HK40048616B - Primary nk car constructs and methods - Google Patents

Description

Primary NK CAR constructs and methods

sequence list

The ASCII text file containing the sequence list is named 104077.00016Pro_ST25, is 42KB in size, was created on November 6, 2019, and is submitted electronically with this application via EFS-Web, incorporated in its entirety by reference.

Technical Field

The field of this invention is recombinant nucleic acids and cells containing them, particularly because they are related to cancer treatment.

Background Technology

The background description includes information that can be used to understand the invention. No information provided herein is acknowledged to be prior art or related to the currently claimed invention, nor is any publication specifically or implicitly referenced considered prior art.

In recent years, cancer immunotherapy based on natural killer (NK) cells has made significant progress. NK cells are cytotoxic lymphocytes that constitute an important component of the innate immune system. In most cases, NK cells account for approximately 4%–10% of circulating lymphocytes and bind to and kill target cells, including virus-infected cells and many malignant cells. NK cell killing is not specific to any particular antigen and can occur without prior immunosensitization. The killing of target cells is usually mediated by cytolytic proteins, including perforin, granzymes, and granzymes.

NK cells have been used as therapeutic agents. For this purpose, NK cells are isolated from peripheral blood lymphocyte fractions of whole blood, expanded in cell cultures to obtain a sufficient number of cells, and then re-infused into the subject. In at least some cases, NK cells have shown moderate efficacy in both ex vivo and in vivo therapies. However, cancer employs various strategies to delay, alter, or even halt anti-tumor immunity, leading to failure to control tumor growth.

The antitumor response of NK cells also faces several limitations. First, the poor ability of NK cells to reach tumor tissue limits their application as a therapy against solid tumors. This is a common problem with cell immunotherapy strategies. Second, changes in NK cell activation receptors and their ligands in tumors can lead to reduced treatment response and tumor progression. For example, high levels of NKG2D (Natural Killer Cell 2, Member D) ligands are detected in the early stages of colorectal cancer, but their expression declines as the disease progresses. Third, the tumor microenvironment (TME) remains a major obstacle to the effectiveness of adoptive NK cells. For example, tumor-infiltrating immune cells embedded in the extracellular matrix (such as dendritic cells (DCs), suppressive or toxic macrophages) and regulatory T (Treg) cells, as well as cancer-associated fibroblasts, may interfere with NK cell activation by secreting immunosuppressive cytokines or by interfering with receptor expression.

Therefore, there is still a need in this field for techniques and methods that can overcome the above problems and modify NK cells to specifically target cancer cells.

Summary of the Invention

The subject of this invention relates to 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. The recombinant nucleic acid may further comprise a sequence encoding CD64. Furthermore, the 5'-UTR sequence portion may further comprise a Kozak sequence.

Preferably, the single-chain antibody fragment sequence portion includes a sequence encoding a single-chain variable fragment suitable for binding to the PDL1 antigen or other tumor antigens. In some embodiments, the recombinant nucleic acid may further include a sequence portion encoding CD16a and/or ER-IL2.

The hinge sequence provides the range of motion for the single-chain antibody fragment sequence, while the transmembrane domain sequence enables the insertion of recombinant nucleic acids into the membrane.

The intracellular domain sequence portion of the recombinant nucleic acid disclosed herein is expected to include a co-stimulatory or signal transduction sequence portion. In one embodiment, the intracellular domain sequence portion includes CD28 and/or CD3ζ. In another embodiment, the intracellular domain sequence portion includes CD28 and/or FcεRIγ. Furthermore, the intracellular domain sequence portion of the recombinant nucleic acid as described in any of the preceding claims can provide enhanced cytotoxic activity against tumor cells.

Preferably, the recombinant nucleic acid disclosed herein 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.

Preferably, the recombinant nucleic acid vector disclosed herein is 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 with 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 with SEQ ID NO: 2. In still other embodiments, the recombinant nucleic acid has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with 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 SEQ ID NO: 4.

In another aspect of the subject matter of this invention, the inventors have disclosed 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 operatively linked to each other as single polynucleotides. It is anticipated that this modified NK cell specifically targets tumor cells.

On the other hand, the inventors have disclosed a method for generating modified NK cells or CAR-NK cells, the method comprising: transfecting primary NK cells with the recombinant nucleic acid disclosed above. Furthermore, compositions comprising modified NK cells and pharmaceutically acceptable excipients have been disclosed. Additionally, modified NK cells can be provided in kits; for example, a kit may contain the NK cells disclosed herein and instructions for use.

