Generation of chimeric antigen receptor mRNA molecules for expression in primary NK cells

Abstract

This invention provides a novel chimeric antigen (CAR) mRNA molecule, a method for generating this molecule, and a method for treating cancer with this molecule. [Solution] A chimeric antigen receptor (CAR) is provided, comprising a T7 promoter, a spacer sequence, a signal peptide, an antigen-binding domain, a hinge region, a transmembrane (TM) domain, and an intracellular domain, wherein the signal peptide comprises differentiation antigen group 64 (CD64) and / or IgG heavy chain variable gene (IgGHv) signal peptide; and the antigen-binding domain binds to an antigen selected from the group consisting of differentiation antigen group 19 (CD19), B cell maturation antigen (BCMA), B7 homolog 4 (B74), SARS-CoV-2 spike, and differentiation antigen group 30α (CD30α).

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 224,100, filed on 21 July 2021 under 119(e) of the U.S. Patent Act. The entire disclosure of U.S. Provisional Patent Application No. 63 / 224,100 is incorporated herein by reference.

[0002] Sequence listing reference This application includes a sequence listing electronically submitted as a text file via EFS-Web. The XML file, named "8774-19-PCT," has a byte size of 154,000 bytes and was recorded on July 11, 2021. The information contained in the XML file is incorporated herein by reference in its entirety in accordance with Section 1.52(e)(5) of the U.S. Patent Law Enforcement Rules (37 CFR). [Background technology]

[0003] Natural killer (NK) cells are the primary mediators of the innate immune system. NK cells can rapidly detect and destroy abnormal cells (such as cancer cells or virus-infected cells) without requiring prior sensitization or HLA matching. The use of immune cells (including NK cells) to treat cancer is a recent trend. This new therapy is expected to be promising for treating tumors that are refractory to conventional surgery, chemotherapy, and radiation therapy.

[0004] Chimeric antigen receptors (CARs) are engineered proteins composed of an extracellular receptor region fused to an intracellular signaling region. Typically, these regions originate from different proteins, but they can also be designed de novo. CAR-expressing T cells utilize a single-stranded variable fragment (scFV), usually the zeta chain of CD3 (CD3ζ), fused to the intracellular signaling domain. Further developments involve the inclusion of secondary costimulatory signals such as CD28 and CD137 to enhance T cell activation. CAR constructs have also been applied to NK cells, particularly in the use of NK-92 CD19-CAR expression for the treatment of CD19+ B-cell tumors, which are also treated with T cell CD19-CARs.

[0005] The inventors have found that while many CAR molecules can be expressed in NK cell lines, they cannot be expressed as either DNA or RNA within primary NK cells. Most CAR technologies use viral vectors for the delivery of DNA molecules into cells. Viral DNA can enter the nucleus and integrate into the host genome, preferentially at transcriptionally active sites. As disclosed herein, the inventors have identified novel combinations of specific domains and stabilizing elements for high expression of CAR RNA molecules within NK cells. [Overview of the project] [Means for solving the problem]

[0006] A chimeric antigen receptor (CAR) comprising a T7 promoter, a spacer sequence, a signal peptide, an antigen-binding domain, a hinge region, a transmembrane (TM) domain, and an intracellular domain is disclosed herein, wherein the signal peptide comprises differentiation antigen group 64 (CD64) and / or IgG heavy chain variable gene (IgGHv) signal peptide; and the antigen-binding domain binds to an antigen selected from the group consisting of differentiation antigen group 19 (CD19), B cell maturation antigen (BCMA), B7 homolog 4 (B7H4), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike, and differentiation antigen group 30α (CD30α).

[0007] In one aspect, the signal peptide comprises SEQ ID NO: 1 or SEQ ID NO: 2.

[0008] In one aspect, the antigen-binding domain that binds to CD19 comprises at least 90%, at least 95% or up to 100% sequence identity to SEQ ID NO: 4.

[0009] In yet another aspect, the antigen-binding domain that binds to BCMA comprises at least 90%, at least 95% or up to 100% sequence identity to SEQ ID NO: 5 or SEQ ID NO: 6.

[0010] In yet another aspect, the antigen-binding domain that binds to B7H4 comprises at least 90%, at least 95% or up to 100% sequence identity to SEQ ID NO: 7.

[0011] In one aspect, the antigen-binding domain that binds to SARS-CoV-2 spike comprises at least 90%, at least 95% or up to 100% sequence identity to SEQ ID NO: 8.

[0012] In one aspect, the antigen-binding domain that binds to CD30α spike comprises at least 90%, at least 95% or up to 100% sequence identity to SEQ ID NO: 57.

[0013] In one aspect, the hinge region is the hinge region of cluster of differentiation 28 (CD28) having SEQ ID NO: 9.

[0014] In one aspect, the TM domain is the CD28 TM domain having SEQ ID NO: 10.

[0015] In one aspect, the co-stimulatory domain comprises the CD28 cytoplasmic domain having SEQ ID NO: 11.

[0016] In one aspect, the intracellular signaling domain comprises the cytoplasmic domain of cluster of differentiation 3ζ (CD3ζ) having SEQ ID NO: 12.

[0017] In yet another embodiment, the CAR comprises an amino acid sequence having at least 90%, at least 95%, or up to 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 15, 16, 17, 18, 19, 20, 21, 22, and 58.

[0018] Disclosed herein are nucleic acid constructs encoding chimeric antigen receptors (CARs), wherein the antigen-binding domain of the CAR binds to an antigen selected from the group consisting of differentiation antigen group 19 (CD19), B cell maturation antigen (BCMA), B7 homolog 4 (B7H4), SARS-CoV-2 spike, and CD30α. In one embodiment, the nucleic acid construct includes a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 24, 25, 26, 27, 28, 29, 30, 31, 32, and 59.

[0019] Disclosed herein are expression vectors encoding a chimeric antigen receptor (CAR), wherein the antigen-binding domain of the CAR binds to an antigen selected from the group consisting of B cell maturation antigen (BCMA), B7 homolog 4 (B7H4), SARS-CoV-2 spike, and CD30α. In one embodiment, the expression vector has a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 33, 34, 35, 36, 37, 38, 39, 40, 41, and 42.

[0020] Primary natural killer (NK) cells modified with an RNA molecule comprising one or more nucleic acids of a T7 promoter, spacer sequence, signal peptide sequence portion, antigen-binding domain sequence portion, hinge region sequence portion, transmembrane (TM) domain sequence portion, and intracellular domain sequence portion, wherein the signal peptide sequence comprises a sequence encoding differentiation antigen group 64 (CD64) and / or IgG heavy chain variable gene (IgGHv); the antigen-binding domain comprises a sequence encoding an antigen-binding portion that binds to an antigen selected from the group consisting of differentiation antigen group 19 (CD19), B cell maturation antigen (BCMA), B7 homolog 4 (B7H4), SARS-CoV-2 spike, and differentiation antigen group 30α (CD30α); and the nucleic acid sequences are operably linked to one another as a single polynucleotide, are further disclosed herein.

[0021] In one embodiment of modified primary NK cells, the intracellular domain sequence portion includes a CD28 cytoplasmic domain having SEQ ID NO: 11 and / or a differentiation antigen group 3ζ(CD3ζ) cytoplasmic domain having SEQ ID NO: 12. In one embodiment, the modified primary NK cells further include a 3'-UTR. In yet another embodiment, the modified primary NK cells further include a poly-A sequence portion.

[0022] A method for generating modified primary CAR-NK cells is also disclosed herein, which includes transfecting primary NK cells with recombinant nucleic acid constructs disclosed herein.

[0023] Also disclosed herein is an immunotherapy method for treating cancer in a subject in need thereof, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising recombinant NK cells disclosed herein to the subject. In one embodiment, cancer is 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, but not limited to, sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endosarcoma, lymphangiosarcoma, lymphangioendothelioma, synoviomas, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma The group is selected 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.

[0024] Modified NK cells disclosed herein for use in the treatment of cancer are also disclosed herein. In one embodiment, cancer is 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, but not limited to, sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endosarcoma, lymphangiosarcoma, lymphangioendothelioma, synoviomas, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma The group is selected 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.

[0025] Modified NK cells for use as pharmaceuticals are also disclosed herein.

[0026] A pharmaceutical composition comprising genetically modified NK cells disclosed herein and a pharmaceutically acceptable carrier is further disclosed herein. [Brief explanation of the drawing]

