First-generation NK CAR structure and method

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 List The contents of the sequence listing ASCII text file named 104077.00016Pro_ST25, which is 42KB in size, was created on November 6, 2019, and submitted electronically via EFS-Web with this application, are incorporated by reference in their entirety.

[0002] The field of the present invention is recombinant nucleic acids and cells containing them, particularly in relation to the treatment of cancer. [Background technology]

[0003] The background information includes information that may be useful in understanding the present invention. None of the information provided herein is considered to be prior art or relating to the invention claimed in this application, nor is any publication explicitly or implicitly referenced considered to be prior art.

[0004] Cancer immunotherapy based on natural killer (NK) cells has made remarkable progress in recent years. NK cells are cytotoxic lymphocytes that are an important component of the innate immune system. In most cases, NK cells make up about 4-10% of circulating lymphocytes and bind to and kill target cells (including virus-infected cells and many malignant cells). Killing by NK cells is nonspecific to specific antigens and can occur without prior immune sensitization. Killing of target cells is typically mediated by cytolytic proteins, including perforin, granzyme, and granulysin.

[0005] NK cells are being used as therapeutic substances. For this purpose, NK cells are isolated from the peripheral blood lymphocyte fraction of whole blood, grown in cell culture to obtain a sufficient number of cells, and then reinjected into the subject. NK cells have shown moderate effectiveness in both ex vivo and in vivo treatments in at least some cases. However, cancer uses various strategies to delay, alter or stop anti-tumor immunity, making it impossible to control tumor growth.

[0006] The anti-tumor response of NK cells also faces many limitations. First, the low ability of NK cells to reach tumor tissue limits their use as a treatment for solid tumors. This is a common problem with cell immunotherapy approaches. Second, changes in NK cell activating receptors and their ligands in tumors can lead to reduced treatment responses and tumor progression. For example, high levels of NKG2D (natural killer group 2, member D) ligands are detected in the early stages of colorectal cancer, but their expression decreases as the disease progresses. Third, the tumor microenvironment (TME) remains a major obstacle to the effectiveness of allogeneically transplanted NK cells. For example, tumor infiltrating immune cells such as dendritic cells (DCs) embedded in the extracellular matrix, inhibitory or tolerogenic macrophages and regulatory T (Treg) cells, and cancer-associated fibroblasts can interfere with NK cell activation by secreting immunosuppressive cytokines or by preventing receptor expression.

Summary of the Invention

[0007] There is still a need in the art for techniques and methods that can modify NK cells for specific targeting of cancer cells in order to overcome the above problems.

[0008] The subject matter 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. Further, the 5'-UTR sequence portion may further comprise a Kozak sequence. [[ID=!]]

[0009] Preferably, 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. In certain embodiments, the recombinant nucleic acid may further comprise a sequence portion encoding CD16a and / or ER-IL2. [[ID=!]]

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

[0011] The intracellular domain sequence portion of the recombinant nucleic acid disclosed herein is envisioned to comprise a co-stimulatory or signaling 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, wherein further, the intracellular domain sequence portion may provide 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 vectors of this disclosure are preferably optimized to target tumor antigens. In one embodiment, 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 another embodiment, 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 another embodiment, 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 another embodiment, the recombinant nucleic acid has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to SEQ ID NO: 4.

[0014] In another aspect of the subject matter of the present invention, the inventors disclose modified NK cells 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 one another as a single polynucleotide. These modified NK cells are intended to specifically target tumor cells.

[0015] In another embodiment, the inventors disclose a method for generating modified NK cells or CAR-NK cells, comprising transfecting primary NK cells with the recombinant nucleic acids disclosed above. Furthermore, compositions comprising modified NK cells and pharmaceutically acceptable excipients are also disclosed. Furthermore, modified NK cells may be provided in a kit, for example, a kit may comprise the NK cells disclosed herein and instructions for use.

[0016] In yet another embodiment, a method for treating cancer or tumor in a subject is disclosed, comprising administering a therapeutically effective amount of modified NK cells or a composition comprising modified NK cells to the subject, wherein the administration treats the cancer or reduces the size of the tumor in the subject. A method for reducing cancer metastasis in a patient is also envisioned, wherein a therapeutically effective amount of modified NK cells or a composition comprising modified NK cells is administered to a subject having cancer metastasis. Preferably, 1 m 2 1x10 3 ~1 × 10 10 Individual NK cells are administered to the target. Administration may be parenteral, intravenous, peritumoral, or by injection. This method may also include further administration of additional therapeutic agents to the target.