In another aspect, a method for treating a subject's cancer or tumor is disclosed, the method comprising administering to the subject a therapeutically effective amount of modified NK cells or a composition containing modified NK cells, wherein the administration treats the cancer or reduces the size of the subject's tumor. A method for reducing cancer metastasis in a patient is also contemplated, wherein a therapeutically effective amount of modified NK cells or a composition containing modified NK cells is administered to a subject with cancer metastasis. Preferably, 1 × ^10³ to 1 × ^10¹⁰ NK cells/ ^m² is administered to the subject. Administration can be parenteral, intravenous, peritumoral, or by infusion. The method may further include administering additional therapeutic agents to the subject.

In another embodiment, the inventors have disclosed a method for treating cancer in a patient in need, the method comprising administering a therapeutically effective amount of any of the genetically modified NK cells disclosed herein to the patient, thereby treating the cancer. The method may further include the step of administering at least one additional therapeutic entity selected from the group consisting of: viral cancer vaccines, bacterial cancer vaccines, yeast cancer vaccines, N-803, antibodies, stem cell grafts, and tumor-targeting cytokines. The cancers mentioned are selected from leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, chronic leukemia, chronic myeloid (granulocytic) leukemia, chronic lymphocytic leukemia, polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's disease, multiple myeloma, Waldenström macroglobulinemia, heavy chain disease, and solid tumors. The solid tumors include, but are not limited to, sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangiolus endothelial sarcoma, synovial tumor, mesothelial carcinoma, Ewing's tumor, leiomyosarcoma, and 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, cystic adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, hepatocellular carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, nephroblastoma, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

Various objects, features, aspects, and advantages of the subject matter of the invention will become clearer from the following detailed description of preferred embodiments and the accompanying drawings (where like reference numerals denote like components).

Attached Figure Description

Figure 1 illustrates an example of the generation of a chimeric antigen targeting the PDL1 protein: pNBS-XL53.

Figure 2 shows an example of the time course of XL53 PDL1 CAR expression after electroporation.

Figure 3 illustrates an example of cytotoxic activity against U251 and MS1 target cells expressing fLuc.

Figure 4 shows an example of a carrier diagram of the XL53 construct.

Figure 5 shows an example of the generation of a chimeric antigen targeting the PDL1 protein, pNBS-XL53-150A.

Figure 6 illustrates an example of the timeline of XL53-150A PDL1 CAR expression after electroporation.

Figure 7 illustrates an example of the cytotoxic activity of NK cells transfected with XL53-150A.

Figure 8 shows an example of a carrier diagram of the XL53-150A construct.

Figure 9 shows an example of the generation of a chimeric antigen targeting the PDL1 protein, NKW29-150A.

Figure 10 illustrates an example of the timeline of NKW29-150A PDL1 CAR expression in NK cells.

Figure 11 illustrates an embodiment of a carrier diagram of the NKW29-150A construct.

Figure 12 illustrates an example of the generation of a chimeric antigen targeting the PDL1 protein, tricistronic XL35.

Figure 13 illustrates an example of XL53-tricistronic PDL1 CAR expression 24 hours after electroporation.

Figure 14 illustrates an example of the cytotoxic activity of CAR-infected cells against MS1 fLuc target cells.

Figure 15 illustrates an embodiment of the carrier diagram of the XL53-tricistronic construct.

Detailed Implementation

All publications and patent applications herein are incorporated by reference to the same extent that each individual publication or patent application is specifically and individually indicated to be incorporated by reference. Where a definition or usage of a term in an incorporated reference is inconsistent with or contrary to the definition of that term provided herein, the definition provided herein shall apply, and not the definition in that reference. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

The inventors here disclose various engineered NK cells as a basis for improving immunotherapies against cancer and tumors. From different perspectives, the inventors have disclosed nucleic acid constructs targeting tumor antigens. Preferably, in some embodiments, 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.

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

In one embodiment, this document discloses 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. In one embodiment, the recombinant nucleic acid comprises, or consists of, or substantially consists 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 NO: 1-4.

The recombinant nucleic acid construct of SEQ ID NO: 1, also referred to herein as XL53, targets a PD1 ligand called PDL1. This molecule is designed for the in vitro synthesis of RNA molecules that will be delivered to natural killer cells for immunotherapy purposes in cancer patients. In vitro transcription can be initiated at the T7 promoter using phage T7 RNA polymerase. The flanking element of the T7 promoter is a 42-bp untranslated sequence (5’UTR: 5’ untranslated), which also contains a Kozak sequence upstream of the CAR gene. The secondary structure of the 5’UTR, together with the Kozak sequence, facilitates 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. These two domains are linked to each other by a 20-aa (amino acid) linker. The hinge and transmembrane domains of the molecule are derived from the CD28 protein. The hinge region provides range of motion and flexibility for the binding domain, while the transmembrane region allows for proper membrane insertion. The cytoplasmic domains of CD28 and CD3ζ are co-stimulatory domains involved in intracellular signaling pathways that enhance the cytotoxic activity of transfected cells. At the 3' untranslated end of the construct, a 94-bp sequence from the 3'UTR of the mouse hemoglobin α gene further stabilizes the construct. This 3'UTR is followed by a 22-bp polyA region. 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: (a) it has a high binding affinity for the PDL1 protein; (b) it uses a combination of the intracellular domains of CD28 and CD3ζ to enhance cytotoxic activity against target cells; and (c) it is RNA-based, thus eliminating concerns about construct integration into the host genome.