[0027] [Figure 1] This is a schematic diagram of a construct for generating the ACE2 CAR molecule (pNKW97). Using the extracellular domain of the ACE2 protein, a CAR molecule with a CD64 signal peptide and a 150-bp poly-A tail was generated. A spacer sequence containing Sequence ID No. 3 was used in this construct. [Figure 2A]This study demonstrates the expression of NKW97-150A (XL53-ACE2-extracellular domain) in cytokine-enriched natural killer (CENK) cells. CENK cells were transfected with pNKW97 RNA using electroporation. After overnight recovery, ACE2 expression was detected using flow cytometry and a conjugated ACE2 antibody. Specificity of the ACE2 antibody was confirmed using isotype controls. As shown, both expression and cell viability are very good for this construct. [Figure 2B] This study demonstrates the expression of NKW97-150A (XL53-ACE2-extracellular domain) in cytokine-enriched natural killer (CENK) cells. CENK cells were transfected with pNKW97 RNA using electroporation. After overnight recovery, ACE2 expression was detected using flow cytometry and a conjugated ACE2 antibody. Specificity of the ACE2 antibody was confirmed using isotype controls. As shown, both expression and cell viability are very good for this construct. [Figure 2C] This study demonstrates the expression of NKW97-150A (XL53-ACE2-extracellular domain) in cytokine-enriched natural killer (CENK) cells. CENK cells were transfected with pNKW97 RNA using electroporation. After overnight recovery, ACE2 expression was detected using flow cytometry and a conjugated ACE2 antibody. Specificity of the ACE2 antibody was confirmed using isotype controls. As shown, both expression and cell viability are very good for this construct. [Figure 2D] This study demonstrates the expression of NKW97-150A (XL53-ACE2-extracellular domain) in cytokine-enriched natural killer (CENK) cells. CENK cells were transfected with pNKW97 RNA using electroporation. After overnight recovery, ACE2 expression was detected using flow cytometry and a conjugated ACE2 antibody. Specificity of the ACE2 antibody was confirmed using isotype controls. As shown, both expression and cell viability are very good for this construct. [Figure 3] The plasmid map of pNKW97 is shown. [Figure 4] This is a schematic diagram of two constructs for generating CAR molecules targeting the B7H4 antigen (pNKW92-93). Two forms of monopeptide B7H4 CARs were designed with different signal peptides (CD64 or IgGHv). A spacer sequence with SEQ ID NO: 3 was used in the pNKW92 construct; a spacer sequence with SEQ ID NO: 60 was used in the pNKW93 construct. [Figure 5A] This study demonstrates the expression of the CD64 or IgGHv B7H4 VH-VL 105A (NKW92, NKW93) CAR molecule in CENK cells. CENK cells were electroporated with pNKW92 or pNKW93 mRNA. After 24 hours, B7H4 CAR expression was detected using flow and biotinylated B7H4, followed by streptavidin-APC. Both constructs showed good expression of the B7H4 CAR. [Figure 5B] This study demonstrates the expression of the CD64 or IgGHv B7H4 VH-VL 105A (NKW92, NKW93) CAR molecule in CENK cells. CENK cells were electroporated with pNKW92 or pNKW93 mRNA. After 24 hours, B7H4 CAR expression was detected using flow and biotinylated B7H4, followed by streptavidin-APC. Both constructs showed good expression of the B7H4 CAR. [Figure 5C] This study demonstrates the expression of the CD64 or IgGHv B7H4 VH-VL 105A (NKW92, NKW93) CAR molecule in CENK cells. CENK cells were electroporated with pNKW92 or pNKW93 mRNA. After 24 hours, B7H4 CAR expression was detected using flow and biotinylated B7H4, followed by streptavidin-APC. Both constructs showed good expression of the B7H4 CAR. [Figure 5D]This study demonstrates the expression of the CD64 or IgGHv B7H4 VH-VL 105A (NKW92, NKW93) CAR molecule in CENK cells. CENK cells were electroporated with pNKW92 or pNKW93 mRNA. After 24 hours, B7H4 CAR expression was detected using flow and biotinylated B7H4, followed by streptavidin-APC. Both constructs showed good expression of the B7H4 CAR. [Figure 5E] This study demonstrates the expression of the CD64 or IgGHv B7H4 VH-VL 105A (NKW92, NKW93) CAR molecule in CENK cells. CENK cells were electroporated with pNKW92 or pNKW93 mRNA. After 24 hours, B7H4 CAR expression was detected using flow and biotinylated B7H4, followed by streptavidin-APC. Both constructs showed good expression of the B7H4 CAR. [Figure 5F] This study demonstrates the expression of the CD64 or IgGHv B7H4 VH-VL 105A (NKW92, NKW93) CAR molecule in CENK cells. CENK cells were electroporated with pNKW92 or pNKW93 mRNA. After 24 hours, B7H4 CAR expression was detected using flow and biotinylated B7H4, followed by streptavidin-APC. Both constructs showed good expression of the B7H4 CAR. [Figure 6] The plasmid map of pNKW92 is shown. [Figure 7] The plasmid map of pNKW93 is shown. [Figure 8A] This is a schematic diagram of four constructs for generating CAR molecules targeting BCMA antigens (pNKW88-91). Four forms of monopeptide BCMA CARs were designed with different signal peptides (CD64 or IgGHv) and variable heavy and light chain sequences. A spacer sequence with SEQ ID NO: 3 was used in the pNKW89 and pNKW91 constructs; a spacer sequence with SEQ ID NO: 60 was used in the pNKW88 and pNKW90 constructs. [Figure 8B]This is a schematic diagram of four constructs for generating CAR molecules targeting BCMA antigens (pNKW88-91). Four forms of monopeptide BCMA CARs were designed with different signal peptides (CD64 or IgGHv) and variable heavy and light chain sequences. A spacer sequence with SEQ ID NO: 3 was used in the pNKW89 and pNKW91 constructs; a spacer sequence with SEQ ID NO: 60 was used in the pNKW88 and pNKW90 constructs. [Figure 9A] This report shows BCMA CAR mRNA expression in CENK cells. Four forms of monocistronic BCMA CAR were designed with different signal peptides (CD64 or IgGHv) and variable heavy and light chain order. CENK cells were electroporated using mRNA from each construct. CAR BCMA expression was detected 24 hours after electroporation using biotinylated BCMA followed by a streptavidin molecule conjugated with APC. As shown, all four constructs exhibit high levels of BCMA CAR. [Figure 9B] This report shows BCMA CAR mRNA expression in CENK cells. Four forms of monocistronic BCMA CAR were designed with different signal peptides (CD64 or IgGHv) and variable heavy and light chain order. CENK cells were electroporated using mRNA from each construct. CAR BCMA expression was detected 24 hours after electroporation using biotinylated BCMA followed by a streptavidin molecule conjugated with APC. As shown, all four constructs exhibit high levels of BCMA CAR. [Figure 9C]This report shows BCMA CAR mRNA expression in CENK cells. Four forms of monocistronic BCMA CAR were designed with different signal peptides (CD64 or IgGHv) and variable heavy and light chain order. CENK cells were electroporated using mRNA from each construct. CAR BCMA expression was detected 24 hours after electroporation using biotinylated BCMA followed by a streptavidin molecule conjugated with APC. As shown, all four constructs exhibit high levels of BCMA CAR. [Figure 9D] This report shows BCMA CAR mRNA expression in CENK cells. Four forms of monocistronic BCMA CAR were designed with different signal peptides (CD64 or IgGHv) and variable heavy and light chain order. CENK cells were electroporated using mRNA from each construct. CAR BCMA expression was detected 24 hours after electroporation using biotinylated BCMA followed by a streptavidin molecule conjugated with APC. As shown, all four constructs exhibit high levels of BCMA CAR. [Figure 9E] This report shows BCMA CAR mRNA expression in CENK cells. Four forms of monocistronic BCMA CAR were designed with different signal peptides (CD64 or IgGHv) and variable heavy and light chain order. CENK cells were electroporated using mRNA from each construct. CAR BCMA expression was detected 24 hours after electroporation using biotinylated BCMA followed by a streptavidin molecule conjugated with APC. As shown, all four constructs exhibit high levels of BCMA CAR. [Figure 10]This study demonstrates the cytotoxicity of CENK cells transfected with CD64-BCMA VL-VH 150A(NKW89)CAR against SUP-B15 BCMA targets and SUP-B15 parent cells. CENK cells were electroporated using mRNA from pNKW89. After 24 hours, the cytotoxicity of CENK-transfected cells against SUP-B15 BCMA targets and control SUP-B15 parent cells was tested using a calcein AM assay. As shown, NKW89-transfected cells exhibit specific cytotoxic activity against SUP-B15 BCMA but no activity against SUP-B15 parent cells. Control untransfected CENK cells show no cytotoxic activity against either SUP-B15 BCMA or parent cells. [Figure 11] The plasmid map of pNKW88 is shown. [Figure 12] The plasmid map of pNKW89 is shown. [Figure 13] The plasmid map of pNKW90 is shown. [Figure 14] The plasmid map of pNKW91 is shown. [Figure 15A] The results of electroporation of PB-NK cells with tripeptide CD19 CAR mRNA are shown. A stable cell line stably expressing CD19 CAR was used as a positive control for CD19 CAR detection. Both GFP and PDL1 CAR mRNA were used as positive controls for electroporation. As shown, tripeptide CAR cannot be expressed as an RNA molecule in PB-NK cells after electroporation. [Figure 15B] The results of electroporation of PB-NK cells with tripeptide CD19 CAR mRNA are shown. A stable cell line stably expressing CD19 CAR was used as a positive control for CD19 CAR detection. Both GFP and PDL1 CAR mRNA were used as positive controls for electroporation. As shown, tripeptide CAR cannot be expressed as an RNA molecule in PB-NK cells after electroporation. [Figure 15C] The results of electroporation of PB-NK cells with tripeptide CD19 CAR mRNA are shown. A stable cell line stably expressing CD19 CAR was used as a positive control for CD19 CAR detection. Both GFP and PDL1 CAR mRNA were used as positive controls for electroporation. As shown, tripeptide CAR cannot be expressed as an RNA molecule in PB-NK cells after electroporation. [Figure 16A] The results for stem memory T cells (Tscm cells) electroporated with tripeptide CD19 CAR mRNA are shown. Tscm cells were electroporated with tripeptide CD19 CAR mRNA using three different electroporation protocols (E1-E3 using increasing electrical pulses). As shown, the tripeptide CAR cannot be expressed as an RNA molecule in memory T cells after electroporation. [Figure 16B] The results for stem memory T cells (Tscm cells) electroporated with tripeptide CD19 CAR mRNA are shown. Tscm cells were electroporated with tripeptide CD19 CAR mRNA using three different electroporation protocols (E1-E3 using increasing electrical pulses). As shown, the tripeptide CAR cannot be expressed as an RNA molecule in memory T cells after electroporation. [Figure 16C] The results for stem memory T cells (Tscm cells) electroporated with tripeptide CD19 CAR mRNA are shown. Tscm cells were electroporated with tripeptide CD19 CAR mRNA using three different electroporation protocols (E1-E3 using increasing electrical pulses). As shown, the tripeptide CAR cannot be expressed as an RNA molecule in memory T cells after electroporation. [Figure 16D]The results for stem memory T cells (Tscm cells) electroporated with tripeptide CD19 CAR mRNA are shown. Tscm cells were electroporated with tripeptide CD19 CAR mRNA using three different electroporation protocols (E1-E3 using increasing electrical pulses). As shown, the tripeptide CAR cannot be expressed as an RNA molecule in memory T cells after electroporation. [Figure 17] These are schematic diagrams of two constructs for generating CAR molecules targeting the CD19 antigen (pNKW87-59). Monopeptide CD19 constructs containing either CD64 or an IgGHv signal peptide were designed to improve the expression of CD19 CAR mRNA molecules in primary NK cells (also known herein as PB-NK cells) and stem memory T cells (Tscm cells). A 150-nucleotide poly(A) tail was added to the end of each molecule to improve mRNA stability. Spacer sequences containing Sequence ID No. 3 were used in these constructs. [Figure 18A] This study demonstrates the expression of the CD19 CAR molecule in PB-NK cells. To improve the expression of the CD19 CAR mRNA molecule in primary NK cells, monopeptide CD19 constructs containing either the CD64 (pNKW87) or IgGHv (pNKW59) signal peptide were designed. Using template DNA, in vitro