WO2024055339A1 - 用于制备和扩增通用型人源化抗cd19 car-nk细胞的方法及其用途 - Google Patents

用于制备和扩增通用型人源化抗cd19 car-nk细胞的方法及其用途 Download PDF

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WO2024055339A1
WO2024055339A1 PCT/CN2022/119604 CN2022119604W WO2024055339A1 WO 2024055339 A1 WO2024055339 A1 WO 2024055339A1 CN 2022119604 W CN2022119604 W CN 2022119604W WO 2024055339 A1 WO2024055339 A1 WO 2024055339A1
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钟晓松
白玥
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卡瑞济(北京)生命科技有限公司
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Definitions

  • the present invention relates to the field of genetic engineering.
  • the present invention relates to genetically engineered feeder cells expressing CD19 and at least one molecule that stimulates immune cell activation and the use of said genetically engineered feeder cells for expanding NK cells.
  • the invention also relates to NK cells engineered to express humanized anti-CD19 CARs, and methods of preparing and expanding them to clinically therapeutic quantities.
  • the engineered NK cells of the invention are non-immunogenic and can be administered to any subject without being rejected by the host immune system (they are "universal").
  • CAR chimeric antigen receptor
  • the CD19 molecule is a potential target for the treatment of B lymphocyte lineage tumors and is also a hot topic in CAR research.
  • the overall effectiveness of CD19-targeting CAR-T cells in lymphoma ranges from 50% to 82% (Castaneda-Puglianini O et al., Assessing and Management of Neurotoxicity After CAR-T Therapy in Diffuse Large B-Cell Lymphoma[J].J Blood Med, 2021,12:775-783.DOI:10.2147/JBM.S281247).
  • CRS cytokine release syndrome
  • CRS neurotoxicity
  • allogeneic CAR-T cells may also cause graft-versus-host disease (GVHD), and the need to use autologous T cells to prepare autologous CAR-T cells makes many patients undergoing clinical enrollment evaluation due to clinical Insufficient production of viable manufactured goods ultimately made them infeasible.
  • GVHD graft-versus-host disease
  • Reasons for insufficient yields of clinically viable finished products include the inability to harvest adequate and sufficient lymphocytes from patients and limited in vitro cell expansion. Therefore, technological development is needed to overcome these problems in CAR-T cell therapy.
  • Natural killer cells are an important class of innate immune cells that perform the body's primary defense (innate immunity) by immediately identifying and eliminating viral, bacterial, and parasitic infections as well as abnormal autologous cells, especially cancer cells. )Function. Unlike T cells that recognize target cells by expressing antigen-specific receptors, NK cells sense abnormal changes in target cells (especially cancer cells) and have contact-dependent cytotoxicity. NK cells do not require antigen specificity or human leukocyte antigen.
  • HLA histocompatibility complex
  • NK cells as effector cells of natural immunity, have strong anti-tumor ability and will not cause strong CRS or graft-versus-host disease
  • cancer antigen-specific CAR genes are introduced into NK cells to develop CAR-NK cell therapy has superiority.
  • NK cells can be obtained from healthy donors unrelated to the patient and can also overcome tumor recurrence due to antigen escape through the natural tumor cell killing mechanism of NK cells (Siegler E L et al., Off-the-Shelf CAR-NK Cells for Cancer Immunotherapy[J].Cell Stem Cell,2018,23(2):160-161.DOI:10.1016/j.stem.2018.07.007).
  • CAR-NK cell therapy has been limited in part by the challenges of manufacturing high doses of CAR-NK cells and the immunogenicity of murine scFv in the expressed CAR molecules that may cause CAR-NK cells to be blocked in vivo. There is a risk of rejection or allergic reaction, resulting in the inability of the CAR-NK cells to survive continuously.
  • a second-generation CD19 CAR containing humanized anti-CD19 scFv and prepared NK cells expressing humanized anti-CD19 CAR in vitro (also referred to as hCAR19 in the present invention).
  • -NK cells which were massively expanded in vitro through genetically engineered feeder cells.
  • the present invention relates specifically to the following aspects.
  • the present invention provides a genetically engineered feeder cell, which is obtained by genetically modifying immortalized cells to express CD19 and at least one molecule that stimulates immune cell activation, and the genetically engineered feeder cell does not Major histocompatibility complex (MHC) I molecules are expressed, and wherein co-culture of the genetically engineered feeder cells with NK cells results in activation and expansion of NK cells.
  • MHC Major histocompatibility complex
  • the invention provides a genetically engineered feeder cell that is genetically modified, such as K562 myeloid leukemia cells, HL-60 human promyelocytic leukemia cells, or OCI-AML3 human acute myeloid leukemia cells.
  • a genetically engineered feeder cell that is genetically modified, such as K562 myeloid leukemia cells, HL-60 human promyelocytic leukemia cells, or OCI-AML3 human acute myeloid leukemia cells.
  • the at least one molecule that stimulates immune cell activation is, for example, selected from the group consisting of 4-1BBL, membrane-bound IL-12, membrane-bound IL-7, membrane Bound IL-15 (mIL15), membrane-bound IL-18 (mIL18), membrane-bound IL-21 (mIL-21), membrane-bound IL-22 (mIL-22).
  • the genetically engineered feeder cells of the invention are K562 cells genetically modified to express CD19, 4-1BBL, and membrane-bound IL-21 (mIL-21).
  • the genetically engineered feeder cells of the present invention are genetically modified using viral vectors (e.g., retroviral vectors) so that they express SEQ ID NO: 13 or are at least 90% or at least identical to SEQ ID NO: 13.
  • viral vectors e.g., retroviral vectors
  • SEQ ID NO: 13 express SEQ ID NO: 13 or are at least 90% or at least identical to SEQ ID NO: 13.