Figure 1 illustrates the generation of a chimeric antigen targeting the PDL1 protein. RNA transcription is initiated by the T7 promoter. The 5'-UTR/Kozak region of the nucleotide provides crucial conditions for translation initiation. The nucleic acid sequence encoding the CD64 signal peptide is located at the 3' end of the UTR/Kozak region and guides the nascent protein to the ER. Following this is the scFv region, which binds to the PDL1 antigen. A hinge region lies adjacent to the scFv region and provides the scFv with a range of motion. Next to the hinge region is a transmembrane domain, which allows the nucleotide construct to insert 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 RNA stability and facilitate translation initiation.

Figure 2 illustrates the timeline of XL53 PDL1 CAR expression after electroporation, while cytotoxic activity against U251 and MS1 target cells expressing fLuc is shown in Figure 3. Cytotoxicity assays, as shown in Figure 3, were set up 2 hours post-transfection and after overnight incubation. A vector diagram of the XL53 construct is shown in Figure 4. Finally, Table 1 below shows the different sequence portions of the XL53 construct.

Table 1

In another embodiment, the inventors have disclosed a molecule XL53-150A comprising the 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 segment is added to the 3' untranslated end of the construct to further stabilize the RNA molecule. Additionally, the internal SapI restriction site is removed from the construct, while the same site is added to the end of the polyA tail. After linearizing the DNA template with SapI, only A nucleotides are retained. This further enhances the translation of the RNA molecule. The main features of this construct compared to XL53 are its longer polyA tail and its longer half-life.

Figure 5 illustrates the generation of the chimeric antigen (pNBS-XL53-150A) targeting the PDL1 protein. Similar to the discussion in Figure 1, RNA transcription is initiated by the T7 promoter. The 5'-UTR/Kozak region of the nucleotide provides crucial conditions for translation initiation. The nucleic acid sequence encoding the CD64 signal peptide is located at the 3' end of the UTR/Kozak region and guides the nascent protein to the ER. Following this is the scFv region, which binds the PDL1 antigen. A hinge region lies adjacent to the scFv region and provides the scFv with a range of motion. After the hinge region is a transmembrane domain, which allows the nucleotide construct to insert 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 RNA stability and translation initiation. The longer poly-A region in this construct provides greater stability and a longer half-life for the RNA construct.

Figure 6 illustrates the timeline of XL53-150A PDL1 CAR expression after electroporation, while the cytotoxic activity of NK cells transfected with XL53-150A is shown in Figure 7. A vector diagram of the XL53-150A construct is shown in Figure 8. Finally, Table 2 below shows the different sequence portions of the XL53-150A construct.

Table 2

In another embodiment, the inventors have disclosed a molecule NKW29 comprising the 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 intracellular domain of XL53-150A is replaced by the intracellular domain of FcεRIγ. The main features of this construct are: (i) it uses a combination of the intracellular domains of CD28 and FcεRIγ to enhance cytotoxic activity against target cells; and (ii) it is relatively stable due to its long poly-A tail.

Figure 9 illustrates the generation of the chimeric antigen (NKW29-150A) targeting the PDL1 protein. Similar to the discussion in Figures 1 and 5, RNA transcription is initiated by the T7 promoter. The 5’-UTR/Kozak region of the nucleotide provides crucial conditions for translation initiation. The nucleic acid sequence encoding the CD64 signal peptide is located at the 3’ end of the UTR/Kozak region and guides the nascent protein to the ER. Following this is the scFv region, which binds to the PDL1 antigen. A hinge region is located adjacent to the scFv region and provides the scFv with a range of motion. After the hinge region is a transmembrane domain, which allows the nucleotide construct to insert into the membrane. This is followed by one or more intracellular domains containing co-stimulatory and/or signal transduction elements. The intracellular domain shown in Figure 9 and SEQ ID NO: 3 is the intracellular domain of Fc□RIγ. Finally, a 3’-UTR and a poly-A region are present to provide RNA stability and translation initiation. The longer poly-A region in this construct provides greater stability and a longer half-life for the RNA construct.

Figure 10 illustrates the timeline of NKW29-150A PDL1CAR expression in NK cells at 24 and 48 hours post-electroporation. NKW29-150A DNA was transcribed in vitro using Sap1 digestion. NK cells were transfected with the in vitro transcribed RNA (2 μg/1e6 cells). PDL1 expression was determined using flow cytometry and biotinylated PDL1/streptavidin APC.