transcribed mRNA molecules were generated for use in electroporation of PB-NK cells. As shown, both constructs exhibited over 94% CD19 CAR expression 24 hours after electroporation. [Figure 18B]This study demonstrates the expression of the CD19 CAR molecule in PB-NK cells. To improve the expression of the CD19 CAR mRNA molecule in primary NK cells, monopeptide CD19 constructs containing either the CD64 (pNKW87) or IgGHv (pNKW59) signal peptide were designed. Using template DNA, in vitro transcribed mRNA molecules were generated for use in electroporation of PB-NK cells. As shown, both constructs exhibited over 94% CD19 CAR expression 24 hours after electroporation. [Figure 18C] This study demonstrates the expression of the CD19 CAR molecule in PB-NK cells. To improve the expression of the CD19 CAR mRNA molecule in primary NK cells, monopeptide CD19 constructs containing either the CD64 (pNKW87) or IgGHv (pNKW59) signal peptide were designed. Using template DNA, in vitro transcribed mRNA molecules were generated for use in electroporation of PB-NK cells. As shown, both constructs exhibited over 94% CD19 CAR expression 24 hours after electroporation. [Figure 18D] This study demonstrates the expression of the CD19 CAR molecule in PB-NK cells. To improve the expression of the CD19 CAR mRNA molecule in primary NK cells, monopeptide CD19 constructs containing either the CD64 (pNKW87) or IgGHv (pNKW59) signal peptide were designed. Using template DNA, in vitro transcribed mRNA molecules were generated for use in electroporation of PB-NK cells. As shown, both constructs exhibited over 94% CD19 CAR expression 24 hours after electroporation. [Figure 18E]This study demonstrates the expression of the CD19 CAR molecule in PB-NK cells. To improve the expression of the CD19 CAR mRNA molecule in primary NK cells, monopeptide CD19 constructs containing either the CD64 (pNKW87) or IgGHv (pNKW59) signal peptide were designed. Using template DNA, in vitro transcribed mRNA molecules were generated for use in electroporation of PB-NK cells. As shown, both constructs exhibited over 94% CD19 CAR expression 24 hours after electroporation. [Figure 18F] This study demonstrates the expression of the CD19 CAR molecule in PB-NK cells. To improve the expression of the CD19 CAR mRNA molecule in primary NK cells, monopeptide CD19 constructs containing either the CD64 (pNKW87) or IgGHv (pNKW59) signal peptide were designed. Using template DNA, in vitro transcribed mRNA molecules were generated for use in electroporation of PB-NK cells. As shown, both constructs exhibited over 94% CD19 CAR expression 24 hours after electroporation. [Figure 19A-B] This study demonstrates the cytotoxic activity of PB-NK cells transfected with CD19 CARs in CD-19-positive, SUP-B15 parental target cell lines after mRNA electroporation. The cytotoxic activity of CD19 CAR-transfected PB-NK cells was determined 24 hours after electroporation using CD19-positive (SUP-B15 parental) and CD19-negative (SUP-B15 mutant) cell lines. As shown, both constructs containing either CD64 (pNKW87) or IgGHv (pNKW59) signal peptides exhibit specific cytotoxicity against CD19-positive cell lines. [Figure 20A-B]This study demonstrates the cytotoxic activity of the CD19 CAR molecule in memory (PB-NK CIML) and control (PB-NK) cells after mRNA transfection. The cytotoxic activity of memory (PB-NK CIML) or control (PB-NK) cells transfected with CD19 CAR was determined 24 hours after electroporation using CD19-positive (SUP-B15 parent) and CD19-negative (SUP-B15 mutant) cell lines. As shown, PB-NK CIML cells transfected with CD19-CAR exhibit comparable cytotoxic activity to control PB-NK cells transfected with CD19-CAR. This further facilitates the specific targeting of CIML cells by CD19-positive tumor cells. [Figure 21A] The results of monitoring CD19 CAR expression 24–72 hours after electroporation of activated T cells with CD19 CAR constructs are shown. To improve the expression of CD19 CAR mRNA molecules in primary lymphocytes, monopeptide CD19 constructs containing either the CD64 (pNKW87 – Figures 21B and 21C) or IgGH (pNKW59 – Figures 21D and 21E) signal peptide were designed. The template DNA was used as a template for in vitro transcribed mRNA molecules, which were further used for electroporation of primary cells. [Figure 21B] The results of monitoring CD19 CAR expression 24–72 hours after electroporation of activated T cells with CD19 CAR constructs are shown. To improve the expression of CD19 CAR mRNA molecules in primary lymphocytes, monopeptide CD19 constructs containing either the CD64 (pNKW87 – Figures 21B and 21C) or IgGH (pNKW59 – Figures 21D and 21E) signal peptide were designed. The template DNA was used as a template for in vitro transcribed mRNA molecules, which were further used for electroporation of primary cells. [Figure 21C]The results of monitoring CD19 CAR expression 24–72 hours after electroporation of activated T cells with CD19 CAR constructs are shown. To improve the expression of CD19 CAR mRNA molecules in primary lymphocytes, monopeptide CD19 constructs containing either the CD64 (pNKW87 – Figures 21B and 21C) or IgGH (pNKW59 – Figures 21D and 21E) signal peptide were designed. The template DNA was used as a template for in vitro transcribed mRNA molecules, which were further used for electroporation of primary cells. [Figure 21D] The results of monitoring CD19 CAR expression 24–72 hours after electroporation of activated T cells with CD19 CAR constructs are shown. To improve the expression of CD19 CAR mRNA molecules in primary lymphocytes, monopeptide CD19 constructs containing either the CD64 (pNKW87 – Figures 21B and 21C) or IgGH (pNKW59 – Figures 21D and 21E) signal peptide were designed. The template DNA was used as a template for in vitro transcribed mRNA molecules, which were further used for electroporation of primary cells. [Figure 21E] The results of monitoring CD19 CAR expression 24–72 hours after electroporation of activated T cells with CD19 CAR constructs are shown. To improve the expression of CD19 CAR mRNA molecules in primary lymphocytes, monopeptide CD19 constructs containing either the CD64 (pNKW87 – Figures 21B and 21C) or IgGH (pNKW59 – Figures 21D and 21E) signal peptide were designed. The template DNA was used as a template for in vitro transcribed mRNA molecules, which were further used for electroporation of primary cells. [Figure 22A]This paper shows the results of monitoring CD19 expression 24–72 hours after electroporation of Tscm cells using CD19 CAR constructs. To improve the expression of CD19 CAR mRNA molecules in memory T cells, monocistronic CD19 constructs were designed containing either the CD64 (pNKW87 – Figures 22B and 22C) or IgGHv (pNKW59 – Figures 22D and 22E) signal peptide. The template DNA was used as a template for in vitro transcribed mRNA molecules, which were further used for electroporation of Tscm cells. As shown, both constructs exhibit high levels of CD19 CAR expression even 72 hours after electroporation. [Figure 22B] This paper shows the results of monitoring CD19 expression 24–72 hours after electroporation of Tscm cells using CD19 CAR constructs. To improve the expression of CD19 CAR mRNA molecules in memory T cells, monocistronic CD19 constructs were designed containing either the CD64 (pNKW87 – Figures 22B and 22C) or IgGHv (pNKW59 – Figures 22D and 22E) signal peptide. The template DNA was used as a template for in vitro transcribed mRNA molecules, which were further used for electroporation of Tscm cells. As shown, both constructs exhibit high levels of CD19 CAR expression even 72 hours after electroporation. [Figure 22C] This paper shows the results of monitoring CD19 expression 24–72 hours after electroporation of Tscm cells using CD19 CAR constructs. To improve the expression of CD19 CAR mRNA molecules in memory T cells, monocistronic CD19 constructs were designed containing either the CD64 (pNKW87 – Figures 22B and 22C) or IgGHv (pNKW59 – Figures 22D and 22E) signal peptide. The template DNA was used as a template for in vitro transcribed mRNA molecules, which were further used for electroporation of Tscm cells. As shown, both constructs exhibit high levels of CD19 CAR expression even 72 hours after electroporation. [Figure 22D] This paper shows the results of monitoring CD19 expression 24–72 hours after electroporation of Tscm cells using CD19 CAR constructs. To improve the expression of CD19 CAR mRNA molecules in memory T cells, monocistronic CD19 constructs were designed containing either the CD64 (pNKW87 – Figures 22B and 22C) or IgGHv (pNKW59 – Figures 22D and 22E) signal peptide. The template DNA was used as a template for in vitro transcribed mRNA molecules, which were further used for electroporation of Tscm cells. As shown, both constructs exhibit high levels of CD19 CAR expression even 72 hours after electroporation. [Figure 22E] This paper shows the results of monitoring CD19 expression 24–72 hours after electroporation of Tscm cells using CD19 CAR constructs. To improve the expression of CD19 CAR mRNA molecules in memory T cells, monocistronic CD19 constructs were designed containing either the CD64 (pNKW87 – Figures 22B and 22C) or IgGHv (pNKW59 – Figures 22D and 22E) signal peptide. The template DNA was used as a template for in vitro transcribed mRNA molecules, which were further used for electroporation of Tscm cells. As shown, both constructs exhibit high levels of CD19 CAR expression even 72 hours after electroporation. [Figure 23] The plasmid map of pNKW59 is shown. [Figure 24] The plasmid map for pNKW87 is shown. [Figure 25] This is a schematic diagram of a construct for generating a CAR molecule targeting the CD30 alpha (CD30α) antigen (pNKW95). A monocistronic CD30α CAR containing a CD64 signal peptide and a 150-bp poly-A tail was designed. A spacer sequence with SEQ ID NO: 3 was used in the construct. [Figure 26A]This study shows the expression of α-CD30 CD64-VL-VH CAR (NKW95-150A) in CENK cells. CENK cells were transfected with pXL46 (short-chain poly(A)) or pNKW95 RNA (150A) using electroporation. After overnight recovery, CD30α CAR expression was detected by flow cytometry and biotinylated CD30, followed by streptavidin-APC. The pNKW95 construct showed higher expression compared to the parent pXL46. [Figure 26B] This study shows the expression of α-CD30 CD64-VL-VH CAR (NKW95-150A) in CENK cells. CENK cells were transfected with pXL46 (short-chain poly(A)) or pNKW95 RNA (150A) using electroporation. After overnight recovery, CD30α CAR expression was detected by flow cytometry and biotinylated CD30, followed by streptavidin-APC. The pNKW95 construct showed higher expression compared to the parent pXL46. [Figure 26C] This study shows the expression of α-CD30 CD64-VL-VH CAR (NKW95-150A) in CENK cells. CENK cells were transfected with pXL46 (short-chain poly(A)) or pNKW95 RNA (150A) using electroporation. After overnight recovery, CD30α CAR expression was detected by flow cytometry and biotinylated CD30, followed by streptavidin-APC. The pNKW95 construct showed higher expression compared to the parent pXL46. [Figure 26D] This study shows the expression of α-CD30 CD64-VL-VH CAR (NKW95-150A) in CENK cells. CENK cells were transfected with pXL46 (short-chain poly(A)) or pNKW95 RNA (150A) using electroporation. After overnight recovery, CD30α CAR expression was detected by flow cytometry and biotinylated CD30, followed by streptavidin-APC. The pNKW95 construct showed higher expression compared to the parent pXL46. [Figure 26E]This study shows the expression of α-CD30 CD64-VL-VH CAR (NKW95-150A) in CENK cells. CENK cells were transfected with pXL46 (short-chain poly(A)) or pNKW95 RNA (150A) using electroporation. After overnight recovery, CD30α CAR expression was detected by flow cytometry and biotinylated CD30, followed by streptavidin-APC. The pNKW95 construct showed higher expression compared to the parent pXL46. [Figure 26F] This study shows the expression of α-CD30 CD64-VL-VH CAR (NKW95-150A) in CENK cells. CENK cells were transfected with pXL46 (short-chain poly(A)) or pNKW95 RNA (150A) using electroporation. After overnight recovery, CD30α CAR expression was detected by flow cytometry and biotinylated CD30, followed by streptavidin-APC. The pNKW95 construct showed higher expression compared to the parent pXL46. [Figure 27] The plasmid map of pNKW95 is shown. [Modes for carrying out the invention]