[0017] In another embodiment, the inventors disclose a method for treating cancer in a patient in need, comprising administering to the patient a therapeutically effective dose of one of the recombinant NK cells disclosed herein, thereby treating the cancer. The method may further include the step of administering at least one additional therapeutic substance selected from the group consisting of viral cancer vaccines, bacterial cancer vaccines, yeast cancer vaccines, N-803, antibodies, stem cell transplants, and tumor-targeting cytokines. Cancer includes leukemia, acute lymphoblastic 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, Waldenström macroglobulinemia, heavy chain disease, and, without limitation, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endosarcoma, lymphangiosarcoma, lymphangioendothelioma, synoviomas, mesothelioma, Ewing's tumor, leiomyosarcoma, and rhabdomyosarcoma. The selection is made from solid tumors including 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, seminomas, embryonic carcinoma, Wilms' tumor, cervical cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal glandoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0018] Various objects, features, aspects and advantages of the subject matter of the present invention will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawings in which similar figures represent similar components. [Brief explanation of the drawing]

[0019] [Figure 1] This describes one embodiment of the generation of a chimeric antigen targeted by the PDL1 protein:pNBS-XL53. [Figure 2] This shows one embodiment of the time course of XL53 PDL1 CAR expression after electroporation. [Figure 3] This describes one embodiment of the cytotoxic activity of fLuc-expressing U251 and MS1 target cells. [Figure 4] This shows one embodiment of a vector map for an XL53 construct. [Figure 5] This document describes one embodiment of generating a chimeric antigen targeted by the PDL1 protein, pNBS-XL53-150A. [Figure 6] This shows one embodiment of the time course of XL53-150A PDL1 CAR expression after electroporation. [Figure 7] This describes one embodiment of the cytotoxic activity of NK cells transfected with XL53-150A. [Figure 8] This shows one embodiment of a vector map of the XL53-150A structure. [Figure 9] This document describes one embodiment of the generation of a chimeric antigen targeted by the PDL1 protein, NKW29-150A. [Figure 10] This shows one embodiment of the time course of NKW29-150A PDL1 CAR expression in NK cells. [Figure 11] This shows one embodiment of the vector map of the NKW29-150A structure. [Figure 12] This document presents one embodiment of the generation of a chimeric antigen targeted by the PDL1 protein and tricistronic XL35. [Figure 13] This shows one embodiment of XL53-tricistronic PDL1 CAR expression 24 hours after electroporation. [Figure 14] This demonstrates one embodiment of the cytotoxic activity of CAR-infected cells in MS1 ​​fLuc-targeted cells. [Figure 15] This shows one embodiment of the vector map of the XL53-tricistronic construct. Detailed description of the invention

[0020] All publications and patent applications herein are invoked by reference to the same extent as each individual publication or patent application is specifically and individually indicated as being invoked by reference. If a definition or use of a term in an invoked reference conflicts with or contradicts a definition of that term provided herein, the definition provided herein shall prevail, and the definition in the reference shall not. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains.

[0021] The present inventors disclose various engineered NK cells as a basis for improving immunotherapy for cancer and tumors. From a different perspective, the 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 may be chimeric antigen receptor constructs for transfecting primary NK cells to generate CAR-NK cells.

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

[0023] In one embodiment, recombinant nucleic acids 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 are disclosed herein. In one embodiment, the recombinant nucleic acid comprises, consists of, or is essentially composed 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 Sequence ID No. 1, also known herein as XL53, is targeted to a PD1 ligand called PDL1. This molecule is designed for the 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) upstream of the CAR gene, which also contains a Kozak sequence. 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 cytosolic signal recognition peptide (SRP) that 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 linked 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 mobility 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 cytotoxic activity in transfected cells. At the 3' untranslated end of the construct, a 94-bp sequence from the 3'UTR of the mouse (Mus musculus) hemoglobin α gene further stabilizes the construct. This 3'UTR is followed by a 22-bp poly(A) extension. The combination of the 3'UTR and poly(A) provides stability to the RNA molecule. The main features of the construct of Sequence ID No. 1 are (a) its high binding affinity to the PDL1 protein, (b) its use of the combination of the intracellular domains of CD28 and CD3ζ for enhanced cytotoxic activity against target cells, and (c) its RNA-based nature, thereby posing no problems regarding the integration of the construct into the host genome.