The vector diagram of the NKW29-150A construct is shown in Figure 11. Finally, Table 3 below shows the different sequence portions of the NKW29-150A construct.

Table 3

Builder Name NKW29-150A size 7493bp SV40 PolyA signal 271-405 Purine resistance gene 1009-410 Ampicillin resistance gene 58-918 rrnG terminator 916-1051 EF1α promoter 2-1183 T7 starter 1191-1209 5’UTR 1210-1252 Kozak sequence 1246-1253 CD64 signal peptide 1254-1299 Rbsc6-VL (Variable Light Chain) 1300-1623 connector 1624-1683 Rbsc6-VH (Variable Heavy Chain) 1684-2037 CD28 hinge area 2038-2154 CD28 transmembrane domain 2155-2235 CD28 cytoplasmic domain 2236-2358 Fce□RIγ cytoplasmic domain 2359-2484 3’UTR mouse hemoglobin α 2492-2585 Poly A 2592-2741

In another embodiment, the inventors have disclosed an XL53-tricistronic molecule comprising a 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. The P2A sequence and EMCV IRES are located prior to the CD16a and ER-IL2 genes, respectively, allowing for independent translation of these genes. The main features of this construct are: (a) it expresses CD16a, which is involved in ADCC (antibody-dependent cytotoxicity) and further triggers NK cell lysis of target cells; and (b) it expresses IL-2, a cytokine that is a key growth factor for NK cell growth and cytotoxic activity.

Figure 12 illustrates the generation of the chimeric antigen (tricistronic XL53) targeting the PDL1 protein. Similar to the discussion in Figure 1, RNA transcription is initiated by the T7 promoter. The 5’-UTR/Kozak region of the nucleotide provides crucial conditions for translation initiation. The nucleic acid sequence encoding the CD64 signal peptide is located at the 3’ end of the UTR/Kozak region and guides the nascent protein to the ER. Following this is the scFv region, which binds the PDL1 antigen. A hinge region lies adjacent to the scFv region and provides the scFv with a range of motion. After the hinge region is a transmembrane domain that allows the nucleotide construct to insert into the membrane. This is followed by one or more intracellular domains containing costimulatory and/or signaling elements. Following the costimulatory and/or signaling elements are P2A for ribosome entry, CD16a, important for ADCC, EMCV as a ribosome entry site, and ER-IL2. Finally, the 3’-UTR and poly-A regions are present to provide RNA stability and translation initiation. The longer poly-A region in this construct provides the RNA construct with greater stability and a longer half-life.

Figure 13 illustrates XL53-tricistinoPDL1 CAR expression 24 hours after electroporation. The cytotoxic activity of CAR-infected cells against MS1fLuc target cells is shown in Figure 14. In this case, transfected cells were mixed with target cells and incubated overnight 2 hours after electroporation. A vector diagram of the XL53-tricistino construct is shown in Figure 15. Finally, Table 4 below shows the different sequence portions of the XL53-tricistino construct.

Table 4.

The most commonly used CAR technology currently employs viral vectors as a means of delivering DNA molecules into cells. Viral DNA enters the cell nucleus and can integrate into the host genome. The inventors have developed a novel method using RNA molecules, as RNA enters only the cytoplasm and is ready for translation. The inventors have overcome the degradation problem of RNA molecules by introducing several elements, such as the 5' and 3' UTRs and long poly-A, to improve the stability of the molecules disclosed herein.

As anticipated by the inventors, some variations of the inventive concept will involve introducing different 5′ or 3′ UTR elements that can improve the stability of RNA molecules. The construct can also be modified by adding (or swapping) more common stimulatory domains. Adding other cytokine genes to the same construct (such as bicistronic or tricistronic) can also improve the activity of the molecule.

In one embodiment, this document discloses 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 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 a Kozak sequence. The Kozak sequence (or Kozak concordant sequence) is a nucleic acid motif that serves as a translation initiation site in most mRNA transcripts. It is considered the optimal sequence for initiating translation in eukaryotes, and the sequence is a constituent aspect of protein regulation. The sequence is generally defined as 5′-(gcc)gccRccAUGG-3′, where R represents a purine (adenine or guanine). Of course, variants of the Kozak sequence are known to those skilled in the art and are contemplated herein by the inventors.

The single-chain antibody fragment sequence portion of the recombinant nucleic acid contains a sequence encoding a single-chain variable fragment suitable for binding the PDL1 antigen. The hinge portion serves to provide a range of motion for the single-chain antibody fragment sequence portion. The transmembrane domain sequence portion enables insertion of the recombinant nucleic acid into the membrane. The intracellular domain sequence portion contains a co-stimulatory 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 towards the 3' end of the recombinant nucleic acid provides stability and translation initiation for the RNA. Additionally, a poly-A sequence portion may be present for further stability reasons. 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.