[0028] Unless otherwise defined herein, scientific and technical terms used in this application have meanings that are generally understood by those skilled in the art. In general, nomenclature and techniques used in relation to chemistry, molecular biology, cell and cancer biology, immunology, microbiology, pharmacology and protein and nucleic acid chemistry are well known and commonly used in the art.

[0029] All publications, patents, and published patent applications referenced herein are incorporated herein by reference. In case of any conflict, this specification, including its specific definitions, shall prevail.

[0030] As disclosed herein, the inventors have determined novel combinations of specific domains and stabilizing elements for high expression of chimeric antigen mRNA molecules in primary NK cells. In particular, as disclosed herein, the novel combinations include an extracellular domain comprising a signal peptide, antigen-binding domain, hinge, and transmembrane (TM), and an intracellular domain comprising at least one costimulatory domain and an intracellular signaling domain. Furthermore, the CAR has modifications in the 3' untranslated region (UTR) for optimal expression of RNA transcribed in vitro in NK cells. Our initial results have shown that many CAR molecules readily expressed in NK cell lines exhibit minimal or no expression after in vitro transcription and electroporation in primary NK cells. Most CAR technologies use viral vectors for delivery of DNA molecules into cells. Viral DNA can enter the nucleus and integrate into the host genome, preferentially at transcriptionally active sites. As disclosed herein, the inventors have employed an alternative method using mRNA molecules that do not integrate into the genome, thereby avoiding the risks associated with the carcinogenic potential of viral gene delivery. In addition to safety, another advantage of using the CAR mRNA molecules disclosed herein (compared to DNA) is the faster response obtained because the mRNA is rapidly translated upon entry into the cytoplasm. One drawback of mRNA is that it is easily degraded, but the inventors have addressed this drawback by extending the half-life of the mRNA constructs disclosed herein by introducing stabilizing elements to the 5' end of each CAR molecule. As further demonstrated in the figures and examples provided herein, the inventors have also shown successful expression of CAR mRNA in memory NK and T cells.

[0031] The CAR molecules described herein target, but are not limited to, cancer surface markers including CD19, BCMA, and CD30, as well as checkpoint inhibitors, or their ligands including, but are not limited to, B7H4. DNA template vectors are used as templates for the in vitro synthesis of mRNA molecules to be delivered to primary NK cells disclosed herein for immunotherapy in cancer patients. In vitro transcription can be initiated at a promoter, such as the T7 promoter, using bacteriophage T7 RNA polymerase. The T7 promoter is upstream of a spacer sequence (SEQ ID NO: 3 or SEQ ID NO: 60) containing the Kozak sequence (SEQ ID NO: 45) required for translation initiation. A short signal peptide (15-amino acids) from CD64 or IgGHv protein forms 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 CAR binding site is a heterodimer of variable light and heavy chain domains. The two domains are linked to each other via a 20-amino acid (aa) linker. The molecular hinge and TM domains are derived from the CD28 protein. The hinge region provides mobility and flexibility for the binding domain, while the TM region / domain allows for precise intramembrane insertion. The intracellular domain includes at least one co-stimulatory domain and an intracellular signaling domain. The co-stimulatory domain contains the cytoplasmic domain of CD28, while the intracellular signaling domain contains the cytoplasmic domain of CD3ζ. The co-stimulatory and intracellular signaling domains are involved in intracellular signaling pathways that promote cytotoxic activity in transfected cells. The 3'UTR of the construct disclosed herein is a 94-bp sequence from the 3'UTR of the mouse (Mus musculus) hemoglobin α gene, followed by a 150-bp polyA elongation that provides stability to the RNA molecule. Substitutes for the 3'UTR region may be used in the CAR construct. These include, but are not limited to, 3'UTRs derived from human β-globin or 3'UTRs derived from genes highly expressed in NK cells.Examples of genes highly expressed in NK cells include, but are not limited to, innate cytotoxic receptors (NCRs) such as NKp46, NKp30, and NKp44; or c-lectins such as activated immune receptors such as NKG2D and 2B4. 3'UTRs (and their substitutes) can be introduced into CAR constructs to improve the stability of mRNA CAR molecules.

[0032] The constructs disclosed herein are novel in that they possess high binding affinity to specific cancer surface markers, checkpoint inhibitors, and / or their ligands. Furthermore, the constructs contain cytoplasmic domains of CD28 and CD3ζ, which result in enhanced cytotoxic activity against target cells. The constructs are also mRNA-based and therefore there are no concerns regarding the integration of the constructs into the host genome.

[0033] The signal peptide comprises CD64 and / or IgGHv signal peptide. In one embodiment, the signal peptide comprises SEQ ID NO: 1 or SEQ ID NO: 2.

[0034] The antigen-binding domain binds to an antigen selected from the group consisting of CD19, BCMA, B7H4, SARS-CoV-2 spike, and CD30α.

[0035] In one embodiment, the antigen-binding domain that binds to CD19 contains at least 90%, at least 95%, or up to 100% sequence identity with respect to Sequence ID No. 4.

[0036] In yet another embodiment, the antigen-binding domain that binds to BCMA contains at least 90%, at least 95%, or up to 100% sequence identity to SEQ ID NO: 5 or SEQ ID NO: 6.

[0037] In yet another embodiment, the antigen-binding domain that binds to B7H4 contains at least 90%, at least 95%, or up to 100% sequence identity to SEQ ID NO: 7.

[0038] In one embodiment, the antigen-binding domain that binds to the SARS-CoV-2 spike contains at least 90%, at least 95%, or up to 100% sequence identity to SEQ ID NO: 8.

[0039] In one embodiment, the antigen-binding domain that binds to CD30α contains at least 90%, at least 95%, or up to 100% sequence identity with respect to SEQ ID NO: 57.

[0040] In one embodiment, the hinge region is the CD28 hinge region having sequence number 9.

[0041] In one embodiment, the TM domain is a CD28 TM domain having sequence number 10.

[0042] In one embodiment, the co-stimulatory domain includes the CD28 cytoplasmic domain having sequence number 11. In yet another embodiment, the co-stimulatory domain includes 2B4, 4-1BB (also known as CD137 or TNFRS9) and / or OX40. Further co-stimulatory domains may be added to the construct. The order / position of the co-stimulatory domains within the construct may also be rearranged / changed.