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

[0026] Figure 2 shows the time course of XL53 PDL1 CAR expression after electroporation, while Figure 3 shows the cytotoxic activity in fLuc-expressing U251 and MS1 target cells. The cytotoxic assays shown in Figure 3 were prepared 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 regions of the XL53 construct.

[0027] [Table 1]

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

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

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

[0031] [Table 2]

[0032] In another embodiment, the inventors disclose a molecule NKW29 comprising the recombinant nucleic acid construct of Sequence ID No. 3. This molecule is very similar to the XL53-150A construct, with 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 the combination of the intracellular domains of CD28 and FcεRIγ for enhanced cytotoxic activity against target cells, and (ii) it is relatively stable due to its long poly-A tail.

[0033] Figure 9 shows the generation of a chimeric antigen targeted in the PDL1 protein, NKW29-150A. As described in Figures 1 and 5, RNA transcription is initiated by the T7 promoter. The 5'-UTR / Kozak region of the nucleotide significantly aids in translation initiation. The nucleic acid sequence encoding the CD64 signal peptide is located at the 3' end of the UTR / Kozak region, which directs the nascent protein to the ER. This is followed by the scFv region, which binds to the PDL1 antigen. A hinge region is located adjacent to the scFv region, providing the scFv with a range of movement. Following the hinge region is a transmembrane domain that allows for the insertion of the nucleotide construct into the membrane. This is followed by one or more intracellular domains containing co-stimulatory and / or signaling elements. The intracellular domains are shown in Figure 9, and Sequence ID No. 3 is the intracellular domain of FcεRIγ. Finally, the 3'-UTR and poly-A regions are present to provide stability to the RNA and translation initiation. The longer poly-A region in this construct provides an RNA construct with higher 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 (2 ug / 1 e6 cells). PDL1 expression was determined by flow cytometry and biotinylated PDL1 / streptavidin APC.

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

[0036] [Table 3]

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

[0038] Figure 12 shows the generation of a chimeric antigen targeted by the PDL1 protein and tricistronic XL53. As described in Figure 1, RNA transcription is initiated by the T7 promoter. The 5'-UTR / Kozak region of the nucleotide plays a significant role in translation initiation. The nucleic acid sequence encoding the CD64 signal peptide is located at the 3' end of the UTR / Kozak region, which directs the nascent protein to the ER. This is followed by the scFv region, which binds to the PDL1 antigen. A hinge region is located adjacent to the scFv region, which provides the scFv with a range of movement. Following the hinge region is a transmembrane domain that allows insertion of the nucleotide construct into the membrane. This is followed by one or more intracellular domains containing co-stimulatory and / or signaling elements. Following the co-stimulatory and / or signaling elements are P2A for ribosome entry, CD16a which is important for ADCC, and EMCV and ER-IL2, which are ribosome entry sites. Finally, the 3'-UTR and poly-A regions are present to provide stability to the RNA and translation initiation. The longer poly-A region in this construct provides an RNA construct with higher 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, transfected cells were mixed with target cells 2 hours after electroporation for overnight incubation. The vector map of the XL53-tricistronic construct is shown in Figure 15. Finally, Table 4 below 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 delivering DNA molecules to cells. Viral DNA can enter the nucleus and fuse with the host genome. We have developed a novel method using RNA molecules, since only RNA enters the cytoplasm and is prepared for translation. We have overcome the problem of RNA molecule degradation by introducing several elements, such as 5' and 3' UTRs and long poly-A, to improve the stability of the molecules disclosed herein.

[0042] Some variations of the concept of the present invention as envisioned by the inventors would be the introduction of various 5' or 3'UTR elements that can improve the stability of RNA molecules. The constructs can also be modified by the addition (or swapping) of further co-stimulatory domains. The addition of other cytokine genes to the same construct (as bicistronic or tricistronic) can also improve the activity of the molecule.

[0043] In one embodiment, 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 is disclosed herein. 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). Naturally, variant forms of the Kozak sequence are known to those skilled in the art and are assumed herein by the inventors.