In another aspect of this disclosure, this document provides modified NK cells 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 said nucleic acid sequences are operatively linked to each other as single polynucleotides.

Natural killer (NK) cells are cells of the immune system that kill target cells in the absence of specific antigenic stimulation and are not restricted by major histocompatibility complex (MHC) class. NK cells are characterized by the presence of CD56 and the absence of the CD3 surface marker. Endogenous NK cells are typically xenogeneic cell populations in which NK cells have been enriched. Endogenous NK cells can be used for autologous or allogeneic therapy in patients.

As used in this article, “immunotherapy” refers to the use of modified or unmodified NK cells, naturally occurring or modified NK cells or T cells, alone or in combination, that are capable of inducing cytotoxicity upon contact with target cells.

Cancer treatment

This article provides a method for treating cancer or tumors in a subject, the method comprising administering to the subject a therapeutically effective amount of modified NK cells as disclosed above, or a composition comprising modified NK cells as disclosed above, to the subject in need. The administration is intended to treat cancer, reduce tumor size in the subject, or reduce cancer metastasis in the subject.

The term "cancer" refers to all types of cancer, growths, or malignant tumors found in mammals, including leukemia, carcinoma, and sarcoma. Exemplary cancers include brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, liver cancer, kidney cancer, lung cancer, non-small cell lung cancer, melanoma, mesothelioma, ovarian cancer, sarcoma, stomach cancer, uterine cancer, and medulloblastoma. Other examples include Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, ovarian cancer, rhabdomyosarcoma, essential thrombocytosis, essential macroglobulinemia, primary brain tumors, cancer, malignant pancreatic islet tumors, malignant carcinoid tumors, bladder cancer, pre-malignant skin lesions, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, endometrial cancer, adrenocortical carcinoma, endocrine and exocrine pancreatic growths, and prostate cancer.

The terms “metastasis,” “metastatic,” and “metastatic cancer” are used interchangeably and refer to a proliferative disease or condition, such as cancer, that spreads from one organ or another non-adjacent organ or part of the body. Cancer occurs at a site of origin, such as breast cancer, which is called the primary tumor, e.g., 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 in the local area, and/or penetrate the walls of the lymphatic or vascular systems, circulating through these systems to other sites and tissues in the body. A second, clinically detectable tumor formed from the cancer cells of the primary tumor is called a metastatic tumor or secondary tumor. When cancer cells metastasize, it is presumed that the metastatic tumor and its cells resemble the original tumor. Therefore, if lung cancer metastasizes to the breast, the secondary tumor in the breast site is composed of abnormal lung cells rather than abnormal breast cells. The secondary tumor in the breast is called metastatic lung cancer. Therefore, 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 no 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 a primary lung tumor and has one or more secondary tumors in a second or more locations (e.g., in the breast).

As used herein, “treatment” for a symptom, disease, or condition, or for symptoms related to the symptom, disease, or condition, refers to a method of achieving a beneficial or desired outcome (including clinical outcomes). Beneficial or desired clinical outcomes may include, but are not limited to, reducing or improving one or more symptoms or conditions, weakening the severity of a symptom, condition, or disease, stabilizing the state of a symptom, condition, or disease, preventing the development of a symptom, condition, or disease, preventing the spread of a symptom, condition, or disease, delaying or slowing the progression of a symptom, condition, or disease, delaying or slowing the onset of a symptom, condition, or disease, improving or alleviating the state of a symptom, condition, or disease, and partial or complete remission. “Treatment” may also mean prolonging the survival of a subject beyond the expected survival without treatment. “Treatment” may also mean temporarily inhibiting or slowing the progression of a symptom, condition, or disease, but in some cases, it involves permanently stopping the progression of a symptom, condition, or disease. As used herein, the term "treatment" refers to a method of reducing the effect of one or more symptoms of a disease or symptom characterized by protease expression. Therefore, in the disclosed methods, treatment can refer to a reduction of the severity of an identified disease, symptom, or symptom by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. For example, if one or more symptoms of a disease are reduced by 10% in a subject compared to a control, then the method of treating the disease is considered treatment. Therefore, the reduction compared to the natural or control level can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percentage between 10% and 100%. It should be understood that treatment does not necessarily refer to a cure or complete ablation of the disease, symptom, or symptom. Furthermore, as used herein, references to reduction, mitigation, or inhibition include changes of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater compared to control levels, and such terms may include, but do not necessarily include, complete elimination.