[0043] In one embodiment, the intracellular signaling domain includes a CD3ζ cytoplasmic domain having Sequence ID No. 12.

[0044] As disclosed herein, a CAR comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, or up to 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, SEQ ID NOs: 15, SEQ ID NOs: 16, SEQ ID NOs: 17, SEQ ID NOs: 18, SEQ ID NOs: 19, SEQ ID NOs: 20, SEQ ID NOs: 21, SEQ ID NOs: 22, and SEQ ID NOs: 58.

[0045] Another embodiment is a nucleic acid construct encoding a CAR disclosed herein, the CAR comprising an extracellular domain including a signal peptide, an antigen-binding domain, a hinge region and a TM domain; and comprising at least one costimulatory domain and an intracellular signaling domain; the signal peptide comprising CD64 and / or IgGHv signal peptide; and the antigen-binding domain binding to an antigen selected from the group consisting of CD19, BCMA, B7H4, SARS-CoV-2 spike and CD30α and their variants. In one embodiment, the nucleic acid construct comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 24, 25, 26, 27, 28, 29, 30, 31, 32 and 59. In another embodiment, the SARS-CoV-2 antigen refers to the SARS-CoV-2 protein and its variants. Suitable SARS-CoV-2 proteins that can be used as or to generate SARS-CoV-2 antigens include, but are not limited to, main proteases (chain A, also known as 3C-like proteinase or 3C-like proteinase M). PRO Examples include the SARS-CoV-2 nucleocapsid protein (N protein), SARS-CoV-2 membrane protein (M protein), SARS-CoV-2 envelope protein (E protein), and SARS-CoV-2 spike protein (S protein).

[0046] Another embodiment disclosed herein is an expression vector encoding a CAR disclosed herein, the CAR comprising an extracellular domain including a signal peptide, an antigen-binding domain, a hinge region and a TM domain; and an intracellular domain including at least one costimulatory domain and an intracellular signaling domain; the signal peptide comprising CD64 and / or an IgGHv signal peptide; and the antigen-binding domain binding to an antigen selected from the group consisting of CD19, BCMA, B7H4, SARS-CoV-2 spike and CD30α. In one embodiment, the expression vector has a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 33, 34, 35, 36, 37, 38, 39, 40, 41 and 42.

[0047] Further embodiments disclosed herein are modified primary NK cells expressing the CAR disclosed herein, the CAR comprising an extracellular domain including a signal peptide, an antigen-binding domain, a hinge region and a TM domain; and an intracellular domain including at least one costimulatory domain and an intracellular signaling domain; the signal peptide comprising CD64 and / or an IgGHv signal peptide; and the antigen-binding domain binding to an antigen selected from the group consisting of CD19, BCMA, B7H4, SARS-CoV-2 spike and CD30α.

[0048] Another embodiment disclosed herein is a method for producing recombinant NK cells, comprising the step of introducing a CAR mRNA molecule transcribed from an expression vector disclosed herein.

[0049] It is also possible to add other cytokine genes to the same construct (as a bipeptide or tripeptide) that can enhance the activity of RNA CAR molecules. IL-15 is one such cytokine gene.

[0050] To enhance activity, the introduction of domains from activated NK receptors such as NKG2D into the CAR molecules disclosed herein is also possible. NKG2D is a transmembrane protein belonging to the NKG2 family of C-type lectin-like receptors.

[0051] Further embodiments disclosed herein are immunotherapeutic methods for treating cancer in subjects in need. These methods involve administering a pharmaceutical composition comprising recombinant NK cells and a pharmaceutically acceptable carrier to a subject. Another embodiment involves the use of recombinant NK cells and a pharmaceutically acceptable carrier disclosed herein for the treatment of cancer.

[0052] Another embodiment is a pharmaceutical composition comprising modified NK cells as disclosed herein and a pharmaceutically acceptable carrier.

[0053] Throughout this specification, variations such as "comprise," "comprises," or "contains" suggest the inclusion of the integer (or component) or group of integers (or components) described, but not the exclusion of any other integer (or component) or group of integers (or components).

[0054] The singular forms "one (a)", "one (an)", and "that" include plurals unless otherwise specified in the context.

[0055] "To include" means "to include, but not limited to." "To include" and "to include, but not limited to" are used synonymously.

[0056] "Pharmacologically acceptable carrier" refers to a non-toxic carrier that can be administered to a patient (together with the compositions described herein) and does not impair the pharmacological activity of the active agents in the compositions. "Excipient" refers to an additive in a formulation or composition that is not a pharmaceutically active ingredient.

[0057] A "pharmaceutically effective dose" refers to a dose that is effective in treating a patient, for example, that brings about a beneficial and / or desirable change in the overall health of a patient suffering from a disease (including, but not limited to, cancer). Treating includes, but is not limited to, killing cells, preventing the growth of new cells, improving the patient's functional abilities, improving the patient's health, reducing pain, improving appetite, improving the patient's weight, and any combination thereof. A "pharmaceutically effective dose" also refers to the dose necessary to improve the patient's clinical symptoms.

[0058] In this specification, "peptide" and "polypeptide" are used synonymously to refer to polymers constructed from amino acid residues. As used herein, "amino acid residue" refers to any natural amino acid (L or D type), a non-natural amino acid, or an amino acid mimetic (such as a peptide monomer).

[0059] With respect to two or more nucleic acid or polypeptide sequences, “identical” or percentage “identical” means that when compared and aligned against the greatest match on a comparison window, two or more sequences or subsequences are the same or have a certain percentage of the same amino acid residues or nucleotides. The degree of amino acid or nucleic acid sequence identity as used in this disclosure is determined using the BLAST algorithm described in Altschul et al. (1990) J.Mol.Biol.215:403-10. This algorithm identifies high-scoring sequence pairs (HSPS) by identifying short word lengths W in a query sequence that, when aligned with words of the same length in a database sequence, match or satisfy a certain positive threshold score T. T is called the neighbor word score threshold (Altschul et al., (1990) J.Mol.Biol.215:403-10). Initial neighbor word hits serve as a seed to initiate a search for longer HSPs that contain them. Next, word hits are extended in both directions along each sequence as long as the cumulative alignment score can be increased. The cumulative score is calculated for nucleotide sequences using parameters M (reward score for pairs of matching residues; always > 0) and N (penalty score for mismatched residues; always < 0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. The extension of word hits in each direction is stopped when the cumulative alignment score decreases by an amount X from its maximum achieved value; for the accumulation of residue alignments with one or more negative scores, the cumulative score becomes zero or less; or reaches the end of either sequence. To determine the percentage identity of amino acid sequences, the BLASTP settings are word length (W), 3; expected value (E), 10; and the BLOSUM62 scoring matrix. For nucleic acid sequence analysis, the BLASTN program settings are word length (W), 11; expected value (E), 10; M=5; N=-4; and comparison of both strands.The TBLASTN program (which queries nucleotide sequence databases using protein sequences) uses a word length (W) of 3, an expected value (E) of 10, and a BLOSUM 62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).

[0060] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin & Altschul (1993) Proc. Nat'l. Acad. Sci. USA 90:5873-87). The minimum sum probability (P(N)) provides an indication of the probability that the match between two nucleotide or amino acid sequences occurs by chance. For example, if the minimum sum probability in the comparison between the test nucleic acid and the reference nucleic acid is less than approximately 0.01, the nucleic acid is considered similar to the reference sequence.

[0061] The "length" of a polypeptide is the number of end-to-end linked amino acid residues that make up the polypeptide, excluding non-peptide linkers and / or modifications that the polypeptide may contain.

[0062] Hydrophobic amino acid residues are characterized primarily by functional groups ("side chains") with nonpolar chemical properties. Such hydrophobic amino acid residues can be natural (L or D type) or unnatural. Alternatively, hydrophobic amino acid residues can be amino acid mimes characterized primarily by side chains with nonpolar chemical properties. Conversely, hydrophilic amino acid residues are characterized primarily by side chains with polar (charged or uncharged) chemical properties. Such hydrophilic amino acid residues can be natural (L or D type) or unnatural. Alternatively, hydrophilic amino acid residues can be amino acid mimes characterized primarily by side chains with polar (charged or uncharged) chemical properties. Suitable unnatural amino acid residues and amino acid mimes are known in the art. See, for example, Liang et al. (2013) PLoS ONE 8(7):e67844.

[0063] Most amino acid residues can be considered either hydrophobic or hydrophilic, but some can behave either hydrophobicly or hydrophilically depending on the context. For example, glycine, proline, and cysteine ​​can function as hydrophilic amino acid residues depending on the context due to their relatively weak nonpolar properties. Conversely, histidine and arginine can function as hydrophobic amino acid residues depending on the context due to their bulky, slightly hydrophobic side chains.

[0064] Unless otherwise specified, each embodiment disclosed herein may be used alone or in combination with any one or more other embodiments herein.

[0065] "Transfection" refers to the introduction of foreign nucleic acids into eukaryotic cells. Transfection can be achieved by various means known in the art, including electroporation, polymers (nanoparticles), calcium phosphate-DNA coprecipitation, DEAE-dextran mediated transfection, polybren mediated transfection, microinjection, liposome fusion, lipofection, plasmofusion, and biolistec methods.

[0066] "Stable transfection" or "stable transfection" refers to the introduction and integration of foreign nucleic acids or DNA into the genome of transfected cells.

[0067] The term "mutant" refers to a protein or fragment thereof having an amino acid sequence similar to, but not identical to, a reference sequence (e.g., the SARS-CoV-2 protein sequence), where the activity of the mutant protein is substantially unchanged. These changes in sequence may occur spontaneously or they may be manipulated using techniques known to those skilled in the art. Suitable examples of changes include, but are not limited to, amino acid deletions, insertions, substitutions, and combinations thereof.