  • Membrane-bound IL-21 mIL-21 having an amino acid sequence of at least 90% or at least 95% sequence identity.
  • the present invention provides a method for preparing the genetically engineered feeder cells of the first aspect of the present invention, the method comprising:
  • Cancer cells are transduced or transfected with nucleic acids encoding CD19 and at least one molecule that stimulates immune cell activation, wherein the at least one molecule that stimulates immune cell activation is, for example, selected from the group consisting of 4-1BBL, membrane-bound IL-12, membrane Bound IL-7, membrane-bound IL-15 (mIL15), membrane-bound IL-18 (mIL18), membrane-bound IL-21 (mIL-21), membrane-bound IL-22 (mIL-22),
  • the cancer cells include K562 myeloid leukemia cells, HL-60 cells, and OCI-AML3 cells;
  • the isolated cells are irradiated to produce genetically engineered feeder cells.
  • the present invention provides the use of the genetically engineered feeder cells of the first aspect of the present invention for expanding NK cells, wherein the NK cells can be naturally occurring NK cells or genetically modified ones.
  • NK cells can be naturally occurring NK cells or genetically modified ones.
  • the naturally occurring NK cells to be expanded are isolated from or contained in peripheral blood, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, infection site tissue, ascites, pleural effusion, spleen tissue, or tumor .
  • the genetically modified NK cells to be expanded are CAR-NK cells, e.g., humanized anti-CD19 CAR-NK cells.
  • the invention provides methods for preparing and expanding humanized anti-CD19 CAR-NK cells, including:
  • irradiated for example, gamma ray irradiated
  • genetically engineered feeder cells of the first aspect of the present invention with naturally occurring NK cells; for example, co-culture for 2-10 days, for example, co-culture for 6 days
  • the naturally occurring NK cells are isolated from or contained in peripheral blood, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, infection site tissue, ascites, pleural effusion, spleen tissue, or tumor;
  • c) Co-culture of humanized anti-CD19 CAR-NK cells with irradiated (e.g., gamma irradiated) genetically engineered feeder cells of the first aspect of the invention and optionally IL-2 is sufficient to expand human activating the anti-CD19 CAR-NK cells for a period of time (e.g., 7-42 days, e.g., 14-28 days), thereby expanding the humanized anti-CD19 CAR-NK cells; e.g., the humanized anti-CD19 CAR-NK cells NK cells expand at least 1500 to 5000 times.
  • the hCAR19-NK cell-expressed humanized anti-CD19 CAR comprises:
  • Humanized anti-CD19 scFv sequence wherein the scFv sequence specifically binds CD19 and includes:
  • a light chain variable region comprising or sharing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% with the sequence of SEQ ID NO: 1 % identity of the sequence, and
  • a heavy chain variable region comprising or sharing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% with the sequence of SEQ ID NO:2 % identity sequence;
  • Hinge region/spacer region which is selected from the CD8a hinge region (SEQ ID NO 5), or a CD8 hinge region with at least 90%, at least 95% sequence identity;
  • Costimulatory signaling domain which is:
  • Stimulating signal domain which is the CD3 ⁇ signaling domain shown in SEQ ID NO: 11 or a variant thereof with 1-5 amino acid modifications;
  • the amino acid modification is the addition, deletion or substitution of amino acids.
  • the humanized anti-CD19 CAR expressed by hCAR19-NK cells includes:
  • Humanized anti-CD19 scFv sequence which contains: the sequence of SEQ ID NO:4 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 % or 99% identity of the sequence,
  • Hinge region/spacer region which is the CD8a hinge region shown in SEQ ID NO 5 or a variant thereof with one amino acid modification
  • Transmembrane region which is the CD28 transmembrane domain shown in SEQ ID NO:8 or a variant thereof with one amino acid modification
  • Costimulatory signal domain which is the CD28 costimulatory domain shown in SEQ ID NO: 9 or a variant thereof with one amino acid modification;
  • Stimulating signal domain which is the CD3 ⁇ signaling domain shown in SEQ ID NO: 11 or a variant thereof with one amino acid modification;
  • the amino acid modification is the addition, deletion or substitution of amino acids.
  • the present invention provides humanized anti-CD19 CAR-NK cells prepared and expanded by the method of the fourth aspect of the present invention, which express IFN- ⁇ and undergo degranulation after contact with CD19-expressing tumor cells, And kill tumor cells expressing CD19.
  • the present invention provides the use of the humanized anti-CD19 CAR-NK cells of the fifth aspect of the present invention for preparing drugs for treating CD19-expressing tumors.
  • the humanized anti-CD19 CAR-NK cells of the present invention can bind to CD19 on tumor cells and destroy the tumor cells.
  • the CD19-expressing tumor is selected from multiple myeloma, leukemia, lymphoma, or a solid tumor
  • the leukemia is acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML)
  • the lymphoma is non-Hodgkin lymphoma
  • the solid tumor is brain, prostate, breast, lung, colon, uterus, skin, liver, bone, pancreas, ovary, testicle, bladder, kidney, head, neck , tumors of the stomach, cervix, rectum, throat, or esophagus.
  • Figure 1 shows a schematic diagram of the constituent domains of the "humanized anti-CD19 scFv-28Z" CAR (also referred to herein as "hCAR19").
  • 5'LTR and 3'LTR represent the long terminal repeat sequences at the 5' and 3' ends respectively
  • CD8a represents the CD8a hinge region
  • CD28 represents the CD28 transmembrane domain and CD28 costimulatory domain
  • ⁇ chain represents the CD3 ⁇ signaling structure. area.
  • Figure 2 shows the results of flow cytometry detection of hCAR19 expression after NK cells were transfected with a retrovirus containing the hCAR19 coding sequence.