[0044] The single-chain antibody fragment sequence portion of the recombinant nucleic acid includes 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 for insertion of the recombinant nucleic acid into the membrane. The intracellular domain sequence portion includes a co-stimulatory or signaling sequence portion such as CD28, CD3ζ, and / or FcεRIγ. The intracellular domain sequence portion is selected to provide enhanced cytotoxic activity against tumor cells. A 3'-UTR region relative to the 3' end of the recombinant nucleic acid provides stability to RNA and translation initiation. Furthermore, a poly-A sequence portion may be present for additional stability. In some embodiments, the poly-A sequence portion contains 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 disclosure of the present invention, modified NK cells are provided herein, 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 one another as a single polynucleotide.

[0046] Natural killer (NK) cells are immune system cells that kill target cells without restriction based on major histocompatibility complex (MHC) class, in the absence of specific antigen stimulation. NK cells are characterized by the presence of CD56 and the absence of the CD3 surface marker. Endogenous NK cells are generally a heterogeneous population of NK-enriched cells. Endogenous NK cells may be intended for patient autologous or allogeneic transplantation therapy.

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

[0048] Cancer treatment A method for treating cancer or tumors in a subject is provided herein, comprising administering to a patient in need of such treatment a therapeutically effective dose of the modified NK cells disclosed above or a composition comprising the modified NK cells disclosed above to the subject. The administration is intended to treat cancer, reduce the size of tumors in the subject, or reduce cancer metastases in the subject.

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

[0050] The terms “metastasis,” “metastatic,” and “metastatic cancer” are synonymous and refer to the spread of a proliferative disease or disorder, such as cancer, from one organ or another non-adjacent organ or part of the body. Cancer originates in a site of origin, such as the breast, which is called the primary tumor, such as primary breast cancer. Some cancer cells in the primary tumor or site of origin acquire the ability to penetrate and infiltrate the surrounding normal tissue locally and / or to penetrate the walls of the lymphatic or vascular system and circulate through the system to other parts 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. Therefore, if lung cancer metastasizes to the breast, the secondary tumor in the breast site consists of abnormal lung cells, not abnormal mammary gland cells. The secondary tumor in the breast is called metastatic lung cancer. Thus, the term metastatic cancer refers to a disease in which the subject has or had a primary tumor and has one or more secondary tumors. The phrase "non-metastatic cancer" or "subjects with non-metastatic cancer" refers to a disease in which a 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 a history of primary lung tumor, and also has one or more secondary tumors in a second or more locations, such as the breast.

[0051] As used herein, “treating” a condition, disease, or disorder, or symptoms associated with a condition, disease, or disorder, or “treatment” thereof, refers to methods for obtaining beneficial or desired outcomes, including clinical outcomes. Beneficial or desired clinical outcomes may include, but are not limited to, relief or improvement of one or more symptoms or conditions, whether partial or whole; reduction of the severity of a condition, disorder, or disorder; stabilization of the condition, disorder, or disorder; prevention of the onset of a condition, disorder, or disorder; prevention of the transmission of a condition, disorder, or disorder; delay or slowing of the progression of a condition, disorder, or disorder; delay or slowing of the onset of a condition, disorder, or disorder; improvement or relief and remission of the condition, disorder, or disorder. “Treatment” may also mean extending the survival of the subject beyond the survival expected without treatment. “Treatment” may also mean inhibiting the progression of a condition, disorder, or disorder, temporarily slowing the progression of a condition, disorder, or disorder, but may include, in some cases, permanently halting the progression of a condition, disorder, or disorder. As used herein, the terms treatment, to treat, or to cure mean the effect of one or more symptoms of a disease or condition characterized by protease expression, or a method of reducing the symptoms of a disease or condition characterized by protease expression. Accordingly, in the methods disclosed, treatment may mean 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. For example, a method for treating a disease is considered a treatment if there is a 10% reduction in one or more symptoms of the disease in the subject compared to a control. Accordingly, the reduction may be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percentage between 10% and 100% compared to untreated or control levels. It is understood that treatment does not necessarily mean the cure or complete elimination of a disease, condition, or symptoms of a disease or condition.Furthermore, as used herein, references to reduction, decrease, or inhibition include changes of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to a control level, and such terms may include, but do not necessarily, complete removal.

[0052] The terms subject, patient, and individual are not intended to be restrictive and are generally interchangeable. That is, an individual described as a patient does not necessarily have a given disease and may simply be receiving a medical examination. When used throughout, a subject may be a vertebrate, more specifically mammals (e.g., humans, horses, cats, dogs, cattle, pigs, sheep, goats, mice, rabbits, rats, and guinea pigs), birds, reptiles, amphibians, fish, and any other animal. The term does not indicate a specific age or sex. Therefore, it is intended to include mature and neonatal subjects, regardless of whether they are male or female. When used herein, patient, individual, and subject may be used synonymously, and these terms are not intended to be restrictive. That is, an individual described as a patient does not necessarily have a given disease and may simply be receiving a medical examination. The terms patient or subject include human and animal subjects.