[0068] Amino acids can be classified into several groups based on their physical properties. Examples of such groups, though not limited to them, include charged amino acids, uncharged amino acids, polar uncharged amino acids, and hydrophobic amino acids. Preferred variants are those in which an amino acid is substituted with an amino acid from the same group. Such substitutions are called conservative substitutions.

[0069] Natural residues can be classified into several types based on their general side-chain properties: 1) Hydrophobic: Met, Ala, Val, Leu, Ile; 2) Neutral hydrophilic: Cys, Ser, Thr; 3) Acidic: Asp, Glu; 4) Basicity: Asn, Gln, His, Lys, Arg; 5) Residues that affect chain orientation: Gly, Pro; and 6) Aromatics: Trp, Tyr, Phe.

[0070] Non-conservative substitutions can involve exchanging a member of one class for a member of another class.

[0071] Methods and uses are also provided for treating or improving the symptoms of cancer and / or treating cancer or tumors in an individual. Methods and / or uses involve administering to a patient in need a therapeutically effective amount of modified NK cells disclosed herein or a composition comprising modified NK cells disclosed herein. The administration is intended to treat cancer, reduce tumor size in the subject, or reduce cancer metastasis in the subject. One embodiment is modified NK cells disclosed herein for use in the treatment of cancer. Yet another embodiment is modified NK cells disclosed herein for use as a pharmaceutical agent.

[0072] 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.

[0073] 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.

[0074] The terms subject, patient, and individual are not intended to be limiting and are generally interchangeable. That is, an individual described as a patient does not necessarily have a given disease 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 limiting. 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.

[0075] References to “treatment” and “prevention” in this specification should be understood in their broadest context. “Treatment” does not necessarily imply that a mammal will be treated to complete recovery. Similarly, “prevention” does not necessarily mean that the subject will not ultimately reduce the condition. The term “prevention” may be understood as reducing the severity of the onset of a particular condition. Treatments may also reduce the severity of an existing condition or the frequency of acute attacks. As used herein, “to treat,” “to cure,” and similar terms mean to stabilize and / or alleviate the symptoms of a disease or condition. In some embodiments, compositions disclosed herein may prevent the onset of a disease or condition, or cure a condition or disease independently of treatment.

[0076] The route, frequency of administration, and dosage of the therapeutic compositions described herein will vary depending on the individual and the disease and can be readily established using standard techniques. In general, pharmaceutical compositions can be administered by injection (e.g., intradermal, intramuscular, intravenous, or subcutaneous), intranasally (e.g., by inhalation), in pill form (e.g., for oral, vaginal, or rectal delivery as a suppository).

[0077] As described herein, the compositions of the present invention are suitable for parenteral administration. These compositions can be administered, for example, intraperitoneally, intravenously, intrathecally, intracranially, intradermally, intramuscularly, intraocularly, intrathecally, intracerebrally, intranasally, transmucosally, by injection, orally, rectally, by intravenous drip, via patches and implants, parenterally, locally, subcutaneously, topically, nasally, orally, sublingually, intraocularly, by implantable depot, by nanoparticle-based delivery systems, microneedle patches, microspheres, beads, osmotic or mechanical pumps, and / or other mechanical means. Optionally, NK cells are administered parenterally. Optionally, NK cells are administered intravenously. Optionally, NK cells are administered peritumorally. One of ordinary skill in the art will understand that the method of administering the compositions of the present invention can depend on factors such as the age, weight, and physical condition of the patient being treated and the disease or condition being treated. Thus, one of ordinary skill in the art will be able to individually select the optimal method of administration for the patient.

[0078] The modified NK cells disclosed herein can be administered to a subject by 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 Any range (including endpoints) between any two of the following NK cell counts (e.g., 1 × 10⁶) may be administered. Optionally, 1 × 10⁶ NK cells may be administered. 8 ~1 × 10 10 Individual cells are administered to the target. Optionally, cells are administered at least once a week for a period of one week or more. Optionally, cells are administered once or twice a week for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks or longer.

[0079] In another embodiment, the total dose is calculated based on the body surface area m². 2 It can also be calculated by injecting approximately 1000 cells / m² into the target. 2 Up to approximately 10 billion cells / injection / m 2 For example, per injection, approximately, at least approximately, or at most approximately 1 × 10 10 pieces / m 2 , 1 x 10 9 pieces / m 2 , 1 x 10 8 pieces / m 2 , 1 x 10 7 pieces / m 2 , 5×10 7 pieces / m 2 , 1 x 10 6 pieces / m 2 , 5×10 6 pieces / m 2 , 1 x 10 5 pieces / m 2 , 5×10 5 pieces / 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.

[0080] 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 comprise 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 may contain about 1% to about 10% human serum or human serum equivalents. Optionally, the culture medium may contain about 1% to about 5% human serum or human serum equivalents. Optionally, the culture medium may contain 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 may be used instead of human serum. Such serum substitutes may be known in the art. Optionally, NK cells are administered in a composition comprising NK cells and an isotonic liquid solution supporting cell viability. Optionally, NK cells are administered in a composition reconstituted from cryopreserved samples.

[0081] In accordance with the methods and uses provided herein, one or more of the agents provided herein, in an effective or therapeutically effective dose, are administered to the subject. The terms effective dose, therapeutically 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 the agent may be determined empirically by those skilled in the art. The dosage range for administration is sufficiently large 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 large as to cause substantial adverse side effects, such as undesirable cross-reactions or anaphylactic reactions. Generally, the dosage may vary depending on age, condition, sex, type of disease, severity of disease or disorder, route of administration, or whether other agents 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, over a day, or over several days in one or more doses. Guidelines for appropriate dosages for a given type of drug can be found in the literature. For example, for a given parameter, an 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, a therapeutically 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 precise dosage 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)).

[0082] A composition suitable for injection comprises a sterile aqueous solution (if water-soluble) and a sterile powder for the immediate preparation of a sterile injection solution. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Prevention of microbial action can be achieved by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. In many cases, it is preferable to include an isotonic agent, such as sugar or sodium chloride. Sustained absorption of the injection composition can be achieved by the use of absorption-delaying agents in the composition, such as aluminum monostearate and gelatin.

[0083] Sterile injectable solutions are prepared by incorporating the required amount of active compound, along with various other components listed above as needed, into a suitable solvent, and then sterilizing by means of filtration or other suitable means, for example. Dispersions can be prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and other necessary components from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods include vacuum drying and freeze-drying techniques to produce a powder of the active ingredient plus any further desired components from a pre-sterilized filtered solution.

[0084] If the active ingredients are suitably protected, they may be administered orally, for example, with an inert diluent or an absorbable edible carrier, or they may be encapsulated in hard or soft-shell gelatin capsules, or they may be compressed into tablets. For oral therapeutic administration, the active compound may be incorporated into an excipient and used in the form of ingestible tablets, oral tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc.

[0085] In connection with any of the methods and uses described above, the composition may be administered in combination with another drug. In any case, the composition may be administered before, concurrently with, or after the administration of the other drug. According to the methods described herein, two or more compounds or compositions may be co-administered with one or more other compounds, such as known chemotherapeutic agents, antiviral compounds or molecules, as well as antibiotics, chloroquine, hydroxychloroquine, known drugs for treating pneumonia, analgesics (such as lidocaine or paracetamol), anti-inflammatory drugs (such as betamethasone, nonsteroidal anti-inflammatory drugs (NSAIDs), acetaminophen, ibuprofen, naproxen), and / or other suitable drugs. The methods provided may be further combined with other oncological treatments such as radiotherapy, surgery, hormone therapy, and / or immunotherapy. Thus, the methods provided may further include administering 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, neuron blockers, 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 can 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.

[0086] In one embodiment, the composition may be administered together with N-803 (also known as "ALT-803"). In one embodiment, N-803 may be administered together with or separately from the pharmaceutical composition comprising the peptide disclosed herein.

[0087] In one embodiment, additional therapeutic substances may be selected from the group consisting of viral cancer vaccines, bacterial cancer vaccines, yeast cancer vaccines, antibodies, stem cell transplants, and tumor-targeting cytokines.

[0088] 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 include, but are not limited to, 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.

[0089] "Co-administration" refers to simultaneous administration of the same formulation or two different formulations via the same or different routes, or consecutive administration via the same or different routes. "Consecutive" administration refers to a time difference of several seconds, minutes, hours, or days between the administration of two or more distinct compounds.

[0090] N-803 is an interleukin-15 (IL-15) superagonist conjugate. In preclinical studies, IL-15 has shown potent antitumor activity against established tumors. Furthermore, N-803 may synergistically enhance the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of therapeutic antibodies, as well as the antitumor activity of checkpoint inhibitors such as anti-PD-1, anti-PD-L1, and anti-CTLA antibodies (Rhode et al., Cancer Immunol Res., 2016).

[0091] A useful concentration of N-803 is a concentration suitable for the subject in question. Those skilled in the art will understand that different individuals may require different total amounts of N-803. In some embodiments, the amount of N-803 is a pharmaceutically effective amount. Those skilled in the art may determine the amount of N-803 in the composition necessary to treat the subject based, for example, on factors such as the subject's age, weight, and physical condition. The pharmaceutically effective amount of N-803 may be approximately 1 pg of compound / kg body weight to approximately 20 μg / kg of compound / kg body weight; or approximately 0.1 μg / kg to 20 μg / kg; or approximately 1 μg / kg body weight to approximately 1 mg of compound / kg body weight or approximately 1 mg / kg body weight to approximately 5000 mg / kg body weight; or approximately 5 mg / kg body weight to approximately 4000 mg / kg body weight or approximately 10 mg / kg body weight to approximately 3000 mg / kg body weight; or approximately 50 mg / kg body weight to approximately 2000 mg / kg body weight; or approximately 100 mg / kg body weight to approximately 1000 mg / kg body weight; or approximately 150 mg / kg body weight to approximately 500 mg / g body weight. Preferably, 100 pk / kg to 20 μg / kg. In other embodiments, the dose may be approximately 0.1, 0.5, 1, 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 mg / kg body weight. In other embodiments, the dose may be in the range of approximately 5 mg of compound / kg body weight to approximately 20 mg of compound / kg body weight. In other embodiments, the dose may be about 8, 10, 12, 14, 16, or 18 mg / kg body weight.