The terms subject, patient, individual, etc., are not intended to be restrictive and are generally used interchangeably. That is, an individual described as a patient does not necessarily have a given disease and may simply be seeking medical advice. As used throughout, a subject can be a vertebrate, more specifically a mammal (e.g., human, horse, cat, dog, cow, pig, sheep, goat, mouse, rabbit, rat, and guinea pig), a bird, reptile, amphibian, fish, and any other animal. The term does not specify a particular age or sex. Therefore, it is intended to cover adult and newborn subjects, whether male or female. As used herein, patient, individual, and subject are used interchangeably, 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 seeking medical advice. The terms patient or subject include both human and mammalian subjects.

As used herein, “administration” means the provision, contact, and/or delivery of one or more compounds to achieve the desired effect through any appropriate route. Administration may include, but is not limited to, oral, sublingual, parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular, intra-articular, intra-articular, intra-synovial, intrasternal, intrathecal, intralesional, intracranial, percutaneous, local, buccal, rectal, vaginal, nasal, ocular, inhalation, and implantation. Optionally, NK cells are administered parenterally. Optionally, NK cells are administered intravenously. Optionally, NK cells are administered peritumorally.

The modified NK cells disclosed herein can be administered to the subject in absolute numbers, for example, from about 1,000 cells per injection to up to about 10 billion cells per injection, such as about, at least about, or at most about 1× ^10¹⁰ , 1× ^10⁹ , 1×10⁸, 1× ^10⁷ , 5× ^10⁷ , 1× ^10⁶ , 5× ^10⁶ , 1× ^10⁵ , 5× ^10⁵ , 1× ^10⁴ , 5× ^10⁴ , 1× ^10³ , 5× ^10³ (and so ^on ) NK cells per injection, or any range (including endpoints) between any two of these numbers. Optionally, 1× ^10⁸ to 1× ^10¹⁰ cells are administered to the subject. Optionally, cells are administered once or more weekly for one or more weeks. Optionally, cells may be applied once or twice a week for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks or more.

In another embodiment, the total dose can also be calculated using ^m² body surface area. NK cells can be administered to subjects at doses ranging from approximately 1,000 cells/injection/ ^m² to up to approximately 10 billion cells/injection/ ^m² , such as approximately, at least approximately, or at most approximately 1 × ^10¹⁰ cells/ ^m² , 1 × ^10⁹ cells/ ^m² , 1 × ^10⁸ cells/ ^m² , 1 × ^10⁷ cells/ ^m² , 5 × ^10⁷ cells/ ^m² , 1 × ^10⁶ cells/ ^m² , ⁵ × ^10⁶ cells/m², 1 × ^10⁵ cells/ ^m² , 5 × ^10⁵ cells/ ^m² , 1 × ^10⁴ cells/ ^m² , 5 × ^10⁴ cells/ ^m² , 1 × ^10³ cells/ ^m² , ⁵ × 10³ cells/ ^m² (and so on), or any range (including endpoints) between any two of these numbers. Optionally, 1× ^10³ to 1× ^10¹⁰ NK cells/m² are administered to the subject. Optionally, 2× ^10⁹ NK cells/ ^m² are administered to the subject.

Optionally, NK cells can be administered to such individuals in relative numbers of cells, for example, from about 1,000 cells/kg individual to up to about 10 billion cells/kg individual, such as about, at least about or at most about 1× ^10¹⁰ , 1× ^10⁹ , 1× ^10⁸ , 1× ^10⁷ , 5×10⁷, 1× ^10⁶ , 5× ^10⁶ , 1× ^10⁵ , 5× ^10⁵ , 1× ^10⁴ , 5× ^10⁴ , 1× ^10³ , 5× ^10³ (and so on) cells/kg individual, or any range (including the endpoints) between any two ^of these numbers.

In some embodiments, NK cells are administered in the form of a composition comprising NK cells and a culture medium, such as human serum or an equivalent thereof. The culture medium may comprise human serum albumin and/or human plasma. Optionally, the culture medium comprises about 1% to about 15% human serum or a human serum equivalent. Optionally, the culture medium comprises about 1% to about 10% human serum or a human serum equivalent. Optionally, the culture medium comprises about 1% to about 5% human serum or a human serum equivalent. Optionally, the culture medium comprises about 2.5% human serum or a human serum equivalent. Optionally, the serum is human AB serum. Optionally, a serum substitute acceptable for human therapeutic agents is used instead of human serum. Such serum substitutes may be known in the art. Optionally, NK cells are administered in the form of a composition comprising NK cells and an isotonic liquid solution supporting cell viability. Optionally, NK cells are administered in the form of a composition reconstituted from a cryopreserved sample.