  • “Mock” represents the Mock control cells obtained after transfecting NK cells with a retrovirus that does not contain the hCAR19 coding sequence.
  • Figure 3 shows the results of detecting the expression of exogenous CD3 ⁇ in the transfected cells by Western blotting using anti-CD3 ⁇ mAb after transfecting NK cells with a retrovirus containing the hCAR19 coding sequence. From left to right in the figure, lane 1: molecular weight markers; lane 2: Mock control cells; lane 3: hCAR19-NK cells.
  • Figure 4 shows the detection of IFN- ⁇ production by flow cytometry after Mock control cells and hCAR19-NK cells were co-cultured with K562, Raji or K562-CD19 cells for 6 hours.
  • Figure 5 shows the detection of CD107a molecules on the surface of NK cells by flow cytometry after Mock control cells and hCAR19-NK cells were co-cultured with K562, Raji or K562-CD19 cells for 6 hours.
  • Figure 9 shows the vehicle group (black line), Mock control cell group (brown line), hCAR19-NK cell group (red line), and FMC63 CAR-NK cell group after inoculating mice with Raji-GFP-LUC tumor cells. (Blue line) The survival percentage of mice, thus showing the therapeutic effect of each NK cell on tumors.
  • CD19 refers to the cluster of differentiation 19 protein, which is an antigenic determinant detectable on leukemia precursor cells.
  • Human and mouse amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt and Swiss-Prot.
  • the amino acid sequence of human CD19 can be found under UniProt/Swiss-Prot accession number P15391 (full length is 556 amino acids), and the nucleotide sequence encoding human CD19 can be found under accession number NM_001178098.
  • CD19 is expressed on most B-lineage cancers, including, for example, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma.
  • chimeric antigen receptor refers to a chimeric fusion protein having an extracellular domain fused via a transmembrane domain to an intracellular signaling domain capable of activating cells .
  • the intracellular signaling domain of CAR molecules includes two different cytoplasmic signaling domains.
  • one signaling domain may be a cytoplasmic effector functional signaling domain and a second signaling domain may be a cytoplasmic costimulatory signaling domain.
  • Linkers can connect domains to each other.
  • feeder cell refers to cells that provide support to another cell type in vitro or in vitro culture.
  • the feeder cells may provide one or more factors required for survival, growth and/or differentiation of cells cultured with the feeder cells.
  • feeder cells are irradiated or otherwise treated to prevent their proliferation in culture.
  • NK cells are cultured with feeder cells, such as irradiated modified K562 cells.
  • expansion refers to an increase in the number of characteristic cell types from an initial cell population.
  • activation or “activation” of an immune cell refers to the ability of an immune cell to respond and exhibit, at a measurable level, the immune function of the corresponding cell known to those skilled in the art. Methods of measuring immune cell activity are also known to those skilled in the art.
  • NK cell refers to a cell of the immune system that kills target cells in the absence of a specific antigenic stimulus and without restriction based on MHC class.
  • the target cells may be tumor cells or virus-bearing cells.
  • NK cells are characterized by the presence of the CD56 surface marker and the absence of the CD3 surface marker.
  • NK cells typically constitute approximately 10% to 15% of normal peripheral blood mononuclear cells.
  • NK cells are thought to provide a “backup” protection mechanism against viruses and tumors that may evade cytotoxic T lymphocyte (CTL) responses by downregulating MHC class I presenting molecules. In addition to participating in direct cytotoxic killing, NK cells function through the production of cytokines.
  • CTL cytotoxic T lymphocyte
  • the nucleic acid sequence encoding the CAR of the invention can be introduced into a cell by "transfection,” “transformation,” or “transduction.”
  • transfection refers to the introduction of one or more exogenous polynucleotides into a host cell through the use of physical or chemical methods.
  • transfection techniques include, for example, calcium phosphate DNA coprecipitation (see, e.g., Murray E.J. (Ed.), Methods in Molecular Biology, Vol.
  • CAR-NK cell refers to an NK cell transduced with a heterologous nucleic acid encoding a CAR.
  • scFv refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are optionally are continuously linked via flexible short polypeptide linkers and can be expressed as a single-chain polypeptide in which the scFv retains the specificity of the intact antibody from which it was derived.
  • a scFv may have a VL variable region and a VH variable region in any order (eg, relative to the N-terminus and C-terminus of the polypeptide), the scFv may comprise a VL-linker-VH or may comprise VH-joint-VL.
  • CD3 ⁇ is defined as the protein provided by GenBan accession number BAG36664.1 or its equivalent
  • CD3 ⁇ intracellular signaling domain is defined as the amino acid residues from the cytoplasmic domain of the CD3 ⁇ chain that are sufficient Functionally propagates the initial signal necessary for cell activation.
  • the cytoplasmic domain of CD3 ⁇ comprises residues 52 to 164 of GenBank accession number BAG36664.1 or as a functional ortholog thereof from a non-human species (e.g., mouse, rodent, equivalent residues of monkeys, apes, etc.).
  • the "CD3 ⁇ stimulating signal domain” is the sequence provided in SEQ ID NO: 11 or a variant thereof.
  • costimulatory molecule refers to a corresponding binding partner on a cell that specifically binds to a costimulatory ligand thereby mediating a costimulatory response (such as, but not limited to, proliferation) of the cell.
  • Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an effective immune response.
  • Costimulatory molecules include, but are not limited to, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activated NK cell receptors, OX40 , CD40, GITR, 4-1BB (ie CD137), CD27 and CD28.
  • the "costimulatory molecule” is 4-1BB (i.e., CD137) or CD28.
  • the costimulatory signaling domain refers to the intracellular part of the costimulatory molecule.
  • signaling pathway refers to the biochemical relationships between various signaling molecules that play a role in propagating signals from one part of a cell to another part of the cell.