[0092] 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).

[0093] In some embodiments, it may be beneficial to include one or more excipients in the composition. Those skilled in the art will understand that the choice of any one excipient may influence the choice of any other excipients. For example, the choice of a particular excipient may exclude the use of one or more further excipients because the combination of excipients may produce undesirable effects. Those skilled in the art will be able to experimentally determine which excipients (if any) should be included in the formulations or compositions disclosed herein. Excipients may include, but are not limited to, cosolvents, solubilizers, buffers, pH adjusters, bulking agents, surfactants, encapsulating agents, isotonic modifiers, stabilizers, protective agents, and viscosity modifiers. In some embodiments, it may be beneficial to include a pharmaceutically acceptable carrier.

[0094] In certain embodiments, the inclusion of a solubilizer may be beneficial. A solubilizer may be useful in increasing the solubility of any component of the formulation or composition, including peptides or excipients disclosed herein. The solubilizers described herein are not intended to constitute an exhaustive list, but are merely illustrative examples of solubilizers that may be used. In certain embodiments, solubilizers may include, but are not limited to, ethyl alcohol, tert-butyl alcohol, polyethylene glycol, glycerol, methylparaben, propylparaben, polyethylene glycol, polyvinylpyrrolidone, and any pharmaceutically acceptable salts and / or combinations thereof.

[0095] The pH can be any pH that gives the composition desirable properties. Desired properties may include, for example, peptide stability, increased peptide retention compared to compositions at other pH levels, and improved filtration efficiency.

[0096] In some embodiments, it may be beneficial to include an isotonic modifier. The isotonicity of a liquid composition is an important consideration, for example, when administering the composition to a patient by parenteral administration. Therefore, isotonic modifiers may be used to facilitate making the composition suitable for administration. Isotonic modifiers are well known in the art. Therefore, the isotonic modifiers described herein are not intended to constitute an exhaustive list, but are merely listed as exemplary isotonic modifiers that may be used. Isotonic modifiers may be ionic or nonionic and may include, but are not limited to, inorganic salts, amino acids, carbohydrates, sugars, sugar alcohols, and carbohydrates. Exemplary inorganic salts may include sodium chloride, potassium chloride, sodium sulfate, and potassium sulfate. An exemplary amino acid is glycine. Exemplary sugars may include glycerol, propylene glycol, glucose, sucrose, lactose, and sugar alcohols such as mannitol.

[0097] In some embodiments, it may be beneficial to include a stabilizer. The stabilizer helps to enhance the stability of the peptide in the composition of the present invention.

[0098] In some embodiments, the inclusion of a protective agent may be beneficial. A protective agent is an agent that protects a pharmaceutically active ingredient (e.g., a peptide disclosed herein) from undesirable conditions (e.g., instability caused by freezing, lyophilization, or oxidation). Protective agents may include, for example, cryoprotectants, lyophilization protectants, and antioxidants. Cryoprotectants are useful in preventing a decrease in the potency of the pharmaceutically active ingredient (e.g., a peptide disclosed herein) when the formulation is exposed to temperatures below its freezing point. For example, a cryoprotectant may be included in a reconstituted lyophilized formulation so that the formulation can be frozen before dilution for intravenous (IV) administration. Cryoprotectants are well known in the art; therefore, the cryoprotectants described herein are not intended to constitute an exhaustive list, but are merely presented as exemplary cryoprotectants that may be used. Examples of cryoprotectants, but not limited to, include solvents, surfactants, encapsulating agents, stabilizers, viscosity modifiers, and combinations thereof. Freezing agents may include, for example, disaccharides (e.g., sucrose, lactose, maltose, and trehalose), polyols (e.g., glycerol, mannitol, sorbitol, and dulcitol), and glycols (e.g., ethylene glycol, polyethylene glycol, and propylene glycol).

[0099] Lyophilization protectants are useful for stabilizing components of lyophilized formulations or compositions. For example, the peptides disclosed herein may be lyophilized with a lyophilization protectant before reconstitution. Lyophilization protectants are well known in the art. Therefore, the lyophilization protectants described herein are not intended to constitute an exhaustive list, but are merely presented as exemplary lyophilization protectants that may be used. Examples of lyophilization protectants include, but are not limited to, solvents, surfactants, encapsulating agents, stabilizers, viscosity modifiers, and combinations thereof. Exemplary lyophilization protectants may include, for example, sugars and polyols, trehalose, sucrose, dextran, and hydroxypropyl-β-cyclodextrin, which are non-exclusive examples of lyophilization protectants.

[0100] Antioxidants are useful for preventing the oxidation of components in a composition. Oxidation can lead to aggregation of drug products or other adverse effects on the purity or potency of drug products. Antioxidants are well known in the art. Therefore, the antioxidants described herein are not intended to constitute an exhaustive list, but are merely presented as exemplary antioxidants that may be used. Antioxidants may include, for example, sodium ascorbate, citrates, thiols, metabisulfites, and combinations thereof.

[0101] Modifications, alterations, and other implementations of those described herein will be conceivable to those skilled in the art without departing from the spirit and scope of this disclosure. Accordingly, the subsequent claims are not limited to the exemplary descriptions set forth herein.

[0102] Each of the embodiments described herein may be used individually or in combination with one or more other embodiments of the present invention.

[0103] Those skilled in the art will be able to recognize or confirm many equivalents of the compounds, compositions, and methods of use described herein by means of simple routine experiments. Such equivalents are considered to fall within the scope of the compositions and methods disclosed herein.

[0104] All references, patents, and published patent applications and their accompanying drawings cited throughout this application are incorporated herein by reference in their entirety. [Examples]

[0105] The following examples are intended to provide those skilled in the art with a complete disclosure and explanation of how to prepare and use the embodiments, and are not intended to limit the scope of what the inventors consider to be the invention, nor to represent that the following experiments are all or only experiments that can be performed. While efforts have been made to ensure accuracy with respect to the numerical values ​​used (e.g., quantity, temperature, etc.), some experimental error and deviation should be taken into consideration. Unless otherwise specified, parts are by weight, molecular weight is weight-average molecular weight, and temperature is in degrees Celsius. Standard abbreviations are used.

[0106] Example 1: This example illustrates the generation of the RNA constructs disclosed herein. Vectors encoding the CAR constructs disclosed herein were generated by Gibson assembly of either PCR fragments or gene blocks. A 150-poly-A tail was inserted at the 3' end of each CAR molecule using a manipulated restriction site. The DNA vector was fully sequenced to determine the length of the poly-A tail. The CAR DNA was then linearized using SapI, and the digested DNA was further purified. RNA was transcribed from the purified DNA template using T7 polymerase. The RNA was then precipitated with lithium chloride and subjected to electrophoresis to determine its size and purity.

[0107] [Table 1]

[0108] [Table 2]

[0109] [Table 3]

[0110] [Table 4]

[0111] Table 5

[0112] Table 6

[0113] Table 7

[0114] Table 8

[0115] Table 9

[0116] Table 10

[0117] Example 2: This example shows the results using ACE pNKW97 (Figures 1, 2A-2D). The extracellular domain of the ACE2 protein was cloned into a vector containing the hinge, TM and CD28 costimulatory domains, as well as the CD3ζ signaling domain and 150p-A (Figure 1). The construct was digested using SapI, and the linearized DNA was used as a template for mRNA generation. PBNK cells were electroporated with pNKW97 mRNA. After overnight recovery, ACE2 expression was detected using flow cytometry and conjugated anti-ACE2 antibody. The specificity of the ACE2 antibody was confirmed using isotype control antibodies (Figures 2A, 2C). The non-transfection control (Figure 2B) did not show endogenous expression of ACE2 in CENK cells. However, electroporated CENK cells showed >90% ACE2 CAR expression (Figure 2D). The vector encoding the ACE2 CAR is shown in Figure 3.

[0118] Example 3: This example shows the results obtained using pNKW92-93 (Figures 4, 5A-5F). A CAR molecule targeting the B7H4 antigen preceded by a CD64 or IgGH signal peptide was designed as shown in Figure 4. The CAR DNA was linearized using SapI and used as a template for in vitro transcription of mRNA. Next, CENK cells were electroporated using mRNA. After overnight recovery, B7H4 CAR expression was detected by flow cytometry and biotinylated B7H4, followed by streptavidin-APC. As a control for the specificity of the detection reagent, staining with streptavidin-APC alone was used for each sample. As shown in Figures 5A and 5B, the non-transfection control did not express the B7H4 CAR. On the other hand, both pNKW92 (CD64 signal peptide) and pNKW93 (IgGHv signal peptide) (Figures 5C, 5D, 5E, and 5F, respectively) showed high expression of the B7H4 CAR. The vectors encoding the B7H4 CAR are shown in Figures 6 and 7.

[0119] Example 4: This example shows the results from pNKW88-91 (Figures 8A, B, 9A-9E, 10-14). Four CAR molecules targeting the BCMA antigen were designed as shown in Figures 8A and 8B. The CAR DNA was linearized using SapI and used as a template for in vitro transcription of mRNA. Next, CENK cells were electroporated using mRNA. After overnight recovery, BCMA CAR expression was detected by flow cytometry and biotinylated BCMA, followed by streptavidin-APC. As a control for the specificity of the detection reagent, staining with streptavidin-APC alone was used for each sample. As shown in Figure 9A, the non-transfection control did not express BCMA CAR. On the other hand, all four different constructs (Figures 9B-9E) showed high levels of BCMA CAR. The constructs were tested for cytotoxic activity against SUP-B15 cell lines that stably express BCMA (data are not shown for all constructs). In Figure 10, one CAR construct (pNKW89) was shown to specifically lyse BCMA-expressing target cell lines, while it did not exhibit cytotoxic activity against BCMA-negative parental cell lines. Non-transfection controls were cytotoxic to either parental SUP-B15 or BCMA-expressing SUP-B15 cell lines. Vectors encoding the BCMA CAR are shown in Figures 11-14.