According to the methods provided herein, an effective amount of one or more of the pharmaceutical agents provided herein is administered to a subject. The terms effective amount and effective dose are used interchangeably. The term effective amount is defined as any amount necessary to produce the desired physiological response (e.g., reduction of inflammation). Those skilled in the art can determine the effective amount and schedule of administration of the pharmaceutical agent based on experience. The range of doses administered is those that are sufficiently large to produce the desired effect, in which one or more symptoms of the disease or condition are affected (e.g., reduced or delayed). The dose should not be so large as to cause serious adverse side effects, such as unwanted cross-reactions, allergic reactions, etc. Generally, the dose will vary with age, condition, sex, type of disease, severity of disease or condition, route of administration, or whether other drugs are included in the regimen, and can be determined by those skilled in the art. If any contraindications occur, the dose may be adjusted by an individual physician. The dose may be varied and may be administered once or more daily for one or several days. For a given class of pharmaceutical products, guidelines for appropriate dosages can be found in the literature. For example, for a given parameter, the effective dose will show 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 an increase or decrease of "how many times". For example, the therapeutically effective dose can have an effect of at least 1.2 times, 1.5 times, 2 times, 5 times, or more relative to the control. The exact dosage and formulation will depend on the purpose of treatment and will be determined 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 ed., Gennaro (2012); and Pickar, Dosage Calculations (1999)).

The provided method can be further combined with other oncology therapies such as radiotherapy, surgery, hormone therapy, and/or immunotherapy. Therefore, the provided method may further include administering one or more additional therapeutic agents to the subject. Suitable additional therapeutic agents include, but are not limited to, analgesics, stimulants, corticosteroids, anticholinergics, anticholinesterases, anticonvulsants, antitumor agents, allosteric inhibitors, anabolic steroids, antirheumatic agents, psychotropic agents, nerve blockers, anti-inflammatory agents, anthelmintics, antibiotics, anticoagulants, antifungals, antihistamines, antimuscarinic agents, antimycobacterial agents, antiantibiotics, antiviral agents, dopaminergic drugs, hematologic agents, immunological agents, muscarinic drugs, protease inhibitors, vitamins, growth factors, and hormones. Based on the given disease being treated, those skilled in the art can readily determine the selection of agents and dosages. Optionally, other treatment agents include octreotide acetate, interferon, pembrolizumab, glucopyranosyl lipid A, carboplatin, etoposide, or any combination thereof.

In some embodiments, the additional therapeutic entity may be selected from the group consisting of: viral cancer vaccines, bacterial cancer vaccines, yeast cancer vaccines, N-803, antibodies, stem cell grafts, and tumor-targeting cytokines.

Optionally, the additional treatment agent is a chemotherapeutic agent. A chemotherapy regimen may include administering a single chemotherapeutic agent or a combination of chemotherapeutic agents to the subject. Chemotherapeutic agents include, but are not limited to, alkylating agents, anthracyclines, taxanes, epothilone, histone deacetylase inhibitors, topoisomerase I inhibitors, topoisomerase II inhibitors, kinase inhibitors, monoclonal antibodies, nucleotide analogs and prodrug analogs, peptide antibiotics, platinum-based compounds, retinoids, vinca alkaloids and their derivatives. Optionally, the chemotherapeutic agent is carboplatin.

Combinations of agents or compositions may be administered concurrently (e.g., as a mixture), alone but simultaneously (e.g., via a separate intravenous line), or sequentially (e.g., administering one agent first, followed by a second agent). Therefore, the term combination is used to refer to the concurrent, simultaneous, or sequential administration of two or more agents or compositions. Treatment procedures are preferably determined on an individual basis, based 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 two weeks, monthly, or on any applicable basis where treatment is effective. 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 therapy, at different times, or on a completely different treatment schedule (e.g., the first therapy may be once daily, while the additional treatment is once weekly).

In some embodiments, the numerical values used to describe and claim certain embodiments of the invention, representing the quantities of expressed components, properties (such as concentration), reaction conditions, etc., should be understood to be modified by the term "about" in some cases. Therefore, in some embodiments, the numerical parameters listed in the written description and appended claims are approximate values that may vary depending on the desired properties sought to be obtained in a particular embodiment. The description of ranges of values herein is intended only as a method of abbreviating each individual value falling within that range. Unless otherwise stated herein, each individual value is incorporated into the description as if it were individually referenced herein.

It should also be noted that the terms “prognosis” or “prediction” of symptoms, susceptibility to disease development, or response to anticipated treatment are intended to encompass actions that predict or forecast symptoms, susceptibility, and/or response (including the rate of progression, improvement, and/or duration of symptoms in a subject) (but not treatment or diagnostic actions). Unless otherwise indicated herein or otherwise clearly contradicted by the context, all methods described herein can be performed in any suitable order. The use of any and all instances or exemplary language (e.g., “such”) provided with respect to certain embodiments herein is intended only to better illustrate the invention and does not constitute a limitation on the originally claimed scope of the invention. No language in this specification should be construed as indicating that any unclaimed element is necessary for practicing the invention.