  • cytokine is a general term for proteins released by one cell population that act as intercellular mediators on another cell.
  • cytokines are lymphokines, monokines, interleukins (IL), such as IL-1, IL-1 ⁇ , IL-2, IL-3, IL-4, IL-5, IL-6, IL- 7.
  • homology refers to the degree of relatedness between two given amino acid sequences or nucleotide sequences, and may be expressed as a percentage.
  • homology and identity are often used interchangeably.
  • the sequences are aligned for optimal comparison purposes (e.g., a first and a second amino acid sequence or nucleic acid sequence may be aligned for optimal alignment). Introducing gaps in one or both may allow non-homologous sequences to be discarded for comparison purposes).
  • the length of the aligned reference sequences is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more preferably at least 70%, 80% , 90%, 100% of the reference sequence length.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. The molecules are identical when a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence.
  • Mathematical algorithms can be used to perform sequence comparison and calculation of percent identity between two sequences.
  • the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm is used which has been integrated into the GAP program of the GCG software package (available at http://www.gcg.com available), determine the distance between two amino acid sequences using the Blossum 62 matrix or the PAM250 matrix with gap weights 16, 14, 12, 10, 8, 6, or 4 and length weights 1, 2, 3, 4, 5, or 6 Percent identity.
  • the GAP program in the GCG software package (available at http://www.gcg.com) is used, using the NWSgapdna.CMP matrix and gap weights 40, 50, 60, 70 or 80 and A length weight of 1, 2, 3, 4, 5, or 6 determines the percent identity between two nucleotide sequences.
  • a particularly preferred parameter set (and one that should be used unless otherwise stated) is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.
  • nucleic acid sequences and protein sequences described herein may further be used as "query sequences" to perform searches against public databases, for example to identify other family member sequences or related sequences.
  • amino acid change and “amino acid modification” are used interchangeably and refer to the addition, deletion, substitution and other modifications of amino acids. Any combination of amino acid additions, deletions, substitutions, and other modifications can be made, provided that the final polypeptide sequence has the desired properties.
  • Amino acid substitutions include substitutions with non-naturally occurring amino acids or naturally occurring amino acid derivatives of the twenty standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxy Lysine) substitution.
  • Amino acid changes can be produced using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, etc. It may be useful to alter amino acid side chain groups by methods other than genetic engineering (eg, chemical modification).
  • conservative sequence modification and “conservative sequence change” refer to amino acid modifications or changes that do not significantly affect or change the binding characteristics of an antibody or antibody fragment containing an amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the antibodies or antibody fragments of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conservative substitution is an amino acid substitution in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art.
  • These families include those with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., Glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), ⁇ -side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenyl Alanine, tryptophan, histidine) amino acids.
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • autologous refers to any substance derived from the same individual to whom the substance is later reintroduced.
  • allogeneic refers to any substance derived from a different animal of the same species as the individual into which the substance is introduced. Two or more individuals are said to be allogeneic to each other when the genes at one or more loci are not identical. In some aspects, allogeneic agents from individuals of the same species can be genetically dissimilar enough to allow for antigenic interaction to occur.
  • xenogeneic refers to a graft derived from an animal of a different species.
  • the present invention provides genetically engineered feeder cells for expanding and activating NK cells.
  • co-culture of genetically engineered feeder cells that do not express major histocompatibility complex (MHC) I molecules with NK cells results in activation and expansion of NK cells.
  • MHC major histocompatibility complex
  • genetically engineered feeder cells are derived from immortal cell lines.
  • a cell line that exhibits one or more characteristics of immortal cells in culture are selected from immortal cell lines.
  • genetically engineered feeder cells are derived from cancer cells that are modified to express one or more molecules that promote and/or enhance NK cell activation and/or expansion.
  • the feeder cells are genetically engineered to express membrane-bound interleukin-21 (mbIL21).
  • the genetically engineered feeder cells are modified to express membrane-bound 4-1BB ligand (4-1BBL) in addition to or instead of mbIL21.
  • the genetically engineered feeder cells are modified to express at least one additional membrane-bound interleukin that stimulates NK cell activation in addition to or instead of mbIL21, 4-1BBL, and/or other activation/expansion promoting factors. .
  • the population of genetically engineered feeder cells includes at least a plurality of first cells expressing mbIL21 and a plurality of second cells expressing 4-1BBL, such that the population of genetically engineered feeder cells collectively express mbIL21 and 4 -1BBL both.
  • the population of genetically engineered feeder cells includes a plurality of cells expressing both mbIL21 and 4-1BBL.
  • the genetically engineered feeder cells include some cells that express mbIL21, some cells that express 4-1BBL, and some cells that express both.
  • other ligands and/or activating factors may be additionally expressed in addition to or instead of mbIL21 and/or 4-1BBL.
  • mbIL21 expressed by genetically engineered feeder cells has the amino acid sequence shown in SEQ ID NO. 15.
  • 4-1BBL expressed by genetically engineered feeder cells has the amino acid sequence shown in SEQ ID NO. 14.
  • the genetically engineered feeder cells are derived from a cell line selected from: K562 cells, HL-60 cells, OCI-AML3 cells.
  • the genetically engineered feeder cells also express other interleukin molecules, e.g., membrane-bound IL-12, membrane-bound IL-7, membrane-bound IL-15 (mIL15), membrane-bound IL- 18 (mIL18), membrane-bound IL-21 (mIL-21), membrane-bound IL-22 (mIL-22). Combinations of various interleukins can be expressed in various combinations.
  • interleukin molecules e.g., membrane-bound IL-12, membrane-bound IL-7, membrane-bound IL-15 (mIL15), membrane-bound IL- 18 (mIL18), membrane-bound IL-21 (mIL-21), membrane-bound IL-22 (mIL-22). Combinations of various interleukins can be expressed in various combinations.
