered intravenously. Optionally, the NK cells are administered peritumorally.

[0054] The modified NK cells disclosed herein can be administered to a subject by absolute number of cells, e.g., from about 1000 cells / infusion to up to about 10 billion cells / infusion, e.g., about, at least about, or at most about 1×10 cells / infusion. 10 , 1×10 9 , 1×10 8 , 1×10 7 , 5×10 7 , 1×10 6 , 5×10 6 , 1×10 5 , 5×10 5 , 1×10 4 , 5×10 4 , 1×10 3 , 5×10 3 NK cells, or any range between any two of the numbers (including the endpoints) may be administered. Optionally, 1×10 8 ~1×10 10 The cells are administered to the subject. Optionally, the cells are administered one or more times per week for one or more weeks. Optionally, the cells are administered once or twice per week for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more weeks.

[0055] In another embodiment, the total dose is 100 mg / m of body surface area. 2 Approximately 1000 cells / injection / m 2 ~Up to approx. 10 billion cells / injection / m 2 , e.g., about, at least about, or at most about 1×10 per injection 10 pieces / m 2 , 1×10 9 pieces / m 2 , 1×10 8 pieces / m 2 , 1×10 7 pieces / m 2 , 5×10 7 pieces / m 2 , 1×10 6 pieces / m 2 , 5×10 6 pieces / m 2 , 1×10 5 pieces / m 2 , 5×10 5 pieces / m 2, 1×10 4 pieces / m 2 , 5×10 4 pieces / m 2 , 1×10 3 pieces / m 2 , 5×10 3 pieces / m 2 (etc.) of NK cells or any range between any two of the values ​​(including the endpoints) may be administered. 2 1x10 3 ~1×10 10 NK cells are administered to the subject. Optionally, 2×10 9 pieces / m 2 of NK cells are administered to the subject.

[0056] Optionally, NK cells can be administered to such an individual by relative number of cells, e.g., to said individual from about 1000 cells per kilogram of the individual up to about 10 billion cells per kilogram of the individual, e.g., about, at least about, or at most about 1×10 cells per kilogram of the individual. 10 , 1×10 9 , 1×10 8 , 1×10 7 , 5×10 7 , 1×10 6 , 5×10 6 , 1×10 5 , 5×10 5 , 1×10 4 , 5×10 4 , 1×10 3 , 5×10 3 (etc.) NK® cells, or any range between any two of the values ​​(including the endpoints) may be administered.

[0057] In one embodiment, the NK cells are administered in a composition comprising the NK cells and a medium, such as human serum or an equivalent thereof. The medium may comprise human serum albumin and / or human plasma. Optionally, the medium comprises about 1% to about 15% human serum or a human serum equivalent. Optionally, the medium comprises about 1% to about 10% human serum or a human serum equivalent. Optionally, the medium comprises about 1% to about 5% human serum or a human serum equivalent. Optionally, the medium comprises about 2.5% human serum or a human serum equivalent. Optionally, the serum is human AB serum. Optionally, a serum replacement acceptable for use in human therapeutics is used instead of human serum. Such serum replacements may be known in the art. Optionally, the NK cells are administered in a composition comprising the NK cells and an isotonic liquid solution that supports cell survival. Optionally, the NK cells are administered in a composition reconstituted from a cryopreserved sample.

[0058] According to the methods provided herein, an effective amount of one or more of the agents provided herein is administered to a subject. The terms effective amount and effective dosage are used interchangeably. The term effective amount is defined as any amount necessary to produce a desired physiological response (e.g., reduced inflammation). Effective amounts and schedules for administering agents can be empirically determined by those of skill in the art. The dosage range for administration is large enough to produce the desired effect of affecting (e.g., reducing or delaying) one or more symptoms of a disease or disorder. The dosage should not be so large as to cause substantial adverse side effects, such as undesirable cross-reactions, anaphylactic reactions, and the like. In general, dosages vary with age, disease state, sex, type of disease, extent of disease or disorder, route of administration, or whether other agents are included in the treatment plan, and can be determined by those of skill in the art. Dosages can be adjusted by the individual physician in the event of any contraindications. Dosages can be varied and can be administered daily, daily, or over several days in one or more doses. Guidance on appropriate dosages for a given type of pharmaceutical agent can be found in the literature. For example, for a given parameter, an effective amount indicates an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90% or at least 100%. Efficacy can also be expressed as a "-fold" increase or decrease. For example, a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold or more effect compared to a control. The exact dosage and formulation will depend on the purpose of the treatment and can be ascertained by one of ordinary skill in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Remington: The Science and Practice of Pharmacy, 22nd Edition, Gennaro, Editor (2012) and Pickar, Dosage Calculations (1999)).