As used herein and throughout the claims, the terms “a,” “an,” and “the” include plural references unless the context clearly indicates otherwise. Furthermore, as used herein, “in” includes both “in” and “on” unless the context clearly indicates otherwise. Also as used herein, and unless the context indicates otherwise, the term “coupled to” is intended to include both direct coupling (where two coupled elements are in contact with each other) and indirect coupling (where at least one additional element is located between the two elements). Therefore, the terms “coupled to” and “coupled with” are used synonymously.

It will be apparent to those skilled in the art that further modifications beyond those already described are possible without departing from the inventive concept described herein. Therefore, the subject matter of this invention is limited only to the scope of the appended claims. Furthermore, in interpreting this specification and the claims, all terms should be interpreted in the broadest possible manner, consistent with the context. In particular, the terms “comprises” and “comprising” should be interpreted as referring to an element, component, or step in a non-exclusive manner, indicating that the mentioned element, component, or step may be present, used, or combined with other elements, components, or steps not explicitly mentioned. Where a claim in this specification refers to at least one of the items selected from the group consisting of A, B, C, ..., and N, that text should be interpreted as requiring only one element from that group, rather than A plus N or B plus N, etc.

Claims (19)

1. A recombinant nucleic acid, said recombinant nucleic acid comprising: The recombinant nucleic acid contains the following sequences: a T7 promoter sequence, a 5' untranslated (5'-UTR) sequence, a signal peptide sequence, a single-chain antibody fragment sequence, a hinge region sequence, a transmembrane domain sequence, and one or more intracellular domain sequences; and the recombinant nucleic acid sequence is as shown in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4. 2. A vector comprising the recombinant nucleic acid as described in claim 1. 3. A primary NK cell, said primary NK cell comprising recombinant nucleic acid, said recombinant nucleic acid encoding: The 5’ untranslated (5’-UTR) sequence portion, the signal peptide sequence portion, the single-chain antibody fragment sequence portion, the hinge region sequence portion, the transmembrane domain sequence portion, and one or more intracellular domain sequence portions; The nucleic acid sequences thereon are operatively linked together as individual polynucleotides; and The recombinant nucleic acid sequence is shown in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4. 4. The primary NK cell of claim 3, wherein the intracellular domain sequence portion comprises a co-stimulatory sequence portion. 5. The primary NK cell of claim 3, wherein the primary NK cell further comprises a 3' untranslated region (3'-UTR). 6. The primary NK cells of claim 3, wherein the recombinant nucleic acid is in a vector. 7. A method for generating CAR-NK cells, the method comprising: transfecting primary NK cells with the recombinant nucleic acid as described in claim 1 or the vector as described in claim 2. 8. A composition comprising primary NK cells as described in any one of claims 3 to 6, and a pharmaceutically acceptable excipient. 9. A kit comprising primary NK cells as described in any one of claims 3 to 6, and instructions for use. 10. Use of primary NK cells as described in any one of claims 3 to 6 in the preparation of a medicament for treating cancer or tumor in a subject, wherein the medicament comprises a therapeutically effective amount of primary NK cells for treating the cancer in the subject or for reducing the size of the tumor in the subject. 11. Use of primary NK cells as described in any one of claims 3 to 6 in the preparation of a medicament for reducing cancer metastasis in a subject, wherein the medicament comprises a therapeutically effective amount of primary NK cells for reducing cancer metastasis in the subject. 12. The use as described in claim 10 or 11, wherein the drug comprises an amount of NK cells sufficient to provide a subject with 1× ^10³ to 1× ^10¹⁰ cells/ ^m² . 13. The use as described in claim 10 or 11, wherein the drug is formulated for administration via parenteral, intravenous, peritumoral, or infusion. 14. The use as described in claim 10 or 11, wherein the medicament further comprises additional therapeutic agents. 15. The use as described in claim 14, wherein the additional therapeutic agent is selected from the group consisting of: viral cancer vaccines, bacterial cancer vaccines, yeast cancer vaccines, N-803, antibodies, stem cell grafts, and tumor-targeting cytokines. 16. The use as described in claim 15, wherein the cancer is selected from leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, chronic leukemia, chronic myeloid (granulocytic) leukemia, chronic lymphocytic leukemia, polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's disease, multiple myeloma, Waldenström macroglobulinemia, heavy chain disease, and solid tumors. 17. The use as described in claim 16, wherein the solid tumor is a sarcoma or carcinoma. 18. The use as described in claim 17, wherein the solid tumor is fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangiolunar endothelial sarcoma, synovial tumor, 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, breast cancer, etc. Cephalic adenocarcinoma, cystic adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, nephroblastoma, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma. 19. A recombinant cell comprising the recombinant nucleic acid of claim 1 or the vector of claim 2, wherein the cell is a bacterial cell, or wherein the cell is an autologous NK cell, or wherein the NK cell is genetically modified.