  • the genetically engineered feeder cells also express CD19, wherein the expressed CD19 specifically binds to the humanized anti-CD19 scFv encoded by hCAR19 after contact with the hCAR19-NK cells of the invention. Activate and/or expand hCAR19-NK cells.
  • the CD19CD19 expressed by the genetically engineered feeder cells has the amino acid sequence shown in SEQ ID NO. 13.
  • the present invention also provides a method for preparing the genetically engineered feeder cells of the present invention.
  • at least one heterologous nucleic acid such as encoding CD19, 4-1BBL, membrane-bound IL-12, membrane-bound IL-7, membrane-bound IL-15 (mIL15), membrane-bound IL-18 (mIL18) , membrane-bound IL-21 (mIL-21), membrane-bound IL-22 (mIL-22), one or more nucleic acids
  • feeder cells e.g., K562 cells
  • the genetically engineered feeder cells include heterologous nucleic acid encoding mIL-21, heterologous nucleic acid encoding CD19, and heterologous nucleic acid encoding 4-1BBL.
  • feeder cells eg, K562 cells
  • vectors eg, lentiviral or retroviral vectors
  • at least one heterologous nucleic acid For example, use at least one, at least 2, at least 3, at least 4 or more selected from the group consisting of encoding CD19, 4-1BBL, membrane-bound IL-12, membrane-bound IL-7, membrane-bound IL- Heterologous nucleic acids of 15 (mIL15), membrane-bound IL-18 (mIL18), membrane-bound IL-21 (mIL-21), and membrane-bound IL-22 (mIL-22) are transduced or transfected into feeder cells ( For example, K562 cells).
  • transduction or transfection method such as viral transduction (eg using retroviruses such as MoMLV or lentivirus) or non-viral transduction, mRNA transfection.
  • viral transduction eg using retroviruses such as MoMLV or lentivirus
  • non-viral transduction eg using mRNA transfection.
  • a viral vector is used to deliver at least one heterologous nucleic acid into feeder cells (eg, K562 cells).
  • suitable viral vectors include retroviral (eg MoMLV or lentiviral) vectors, adenoviral vectors, adeno-associated virus vectors and vaccinia virus vectors.
  • a retroviral system is used to introduce one or more heterologous nucleic acids into feeder cells (eg, K562 cells).
  • MoMLV vectors such as SFG retroviral vectors, can be used.
  • the SFG vector is derived from the murine leukemia virus (MLV) backbone. This type of murine leukemia virus (MLV)-based retroviral vector is a commonly used gene delivery vehicle and has been widely used in clinical trials. Current SFG vectors are fully optimized for gene expression for lymphocyte genetic modification, protein expression, and viral titers.
  • cells are transduced with a first construct encoding mbIL21, thereby generating a first population of transduced cells and expanding the first population of transduced cells; and with a second construct encoding 4-1BBL
  • the first transduced cell population is transduced in vivo, thereby generating a second transduced cell population; the second transduced cell population is transduced with a third construct encoding CD19, thereby generating a third transduced cell population.
  • cell population and expand a third transduced cell population.
  • the genetically engineered feeder cells of the invention are prepared by simultaneously transducing a population of cells with a first construct encoding mbIL21, a second construct encoding 4-1BBL, and a third construct encoding CD19.
  • the feeder cells can be genetically engineered by including cells encoding mbIL21, 4-1BB, CD19, as well as membrane-bound IL-12, membrane-bound IL-7, membrane-bound IL-15 (mIL15), membrane It is prepared by transducing cells with one or more constructs of conjugated IL-18 (mIL18) and membrane-bound IL-22 (mIL-22).
  • the present invention also provides for the production of genetically engineered feeder cells by transducing K562 cells with a retroviral vector comprising cDNA sequences encoding CD19, membrane-bound IL-21 (mIL-21), and 4-1BBL.
  • NK cells have the ability to use their own advantages to overcome the shortcomings of CAR-T cells.
  • allogeneic NK cell infusion will neither produce GVHD nor obvious toxic side effects. Compared with CAR-T, it will be safer and will not be limited by autologous cells.
  • CAR-NK cells can also more effectively kill residual tumor cells through the NK cell receptor-dependent pathway and reduce the recurrence rate.
  • NK cells are diverse and can be generated from peripheral blood (PB), umbilical cord blood (UCB), human embryonic stem cells (HESC), induced pluripotent stem cells (IPSC), and even NK-92 cell lines (Marofi F et al. , CAR-engineered NK cells; a promising therapeutic option for treatment of hematological malignancies[J].Stem Cell Res Ther, 2021,12(1):374.DOI:10.1186/s13287-021-02462-y), therefore, construct CAR-NK cells can open up broader prospects for tumor immunotherapy.
  • PB peripheral blood
  • ULB umbilical cord blood
  • HESC human embryonic stem cells
  • IIPSC induced pluripotent stem cells
  • NK-92 cell lines Marofi F et al. , CAR-engineered NK cells; a promising therapeutic option for treatment of hematological malignancies[J].Stem Cell Res Ther, 2021,12(1):374.DOI:10.1186/s13287-021-024
  • CAR-NK cell immunotherapy One of the major limitations of CAR-NK cell immunotherapy is the lack of in vivo persistence of the infused cells without the support of cytokines, which will affect the efficacy of CAR-NK cell therapy.
  • Some studies have shown that the immunogenicity of mouse scFv may cause rejection of CAR-T cells in the body or cause allergic reactions, resulting in the inability of CAR-T cells to survive continuously (Turtle C J et al., CD19 CAR-T cells of defined CD4+: CD8+composition in adult B cell ALL patients[J].J Clin Invest,2016,126(6):2123-2138.DOI:10.1172/JCI85309), affects the therapeutic effect.