[0053] As used herein, “administer” or “to administer” means to provide, bring into contact with and / or deliver one or more compounds by any suitable route to achieve the desired effect. Administration may include, but is not limited to, oral, sublingual, parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular, intra-articular, intra-arterial, intra-bursal, intrasternal, intrathecal, intrafocal, or intracranial injection), percutaneous, topical, oral, rectal, vaginal, nasal, intraocular, inhalation, and implantation. Optionally, NK cells are administered parenterally. Optionally, NK cells are administered intravenously. Optionally, NK cells are administered peritumorally.

[0054] The modified NK cells disclosed in this specification can be administered to a subject based on the absolute number of cells, for example, from about 1000 cells / injection to a maximum of about 10 billion cells / injection to the subject, for example, per injection, about, at least about, or at most about 1×10 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 (etc.) or any range (including endpoints) between any two of the numerical values can be administered. Optionally, 1×10 8 ~ 1×10 10 cells are administered to the subject. Optionally, the cells are administered more than once a week over a period of at least one week. Optionally, the cells are administered once or twice a week for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks or more.

[0055] In another embodiment, the total dose can also be calculated by the m 2 of the body surface area. From about 1000 cells / injection / m 2 to a maximum of about 10 billion cells / injection / m 2 to the subject, for example, per injection, about, at least about, or at most about 1×10 10 cells / m 2 、1×10 9 cells / m 2 、1×10 8 cells / m 2 、1×10 7 cells / m 2 、5×10 7 cells / m 2 、1×10 6 cells / m 2 、5×10 6 cells / m 2 、1×10 5 cells / m 2 、5×10 5 cells / m 2, 1 x 10 4 pieces / m 2 , 5×10 4 pieces / m 2 , 1 x 10 3 pieces / m 2 , 5×10 3 pieces / m 2 Any range (including endpoints) between any two of the following NK cells or numerical values ​​may be administered. Optionally, 1m 2 1x10 3 ~1 × 10 10 Individual NK cells are administered to the target. 2 × 10⁶ cells are administered randomly. 9 pieces / m 2 NK cells are administered to the target.

[0056] NK cells may be administered to such an individual by a relative number of cells, for example, about 1,000 cells per kilogram of the individual to a maximum of about 10 billion cells, for example, about, at least about or at most about 1 × 10¹⁶ cells per kilogram of the individual. 10 , 1 x 10 9 , 1 x 10 8 , 1 x 10 7 , 5×10 7 , 1 x 10 6 , 5×10 6 , 1 x 10 5 , 5×10 5 , 1 x 10 4 , 5×10 4 , 1 x 10 3 , 5×10 3 Any range (including endpoints) between any two of the following NK® cells or numerical values ​​may be administered.

[0057] In one embodiment, NK cells are administered in a composition comprising NK cells and a culture medium, such as human serum or its equivalents. The culture medium may contain human serum albumin and / or human plasma. Optionally, the culture medium may contain about 1% to about 15% human serum or human serum equivalents. Optionally, the culture medium contains about 1% to about 10% human serum or human serum equivalents. Optionally, the culture medium contains about 1% to about 5% human serum or human serum equivalents. Optionally, the culture medium contains about 2.5% human serum or human serum equivalents. Optionally, the serum is human AB serum. Optionally, a serum substitute acceptable for use in human therapeutics is used instead of human serum. Such serum substitutes may be known in the art. Optionally, NK cells are administered in a composition containing NK cells and an isotonic liquid solution to support cell survival. Optionally, NK cells are administered in a composition reconstituted from cryopreserved samples.