[0059] The provided method may further be combined with other tumor treatments, such as radiation therapy, surgery, hormone therapy and / or immunotherapy. Thus, the provided method may further comprise administering one or more additional therapeutic agents to the subject. Suitable additional therapeutic agents include, but are not limited to, analgesics, anesthetics, analgesics, corticosteroids, anticholinergics, anticholinesterases, anticonvulsants, antineoplastics, allosteric inhibitors, anabolic steroids, antirheumatics, psychotherapeutic drugs, neuron blocking drugs, anti-inflammatory drugs, anthelmintics, antibiotics, anticoagulants, antifungal drugs, antihistamines, antimuscarinic drugs, antimycobacterial drugs, antiprotozoal drugs, antiviral drugs, dopaminergic drugs, hemodynamic drugs, immune drugs, muscarinic drugs, protease inhibitors, vitamins, growth factors and hormones. The choice of drug and dosage can be easily determined by those skilled in the art based on the given disease to be treated. Optionally, the additional therapeutic agent is octreotide acetate, interferon, pembrolizumab, glucopyranosyl lipid A, carboplatin, etoposide, or any combination thereof.

[0060] In certain embodiments, the additional therapeutic agent may be selected from the group consisting of viral cancer vaccines, bacterial cancer vaccines, yeast cancer vaccines, N-803, antibodies, stem cell transplants, and tumor-targeted cytokines.

[0061] Optionally, the additional therapeutic agent is a chemotherapeutic agent. The chemotherapeutic treatment plan may include administering to the subject one or a combination of chemotherapeutic agents. Chemotherapeutic agents include, but are not limited to, alkylating agents, anthracyclines, taxanes, epothilones, histone deacetylase inhibitors, inhibitors of topoisomerase I, inhibitors of topoisomerase II, kinase inhibitors, monoclonal antibodies, nucleotide analogs and precursor analogs, peptide antibiotics, platinum-based compounds, retinoids and vinca alkaloids and derivatives. Optionally, the chemotherapeutic agent is carboplatin.

[0062] Combinations of agents or compositions can be administered either concomitantly (e.g., as a mixture), separately but simultaneously (e.g., via separate intravenous lines) or sequentially (e.g., one agent is administered first, followed by the second agent). Thus, the term combination is used to refer to the combined, simultaneous, or sequential administration of two or more agents or compositions. The course of treatment is best determined individually, depending on the particular characteristics of the subject and the type of treatment selected. Treatments such as those disclosed herein can be administered to the subject once a day, twice a day, every other week, once a month, or on any applicable basis that is therapeutically effective. Treatments can be administered alone or in combination with any other treatments disclosed herein or known in the art. The additional treatments can be administered simultaneously with the first treatment, at different times, or on entirely different treatment schedules (e.g., the first treatment can be once a day, while the additional treatment is once a week).

[0063] In certain embodiments, numerical values ​​expressing quantities of ingredients, properties such as concentrations, reaction conditions, and the like, used to describe and claim certain embodiments of the present invention, should be understood to be modified in some cases by the term "about". Thus, in certain embodiments, the numerical parameters set forth in this specification and the appended claims are approximations, which may vary depending on the desired properties sought to be obtained by a particular embodiment. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of individually describing each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated herein as if it were individually set forth herein.

[0064] It should further be noted that the terms "prognosing" or "predicting" a condition, susceptibility to developing a disease, or response to an intended treatment are intended to encompass the act or prediction (rather than treatment or diagnosis) of predicting a condition, susceptibility, and / or response, including the rate of progression, improvement, and / or duration of a condition in a subject. All methods described herein may be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any examples or exemplary language (e.g., "etc.") presented with respect to specific embodiments herein is intended only to better illustrate the invention and does not impose limitations on the scope of the invention as otherwise claimed. No language in this specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0065] As used throughout this description and the claims that follow, the meanings of the singular forms ("a," "an," and "the") include plural references unless the context clearly dictates otherwise. Also, as used in this description, the meaning of "in" includes "in" and "on," unless the context clearly dictates otherwise. Similarly, as used herein and unless the context clearly dictates otherwise, the term "coupled" is intended to include both direct coupling (where the two elements coupled to each other contact each other) and indirect coupling (where at least one additional element is located between the two elements). Thus, the terms "coupled to" and "coupled with" are used interchangeably.