  • the present invention constructs humanized anti-CD19 scFv to reduce the body's immune response to CAR, and at the same time activates and/or amplifies hCAR19-NK cells in vitro to improve the persistence of hCAR19-NK cells in the body, thereby improving Efficacy.
  • the present invention successfully separates and purifies NK cells from peripheral blood, and expands them in vitro under the stimulation of genetically engineered feeder cells (for example, K562-CD19-4-1BBL-mIL21 cells) after irradiation (for example, after 100Gy irradiation). increase.
  • the present invention also constructed a CAR containing a humanized anti-CD19 scFv targeting CD19.
  • the CAR was packaged by retrovirus, transduced into NK cells, and hCAR19-NK cells were amplified to obtain a sufficient amount of hCAR19-NK.
  • hCAR19-NK cells can effectively kill CD19+ tumor cells and secrete large amounts of IFN- ⁇ , and CD19+ tumor cells can specifically stimulate hCAR19-NK cells to express IFN- ⁇ and degranulate, forming hCAR19-NK cells. It lays the foundation for functional verification in vivo.
  • the present invention uses NK cells isolated from peripheral blood to construct humanized CAR-NK cells targeting CD19 for the first time, and verifies that they can specifically kill lymphoma cells.
  • the humanized anti-CD19 CAR of the present invention includes
  • Humanized anti-CD19 scFv sequence wherein the scFv sequence specifically binds CD19 and includes:
  • a light chain variable region comprising or sharing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% with the sequence of SEQ ID NO: 1 % identity of the sequence, and
  • a heavy chain variable region comprising or sharing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% with the sequence of SEQ ID NO:2 % identity sequence;
  • Hinge region/spacer region which is selected from the CD8a hinge region (SEQ ID NO 5), or a CD8 hinge region with at least 90%, at least 95% sequence identity;
  • Costimulatory signaling domain which is:
  • Stimulating signal domain which is the CD3 ⁇ signaling domain shown in SEQ ID NO: 11 or a variant thereof with 1-5 amino acid modifications;
  • amino acid modification is the addition, deletion or substitution of amino acids
  • the humanized anti-CD19 CAR of the invention comprises
  • (3SEQ ID NO:8 shows the CD28 transmembrane domain
  • CAR polypeptides of the invention may also be modified so as to vary in the amino acid sequence but not in the desired activity.
  • a CAR polypeptide can be subjected to additional nucleotide substitutions that result in amino acid substitutions at "non-essential" amino acid residues.
  • a non-essential amino acid residue in a molecule can be replaced with another amino acid residue from the same side chain family.
  • one amino acid fragment can be replaced with a structurally similar fragment that differs in the order and composition of the side chain family members, e.g., a conservative substitution can be made in which the amino acid residues are Replace with amino acid residues with similar side chains.
  • humanized anti-CD19 CAR-NK cells By introducing the nucleic acid sequence encoding the humanized anti-CD19 CAR of the present invention into NK cells, humanized anti-CD19 CAR-NK cells can be obtained.
  • retroviruses provide convenient platforms for gene delivery systems.
  • the selected genes can be inserted into the vector and packaged in retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to the subject's cells in vivo or ex vivo.
  • Numerous retroviral systems are known in the art.
  • Retroviral vectors derived from retroviruses are suitable tools for long-term gene transfer because they allow long-term, stable integration of the transgene and its propagation in progeny cells.
  • the retroviral vector may be, for example, a gamma retroviral vector.
  • a retroviral vector may, for example, comprise a promoter, a packaging signal ( ⁇ ), a primer binding site (PBS), one or more (eg, two) long terminal repeats (LTR), and a transgene of interest, e.g., encoding a CAR genes.
  • ⁇ Retroviral vectors can lack viral structural genes such as gag, pol and env.
  • the invention provides methods of expressing the CARs of the invention in mammalian NK cells and hCAR19-NK cells generated thereby.
  • the present invention constructs a humanized second-generation CD19 CAR retroviral vector; uses irradiated K562-CD19-4-1BBL-mIL21 cells to stimulate peripheral blood-derived NK cells, and then hCAR19-NK cells were obtained by retroviral transduction of NK cells.
  • the present invention detects the transduction efficiency of CAR on NK cells through flow cytometry and Western blot, which shows that the CAR constructed in the present invention can successfully transduce NK cells derived from peripheral blood.
  • the present invention further provides a method for expanding hCAR19-NK cells in vitro.
  • the genetically engineered feeder cells of the present invention are lethally irradiated to ensure no proliferation and no viable feeder cells; and then co-cultured with hCAR19-NK cells for a period of time (for example, , 7-42 days, for example, 14-28 days), thereby expanding hCAR19-NK cells.
  • hCAR19-NK cells of the invention are capable of at least 1500- to 5000-fold expansion in vitro.
  • CD107a degranulation assay and IFN- ⁇ intracellular staining were used to evaluate the specific activation of hCAR19-NK cells by CD19-expressing lymphoma cells; and fluorescence killing assay and ELISA were used to detect the killing of lymphoma cells by hCAR19-NK cells. capacity and cytokine release levels. The results showed that IFN- ⁇ was released and CD107a was degranulated after hCAR19-NK was co-cultured with different CD19-positive cell lines. Fluorescence killing experiments and detection of IFN- ⁇ release showed that hCAR19-NK cells had the killing ability of CD19-positive lymphoma cells and IFN- The ⁇ release levels were significantly higher than those in the control group.
  • the invention provides the use of the hCAR19-NK cells of the invention for treating diseases associated with expression of CD19 in a subject.
  • hCAR19-NK cells of the invention are used to treat CD19-expressing cancer in a subject and are capable of reducing the severity of at least one symptom or indication of cancer or inhibiting cancer cell growth.