[0058] An effective dose of one or more of the drugs provided herein is administered to the subject according to the method provided herein. The terms effective dose and effective dosage are used synonymously. The term effective dose is defined as any amount necessary to produce a desired physiological response (e.g., a reduction in inflammation). The effective dose and schedule for administering a drug may be determined empirically by those skilled in the art. The dosage range for administration is sufficient to produce a desired effect that affects (e.g., reduces or delays) one or more symptoms of a disease or disorder. The dosage should not be so high as to cause substantial adverse side effects, such as undesirable cross-reactions or anaphylactic reactions. In general, the dosage may vary depending on age, condition, sex, type of disease, severity of disease or disorder, route of administration, or whether other drugs are included in the treatment plan, and may be determined by those skilled in the art. The dosage may be adjusted by individual physicians if there are any contraindications. The dosage may be varied and may be administered daily, daily, or over several days in one or more doses. Guidelines for appropriate dosages for a given type of drug may be found in the literature. For example, for a given parameter, the effective dose shows 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 "-times" increase or decrease. For example, the therapeutic effective dose may have an effect of at least 1.2 times, 1.5 times, 2 times, 5 times, or more compared to the control. The exact dose and formulation are determined according to the therapeutic purpose and can be verified by those skilled 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 methods provided may be further combined with other tumor treatments such as radiotherapy, surgery, hormone therapy, and / or immunotherapy. Therefore, the methods provided may further include the administration of one or more additional therapeutic agents. Suitable additional therapeutic agents include, but are not limited to, analgesics, anesthetics, resuscitation agents, corticosteroids, anticholinergics, anticholinesterases, anticonvulsants, antitumor agents, allosteric inhibitors, anabolic steroids, antirheumatic drugs, psychotropic agents, neuroleptics, anti-inflammatory agents, anthelmintics, antibiotics, anticoagulants, antifungal agents, antihistamines, antimuscarinic agents, anti-mycobacterial agents, antiprotozoal agents, antiviral agents, dopamine agonists, hematological agents, immunologists, muscarinic agents, protease inhibitors, vitamins, growth factors, and hormones. The selection of agents and dosages may be readily determined by those skilled in the art based on the given disease being treated. Optionally, additional therapeutic agents include octreotide acetate, interferon, pembrolizumab, glucopyranosyllipid A, carboplatin, etoposide, or any combination thereof.

[0060] In one embodiment, additional therapeutic substances 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-targeting cytokines.

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

[0062] A combination of drugs or compositions may be administered in combination (e.g., as a mixture), separately but simultaneously (e.g., via separate intravenous lines), or sequentially (e.g., one drug is administered first, followed by the second drug). Therefore, the term "combination" is used to refer to the combined, simultaneous, or sequential administration of two or more drugs or compositions. The treatment course is best determined individually depending on the specific characteristics of the subject and the type of treatment chosen. Treatments such as those disclosed herein may be administered to the subject once daily, twice daily, every other week, once a month, or to any therapeutically effective and applicable standard. Treatments may be administered alone or in combination with any other treatments disclosed herein or known in the art. Additional treatments may be administered concurrently with the first treatment, at different time points, or on entirely different treatment schedules (e.g., the first treatment may be once daily while the additional treatment is once weekly).

[0063] In some embodiments, numerical values ​​representing properties such as components and concentrations, and quantities such as reaction conditions, used to describe and claim specific embodiments of the present invention, should be understood to be modified, in some cases, by the term "approximately." Therefore, in some embodiments, the numerical parameters described herein and in the appended claims are approximations, which may vary depending on the desired properties to be obtained by the particular embodiment. The description of ranges of values ​​herein is intended merely as a simple way to indicate each distinct value within that range individually. Unless otherwise specifically indicated herein, each individual value is incorporated herein as if it were individually described herein.

[0064] It should be further noted that the terms “to prognose” or “to predict” a condition, susceptibility to the onset of 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 the condition in the subject. All methods described herein may be performed in any preferred order unless otherwise specifically indicated herein or unless it is clearly inconsistent with the context. The use of any examples or illustrative language (e.g., “etc.”) provided herein in reference to a particular embodiment is intended solely to better illustrate the invention and does not impose any limitation on the scope of the invention as separately claimed. Nothing in this specification should be construed as indicating an unclaimed element essential to the practice of the invention.

[0065] As used herein and throughout the claims, the singular forms ("a," "an," and "it") include plural references unless otherwise specified in the context. Similarly, as used herein, the meaning of "inside" includes "inside" and "on top of" unless otherwise specified in the context. Likewise, as used herein and unless otherwise specified in the context, the term "combined" is intended to include both direct combination (where the two elements being combined are in contact with each other) and indirect combination (where at least one additional element is located between the two elements). Therefore, the terms "combined with" and "combined with" are used synonymously.