[0066] It should be apparent to one skilled in the art that many further modifications besides those already described are possible without departing from the inventive concept herein. Thus, the subject matter of the present invention is not limited except as set forth in the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms "comprise" and "comprising" should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that a referenced element, component, or step may be present, used, or combined with other elements, components, or steps not expressly referenced. When a claim herein refers to at least one selected from the group consisting of A, B, C... and N, the text should be interpreted as requiring only one element from that group, not A plus N or B plus N, etc.

Claims

1. A recombinant nucleic acid comprising a T7 promoter sequence portion, a 5' untranslated (5'-UTR) sequence portion, a signal peptide sequence portion, a single-chain antibody fragment sequence portion, a hinge region sequence portion, a transmembrane domain sequence portion, and one or more intracellular domain sequence portions.

2. The recombinant nucleic acid of claim 1 , wherein the signal peptide sequence portion further comprises a sequence encoding CD64.

3. The recombinant nucleic acid according to claim 1 or 2, wherein the 5'-UTR sequence portion further comprises a Kozak sequence.

4. The recombinant nucleic acid according to any one of claims 1 to 3, wherein the single chain antibody fragment sequence portion comprises a sequence encoding a single chain variable fragment adapted to bind to a PDL1 antigen or other tumor antigen.

5. The recombinant nucleic acid according to any one of claims 1 to 4, wherein the hinge sequence portion provides a range of movement for the single-chain antibody fragment sequence portion.

6. The recombinant nucleic acid according to any one of claims 1 to 5, wherein the transmembrane domain sequence portion enables insertion of the recombinant nucleic acid into a membrane.

7. The recombinant nucleic acid of any one of claims 1 to 6, wherein the intracellular domain sequence portion comprises a costimulatory or signal transduction sequence portion.

8. The recombinant nucleic acid according to any one of claims 1 to 7, wherein the intracellular domain sequence portion comprises CD28 and / or CD3ζ.

9. The recombinant nucleic acid according to any one of claims 1 to 8, wherein the intracellular domain sequence portion comprises CD28 and / or FcεRIγ.

10. The recombinant nucleic acid of any one of claims 1 to 9, wherein the intracellular domain sequence portion provides enhanced cytotoxic activity against tumor cells.

11. The recombinant nucleic acid according to any one of claims 1 to 10, further comprising a 3'-untranslated region (3'-UTR).

12. The recombinant nucleic acid according to any one of claims 1 to 11, wherein the 3'-UTR sequence portion provides RNA stability and translation initiation.

13. The recombinant nucleic acid according to any one of claims 1 to 12, further comprising a poly-A sequence portion.

14. The recombinant nucleic acid according to any one of claims 1 to 13, wherein the poly-A sequence portion comprises at least 150 adenine nucleotides.

15. The recombinant nucleic acid of any one of claims 1 to 14, wherein the poly-A sequence portion provides RNA stability and translation initiation.

16. The recombinant nucleic acid of any one of claims 1 to 15, wherein the vector targets a tumor antigen.

17. A recombinant nucleic acid according to any one of claims 1 to 16, further comprising a sequence portion encoding CD16a.

18. The recombinant nucleic acid according to any one of claims 1 to 17, further comprising a sequence portion encoding ER-IL2.

19. 2. The recombinant nucleic acid of claim 1, having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:

1.

20. 2. The recombinant nucleic acid of claim 1, having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:

2.

21. 2. The recombinant nucleic acid of claim 1, having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:

3.

22. 2. The recombinant nucleic acid of claim 1, having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:

4.

23. A modified NK cell comprising one or more nucleic acids encoding a 5' untranslated (5'-UTR) sequence portion, a signal peptide sequence portion, a single-chain antibody fragment sequence portion, a hinge region sequence portion, a transmembrane domain sequence portion, and one or more intracellular domain sequence portions, wherein the nucleic acid sequences are operably linked to each other as a single polynucleotide.

24. The modified NK cell of claim 23, which specifically targets tumor cells.

25. The modified NK cell of claim 23 or 24, which specifically targets PDL1 or other tumor antigen.

26. The modified NK cell of any one of claims 23 to 25, wherein the signal peptide sequence portion further comprises a sequence encoding CD64.