  • the invention provides methods of treating a disease associated with CD19 expression (eg, CD19-expressing cancer) in a subject, comprising administering to an individual in need thereof a therapeutically effective amount of an hCAR19-NK cell of the invention.
  • a disease associated with CD19 expression eg, CD19-expressing cancer
  • the present invention provides the use of the aforementioned hCAR19-NK cells of the present invention in the preparation of medicaments for treating diseases related to expression of CD19 (eg, cancers that express or overexpress CD19).
  • diseases related to expression of CD19 eg, cancers that express or overexpress CD19.
  • the hCAR19-NK cells of the invention can also be administered to individuals whose cancer has been treated with one or more prior therapies and subsequently relapsed or metastasized.
  • hCAR19-NK cells of the invention are used for parenteral, transdermal, intraluminal, intraarterial, intravenous, intrathecal administration, or direct injection into tissue or tumors.
  • hCAR19-NK cells of the invention are NK cells prepared from autologous NK cells or allogeneic NK cells that express the CAR polypeptide of the invention.
  • the hCAR19-NK cells of the present invention can be administered to a subject at an appropriate dose.
  • the dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, the dosage for any given patient depends on many factors, including the patient's weight, body surface area, age, the specific compound to be administered, sex, time and route of administration, general health, and concurrent medications to be administered. Other medications administered.
  • hCAR19-NK cells of the present invention are expressed as 1 ⁇ 10 6 cells-1 ⁇ 10 12 cells, preferably 5 ⁇ 10 6 cells-1 ⁇ 10 11 cells, more preferably 1 ⁇ Doses of 10 7 cells - 1 x 10 10 immune effector cells, for example 5 x 10 7 cells, 2.5 x 10 8 cells, 1.25 x 10 9 immune effector cells are administered intravenously in single or multiple doses.
  • administration of hCAR19-NK cells of the invention to an individual with cancer results in complete disappearance of the tumor. In some embodiments, administration of hCAR19-NK cells of the invention to an individual with cancer results in a reduction in tumor cells or tumor size of at least 85% or greater. Tumor reduction can be measured by any method known in the art, such as X-ray, positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), cytology, histology, or molecular genetics analyze.
  • PET positron emission tomography
  • CT computed tomography
  • MRI magnetic resonance imaging
  • cytology histology
  • histology histology
  • molecular genetics analyze molecular genetics analyze.
  • CD19-expressing cancers such as hematological malignancies, including but not limited to acute and chronic leukemias, lymphomas, multiple myeloma and Myelodysplastic syndromes; solid tumors, including but not limited to brain, prostate, breast, lung, colon, uterus, skin, liver, bone, pancreas, ovary, testicle, bladder, kidney, head, neck, Tumors of the stomach, cervix, rectum, throat, or esophagus.
  • hematological malignancies including but not limited to acute and chronic leukemias, lymphomas, multiple myeloma and Myelodysplastic syndromes
  • solid tumors including but not limited to brain, prostate, breast, lung, colon, uterus, skin, liver, bone, pancreas, ovary, testicle, bladder, kidney, head, neck, Tumors of the stomach, cervix, rectum, throat, or esophagus.
  • Raji human B lymphocyte; Burkitt lymphoma cell line, CD19 + tumor cell line
  • PG-13 cell lines were purchased from ATCC.
  • K562-GL, Raji-GL, and K562-CD19 cells were constructed by our laboratory.
  • the construction method refers to Kochenderfer J N et al., Construction and preclinical evaluation of an anti-CD19 chimeric antigen receptor[J].J Immunother, 2009, 32(7 ):689-702.DOI:10.1097/CJI.0b013e3181ac6138. All cell lines tested negative for mycoplasma.
  • RPMI 1640 medium and DMEM medium were purchased from Lonza Company. Lymphocyte separation solution was purchased from MP Company of the United States. Fetal bovine serum was purchased from Thermo. Recombinant human interleukin-2 was purchased from Shuanglu Pharmaceutical.
  • Human NK cell isolation kit was purchased from Miltenyi Company.
  • Example 1 Synthesis of CAR gene targeting CD19 and containing humanized scFv and construction of viral expression vector
  • the MSGV-FMC63-28Z recombinant retroviral vector previously constructed in our laboratory was used, which encodes the MSGV (mouse stem cell virus-based splice-gag vector) retroviral backbone and FMC63-28Z CAR.
  • the FMC63-28Z CAR consists of an anti-CD19 scFv derived from FMC63 mouse hybridoma 39; part of the extracellular region, the entire transmembrane and cytoplasmic portion of the human CD28 molecule; and the intracellular component of the human TCR- ⁇ molecule.
  • the exact sequence of the CD28 molecule contained in the FMC63-28Z CAR corresponds to Genbank accession number NM_006139, including all amino acids starting from the amino acid sequence IEVMYPPY to the carboxyl terminus of the protein (Kochenderfer J N, Feldman S A, Zhao Y, et al.
  • CD19 scFv-28Z recombinant retroviral vector the "humanized anti-CD19 scFv-28Z” is also called “hCAR19” herein ( Figure 1), which contains human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor signal sequence (SEQ ID NO:12), humanized anti-CD19 scFv (SEQ ID NO:4), modified CD8a hinge region (SEQ ID NO:5), CD28 transmembrane domain (SEQ ID NO:8) and the coding nucleotide sequences of the CD28 costimulatory domain (SEQ ID NO:9) and the CD3 ⁇ signaling domain (SEQ ID NO:11).