[0066] It will be apparent to those skilled in the art that many further modifications are possible without departing from the concept of the invention as described herein. Therefore, the subject matter of the invention is not limited to the appended claims. Furthermore, in interpreting both this specification and the claims, all terms should be interpreted in the broadest possible way consistent with the context. In particular, the terms “includes” and “contains” should be interpreted as referring to an element, component, or process in a non-exclusive manner, indicating that the element, component, or process mentioned may exist, be used, or be combined with other elements, components, or processes not explicitly mentioned. Where the claims of this specification refer 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, and not A plus N or B plus N, etc.

Claims

1. Primary NK cells comprising recombinant 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, Primary NK cells, wherein the recombinant nucleic acid has at least 90% identity with any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO:

4.

2. Primary NK cells according to claim 1, which specifically target tumor cells.

3. Primary NK cells according to claim 1, which specifically target PDL1 or other tumor antigens.

4. The primary NK cell according to claim 1, wherein the signal peptide sequence portion further comprises a sequence encoding CD64.

5. The primary NK cell according to claim 1, wherein the single-chain antibody fragment sequence portion includes a sequence encoding a single-chain variable fragment adapted to bind to the PDL1 antigen or other tumor antigen.

6. The primary NK cell according to claim 1, wherein the hinge region sequence portion provides a range of movement for the single-chain antibody fragment sequence portion.

7. The primary NK cell according to claim 1, wherein the transmembrane domain sequence portion enables insertion of the peptide encoded by the recombinant nucleic acid into the membrane.

8. The primary NK cell according to claim 1, wherein the intracellular domain sequence portion includes a co-stimulatory or signal transduction sequence portion.

9. The primary NK cell according to claim 1, wherein the intracellular domain sequence portion includes CD28 and / or CD3ζ.

10. The primary NK cell according to claim 1, wherein the intracellular domain sequence portion comprises CD28 and / or FcεRIγ.

11. The primary NK cell according to claim 1, further comprising a 3'-untranslated region (3'-UTR).

12. The primary NK cell according to claim 11, wherein the 3'-UTR sequence portion provides RNA stability and translation initiation.

13. The primary NK cell according to claim 1, further comprising a poly-A sequence portion.

14. The primary NK cell according to claim 13, wherein the poly-A sequence portion comprises at least 150 adenine nucleotides.

15. The primary NK cell according to claim 13, wherein the poly-A sequence portion provides RNA stability and translation initiation.

16. Primary NK cells according to claim 1, wherein recombinant nucleic acid is present in the vector.

17. The primary NK cell according to claim 1, further comprising a sequence portion encoding CD16a.

18. The primary NK cell according to any one of claims 1 to 17, further comprising a sequence portion encoding ER-IL2.

19. A composition comprising primary NK cells according to any one of claims 1 to 18 and a pharmaceutically acceptable excipient.

20. Use of the composition according to claim 19 in the manufacture of a pharmaceutical for treating cancer or tumor in a subject, wherein a therapeutically effective amount of primary NK cells is used to treat the cancer or reduce the size of the tumor in the subject.

21. Use of the composition according to claim 19 in the manufacture of a pharmaceutical product for reducing cancer metastasis in a subject, wherein a therapeutically effective amount of primary NK cells reduces cancer metastasis in the subject.

22. 1m ２ 1 x 10 ３ ~1 x 10 １０ The use according to claim 20 or 21, wherein the individual NK cells are administered to the subject.

23. The use according to claim 22, wherein the NK cells are administered parenterally, intravenously, around the tumor, or by injection, possibly together with additional therapeutic agents.

24. Use of primary NK cells according to any one of claims 1 to 18 in the manufacture of a pharmaceutical for the treatment of cancer in a patient in need thereof, wherein a therapeutically effective amount of primary NK cells is used to treat the cancer.

25. The use according to claim 24, wherein at least one additional therapeutic substance selected from the group consisting of viral cancer vaccines, bacterial cancer vaccines, yeast cancer vaccines, N-803, antibodies, stem cell transplants, and tumor-targeting cytokines is administered to the patient.

26. The aforementioned cancers include leukemia, acute lymphoblastic 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, Waldenström macroglobulinemia, heavy chain disease, sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endosarcoma, lymphangiosarcoma, lymphangioendothelioma, synoviomas, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, and breast cancer. The use according to claim 24, selected from solid tumors including 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, seminomas, embryonic carcinoma, Wilms' tumor, cervical cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal glandoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

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