27. The modified NK cell of any one of claims 23 to 26, wherein the single chain antibody fragment sequence portion comprises a sequence encoding a single chain variable fragment adapted to bind to a PDL1 antigen or other tumor antigen.

28. The modified NK cell of any one of claims 23 to 27, wherein the hinge sequence portion provides a range of movement for the single chain antibody fragment sequence portion.

29. The modified NK cell of any one of claims 23 to 28, wherein the transmembrane domain sequence portion allows insertion of the recombinant nucleic acid into a membrane.

30. The modified NK cell of any one of claims 23 to 29, wherein the intracellular domain sequence portion comprises a costimulatory or signaling sequence portion.

31. The modified NK cell according to any one of claims 23 to 30, wherein the intracellular domain sequence portion comprises CD28 and / or CD3ζ.

32. The modified NK cell according to any one of claims 23 to 31, wherein the intracellular domain sequence portion comprises CD28 and / or FcεRIγ.

33. The modified NK cell of any one of claims 23 to 32, wherein the intracellular domain sequence portion provides enhanced cytotoxic activity against tumor cells.

34. The modified NK cell of any one of claims 23 to 33, further comprising a 3'-untranslated region (3'-UTR).

35. The modified NK cell of any one of claims 23 to 34, wherein the 3'-UTR sequence portion provides RNA stability and translation initiation.

36. The modified NK cell of any one of claims 23 to 35, further comprising a poly-A sequence portion.

37. The modified NK cell of any one of claims 23 to 36, wherein the poly-A sequence portion comprises at least 150 adenine nucleotides.

38. The modified NK cell of any one of claims 23 to 37, wherein the poly-A sequence portion provides RNA stability and translation initiation.

39. The modified NK cell of any one of claims 23 to 38, wherein the vector targets a tumor antigen.

40. The modified NK cell according to any one of claims 23 to 39, further comprising a sequence portion encoding CD16a.

41. The modified NK cell according to any one of claims 23 to 40, further comprising a sequence portion encoding ER-IL2.

42. A method for generating a CAR-NK cell, the method comprising transfecting a primary NK cell with a recombinant nucleic acid according to any one of claims 1 to 22.

43. A composition comprising the modified NK cells of any one of claims 1 to 22 and a pharma- ceutically acceptable excipient.

44. A kit comprising the NK cell according to any one of claims 23 to 43 and instructions for use.

45. 44. A method of treating cancer or tumor in a subject, comprising administering to the subject a therapeutically effective amount of the modified NK cells of any one of claims 23-42 or the composition of claim 43, wherein administration treats the cancer or reduces the size of the tumor in the subject.

46. 44. A method of reducing cancer metastasis in a subject, comprising administering to the subject a therapeutically effective amount of the modified NK cell of any one of claims 23 to 42 or the composition of claim 43, thereby reducing cancer metastasis in the subject.

47. 1m ２ 1x10 ３ ~1×10 １０ 47. The method of claim 45 or 46, wherein the NK cells are administered to the subject.

48. The method of any one of claims 45 to 47, wherein the NK cells are administered parenterally, intravenously, peritumorally or by injection.

49. The method of any one of claims 45 to 48, further comprising administering to the subject an additional therapeutic agent.

50. A recombinant cell comprising a recombinant nucleic acid according to any one of claims 1 to 22.

51. 51. The recombinant cell of claim 50, which is a bacterial cell.

52. 51. The recombinant cell of claim 50, which is an autologous NK cell.

53. 53. The recombinant cell of claim 52, wherein the NK cell is genetically modified.

54. A method of treating cancer in a patient in need thereof, comprising administering to said patient a therapeutically effective amount of a genetically modified NK cell according to any one of claims 23 to 41, thereby treating said cancer.

55. 55. The method of claim 54, further comprising administering at least one additional therapeutic agent selected from the group consisting of a viral cancer vaccine, a bacterial cancer vaccine, a yeast cancer vaccine, N-803, an antibody, a stem cell transplant, and a tumor-targeted cytokine.

56. The cancers include, but are not limited to, leukemia, acute lymphocytic leukemia, acute myeloid leukemia, chronic leukemia, chronic myeloid (granular) leukemia, chronic lymphocytic leukemia, polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's disease, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, sarcomas and carcinomas, such as, but not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelioma, lymphangiosarcoma, lymphangioendothelioma, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, and the like.

56. The method of claim 54 or 55, wherein the cancer is selected from solid tumors including breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

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