[0120] Example 5: This example demonstrates the rationale for developing the monopeptide CD19 CAR. mRNA for electroporation of PB-NK cells was generated using the tripeptide CD19 CAR previously used to create a stable CD19-CAR NK92 cell line. After overnight recovery from electroporation, CD19 CAR was detected using flow cytometry and biotinylated anti-CAR (F(ab')2), followed by streptavidin-APC. As a control for the specificity of the detection reagent, staining with streptavidin-APC alone was used for each sample. As shown in Figure 15A, the non-transfection-negative control did not express CD19 CAR, while the positive control cell line showed high expression of CD19 CAR. To evaluate the electroporation protocol, two different RNA templates (GFP and PDL1) were used, both of which showed high expression of the corresponding encoded protein 24 hours after electroporation (Figure 15B). The tripeptide CD19 CAR mRNA could not be expressed in PB-NK cells (Figure 15C). To ensure that the lack of CD19 CAR expression was not specific to PB-NK cells, memory T cells (Tscm) were electroporated with the same CD19 CAR mRNA using three different protocols with increasing electrical pulses. Neither untransfected negative controls (Figure 16A) nor transfected Tscm cells (Figures 16B-D) expressed CD19 CAR. The next step was to design a monopeptide CD19 CAR using a domain optimized for mRNA expression in primary NK cells. Two CAR molecules targeting the CD19 antigen (with different signal peptides) were designed as shown in Figure 17. The CAR DNA was linearized using SapI and used as a template for in vitro transcription of mRNA. Next, PB-NK cells were electroporated with the CAR mRNA. After overnight recovery, CD19 CARs were detected using flow cytometry and biotinylated CD19, followed by streptavidin-APC. As a control for the specificity of the detection reagents, staining with streptavidin-APC alone was used for each sample.As shown in Figures 18A–18B, the untransfected control did not express the CD19 CAR. On the other hand, PB-NK cells transfected with both constructs (Figures 18C, 18D, and 18E, 18F) showed high levels of the CD19 CAR. The CAR molecule showed cytotoxic activity against the CD19 antigen-expressing SUP-B15 cell line (parent), but not against CD19-negative mutants of the same cell line (Figures 19A and B). The use of memory NK cells (CIMLs) is promising for extending the survival and activity of NK cells in the tumor microenvironment. PB-NK cells were subjected to cytokine treatment (IL12 / IL18 / N-803) for 16 hours. After cytokine removal, both control and CIML cells were electroporated with pNKW87 mRNA. As shown in Figures 20A-20B, transfected cells exhibited cytotoxic activity against CD19-positive target cells only in both memory (Figure 20A) and control (Figure 20B) PBNK cells. Next, the monopeptide CD19 CAR was tested in activated T cells (control to Tscm) and Tscm cells. For this experiment, the stability of CD19 CAR expression was also tested over a 72-hour period. As shown in Figures 21B and 21C (pNKW87) and Figures 21D and 21E (pNKW59), CD19 CAR levels remained high even 72 hours after electroporation of activated T cells. The non-transfection-negative control (Figure 21A) did not show CD19 CAR expression. Similar stability tests were performed using electroporation of Tscm cells with the same CAR RNA molecule (Figures 22B and 22C and Figures 22D and 22E: pNKW87 and pNKW59, respectively). The results show high stability of CD19 CAR mRNA in transfected Tscm cells even 72 hours after transfection. The non-transfection-negative control (Figure 22A) does not show CD19 CAR expression. The vector encoding CD19 CAR is shown in Figures 23 and 24.

[0121] Example 6: This example shows the results using CD30 pNKW95 (Figures 25, 26A-26F and 27). A CAR molecule targeting the CD30α antigen was designed in a vector containing the hinge, TM and CD28 costimulatory domains, as well as the CD3ζ signaling domain and 150p-A (Figure 25). The vector was digested using SapI, and the linearized DNA was used as a template for mRNA generation. CENK cells were electroporated with pNKW95 mRNA. After overnight recovery, CD30 expression was detected by flow cytometry and biotinylated CD30, followed by streptavidin-APC. Specificity of the detection method was confirmed using streptavidin-APC staining alone. Non-transfection controls (Figures 26A, 26B) did not show endogenous expression of CD30 in CENK cells. The original plasmid from which pNKW95 originated (pXL46) was used for comparison. Since pXL46 lacks a long poly(A) tail, a poly(A) tail (less than 50 nucleotides) was added during in vitro transcription. As shown in Figures 26E and 26F, pNK95 exhibits higher CD30 CAR expression compared to the original construct (pXL46: Figures 26C and 26D). The vector encoding the CD30 CAR is shown in Figure 27.

Claims

1. A chimeric antigen receptor (CAR) comprising a T7 promoter, a spacer sequence, a signal peptide, an antigen-binding domain, a hinge region, a transmembrane (TM) domain, and an intracellular domain, The signal peptide comprises a differentiation antigen group 64 (CD64) and / or an IgG heavy chain variable gene (IgGv) signal peptide; The antigen-binding domain is a chimeric antigen receptor (CAR) that binds to an antigen selected from the group consisting of differentiation antigen group 19 (CD19), B cell maturation antigen (BCMA), B7 homolog 4 (B7H4), SARS-CoV-2 spike, and differentiation antigen group 30α (CD30α).

2. The CAR according to claim 1, wherein the signal peptide comprises SEQ ID NO: 1 or SEQ ID NO:

2.

3. The CAR according to claim 1, wherein the antigen-binding domain that binds to CD19 has at least 90%, at least 95%, or up to 100% sequence identity with respect to SEQ ID NO:

4.

4. The CAR according to claim 1, wherein the antigen-binding domain that binds to BCMA contains at least 90%, at least 95%, or up to 100% sequence identity to SEQ ID NO: 5 or SEQ ID NO:

6.

5. The CAR according to claim 1, wherein the antigen-binding domain that binds to B7H4 contains at least 90%, at least 95%, or up to 100% sequence identity with respect to SEQ ID NO:

7.

6. The CAR according to claim 1, wherein the antigen-binding domain that binds to the SARS-CoV-2 spike contains at least 90%, at least 95%, or up to 100% sequence identity with respect to SEQ ID NO:

8.

7. The CAR according to claim 1, wherein the antigen-binding domain that binds to CD30α contains at least 90%, at least 95%, or up to 100% sequence identity with respect to SEQ ID NO:

57.

8. The CAR according to claim 1, wherein the hinge region is a differentiated antigen group 28 (CD28) hinge region having sequence number 9.

9. The CAR according to claim 1, wherein the TM domain is a CD28 TM domain having sequence number 10.

10. The CAR according to claim 1, wherein the intracellular domain includes a co-stimulatory domain and includes a CD28 cytoplasmic domain having sequence number 11.

11. The CAR according to claim 1, wherein the intracellular signaling domain comprises a differentiation antigen group 3ζ (CD3ζ) cytoplasmic domain having Sequence ID No.

12.

12. The CAR according to claim 1, comprising an amino acid sequence having at least 90%, at least 95%, or up to 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 15, 16, 17, 18, 19, 20, 21, 22, and 58.

13. A nucleic acid construct encoding a CAR as described in claim 1.

14. A nucleic acid construct according to claim 13, comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO:

59.

15. An expression vector encoding the CAR according to claim 1.

16. The expression vector according to claim 15, having a nucleic acid sequence selected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO:

42.

17. Modified primary natural killer (NK) cells modified with an RNA molecule containing one or more nucleic acids of the following: T7 promoter, spacer sequence, signal peptide sequence portion, antigen-binding domain sequence portion, hinge region sequence portion, transmembrane (TM) domain sequence portion, and intracellular domain sequence portion, The signal peptide sequence includes a sequence encoding differentiation antigen group 64 (CD64) and / or IgG heavy chain variable gene (IgGv); The antigen-binding domain includes a sequence encoding an antigen-binding portion that binds to an antigen selected from the group consisting of differentiation antigen group 19 (CD19), B cell maturation antigen (BCMA), B7 homolog 4 (B7H4), SARS-CoV-2 spike, and differentiation antigen group 30α (CD30α); Modified primary natural killer (NK) cells in which the nucleic acid sequences are operably linked to one another as a single polynucleotide.

18. The modified primary NK cell according to claim 17, wherein the intracellular domain sequence portion comprises a CD28 cytoplasmic domain having SEQ ID NO: 11 and / or a differentiation antigen group 3ζ (CD3ζ) cytoplasmic domain having SEQ ID NO:

12.

19. The modified primary NK cell according to claim 17, further comprising a 3' untranslated region (3'-UTR).

20. The modified primary NK cell according to claim 17, further comprising a poly-A sequence portion.

21. A method for generating modified primary CAR-NK cells, comprising transfecting primary NK cells with the recombinant nucleic acid construct described in claim 13.

22. A method of immunotherapy for treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition containing recombinant NK cells according to claim 17.

23. 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, and, without limitation, sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endosarcoma, lymphangiosarcoma, lymphangioendothelioma, synoviomas, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, and pancreatic cancer. The method according to claim 22, selected from the group consisting of solid tumors including breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, 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.

24. Modified NK cells according to claim 17 for use in the treatment of cancer.

25. 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, and, without limitation, 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. Modified NK cells according to claim 24, selected from the group consisting of 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.

26. Modified NK cells according to claim 17, for use as a pharmaceutical agent.

27. A pharmaceutical composition comprising the modified NK cells described in claim 17 and a pharmaceutically acceptable carrier.

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