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • SEQ ID NO:4 humanized anti-CD19 scFv
  • modified CD8a hinge region SEQ ID NO:5
  • CD28 transmembrane domain SEQ ID NO:8 and the
  • the "humanized anti-CD19 scFv-28Z" construct in the MSGV-humanized anti-CD19 scFv-28Z recombinant retroviral vector was deleted to obtain an empty vector without hCAR19 construct, which was used as a negative control in the following experiments. Also called Mock control.
  • a plasmid encoding hCAR19 and a plasmid encoding the RD114 envelope protein were used using LIPOFECTAMINE TM 2000 (Life Technologies, Carlsbad, CA). 1996)) transfected PG13 packaging cell system (Kochenderfer J N et al., Construction and preclinical evaluation of an anti-CD19 chimeric antigen receptor [J]. J Immunother, 2009, 32(7): 689-702. DOI: 10.1097/CJI .0b013e3181ac6138). The transfected cells were incubated in antibiotic-free D10 medium at 37°C for 6-8 hours.
  • the medium used for transfection was then replaced with fresh D10 medium and the cells were incubated for an additional 36-48 hours.
  • PG13 cells were cultured on poly-D-lysine-coated dishes (BD Biosciences, San Jose, CA) during and after transfection.
  • the supernatant containing the retrovirus is removed from the plate and centrifuged to remove cellular debris. Store the supernatant at -80°C.
  • the generated retrovirus encoding hCAR19 or Mock control retrovirus was used for subsequent transduction of NK cells.
  • K562 cells were engineered to co-express the following molecules: CD19, 4-1BBL, and membrane-bound IL-21 (mIL-21) to obtain engineered feeder cells, namely, K562 -CD19-4-1BBL-mIL21 cells.
  • K562 human myeloid leukemia cell line
  • ATCC American Type Culture Collection
  • VA Manassas, VA, USA
  • K562 cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco, US), 100 units/mL penicillin and 100 ⁇ g/mL streptomycin (Invitrogen, CA, USA) at 37 °C in a humidified 5% CO2 incubator.
  • FBS heat-inactivated fetal bovine serum
  • Penicillin 100 ⁇ g/mL streptomycin
  • mIL-21 membrane-bound IL-21 containing CD19 encoding the amino acid sequence shown in SEQ ID NO: 13, 4-1BBL encoding the amino acid sequence shown in SEQ ID NO: 14, and the amino acid sequence shown in SEQ ID NO: 15 cDNA sequence, in which the coding nucleotides are connected by the nucleotide coding for the P2A self-cleaving peptide (SEQ ID NO: 16).
  • K562-CD19-4-1BBL-mIL21 cells were generated by transducing K562 cells with a retroviral vector containing cDNA sequences encoding CD19, 4-1BBL, and membrane-bound IL-21 (mIL-21).
  • the FMC63-28Z CAR coding sequence in the MSGV-FMC63-28Z recombinant retroviral vector previously constructed in our laboratory was replaced with one encoding CD19, 4-1BBL and membrane-bound IL-21 (mIL- 21) cDNA sequence to obtain MSGV-CD19-4-1BBL-mIL21 recombinant retroviral vector; and generate retroviral supernatant encoding CD19-4-1BBL-mIL21.
  • Fresh retroviral supernatant encoding CD19-4-1BBL-mIL21 was added to cultured K562 cells for two consecutive days. Cells were then maintained in RPMI-1640 containing 10% fetal calf serum and antibiotics.
  • CD19, 4-1BBL and mIL21 The surface expression of CD19, 4-1BBL and mIL21 was analyzed by flow cytometry, and subcloned cells expressing high levels of CD19, 4-1BBL and mIL21 were enriched as engineered feeder cells to stimulate NK cell expansion.
  • This example illustrates methods of generating hCAR19-expressing NK cells.
  • Peripheral blood samples were collected from multiple healthy volunteers. Peripheral blood mononuclear cells were isolated by centrifugation on a Lymphoprep density gradient (Nycomed, Oslo, Norway) and washed twice in RPMI-1640. To purify primary NK cells from peripheral blood mononuclear cells, CD56 + NK cells were enriched using a human NK cell isolation kit (Miltenyi Biotec).
  • the K562-CD19-4-1BBL-mIL21 cells prepared in Example 2 irradiated with 100Gy gamma rays were placed in a 24-well plate as feeders, and were mixed with enriched CD56 + NK cells by cell.
  • the amplified NK cells were transfected with the hCAR19-encoding retrovirus or Mock control retrovirus prepared in Example 1 to obtain hCAR19-NK cells or Mock control cells.
  • the cell culture medium was replaced with RPMI-1640 complete medium containing IL-2 (400IU/ml), and the K562-CD19-4-1BBL-mIL21 cells prepared in Example 2 irradiated with 100Gy gamma rays were used.
  • As feeder cells hCAR19-NK cells or Mock control cells were cultured.
  • anti-CD19 Fab antibody was used to detect the expression of hCAR19 on the surface of hCAR19-NK cells by flow cytometry, and Western blotting was used to verify whether the transfected hCAR19-NK cells expressed exogenous CD3 ⁇ .
  • NK cells can be engineered to stably express hCAR19 of the present invention.
  • the hCAR19-NK cells of Example 3 were used to detect the specific activation of hCAR19-NK cells by CD19 + tumor cells through the following experiments.
  • IFN- ⁇ intracellular staining experiment Co-culture effector cells (Mock control cells or hCAR19-NK cells) with three types of tumor cells at an effect-to-target ratio of 5:1, and add 1 ⁇ l of GolgiStop TM Protein Transport Inhibitor (Fisher Scientific, BD 554724), cells were collected 6 hours later, and IFN- ⁇ was detected by flow cytometry according to the standard procedure for intracellular staining of IFN- ⁇ .
  • Effector cells (Mock control cells or hCAR19-NK cells) were co-cultured with three types of tumor cells at an effect-to-target ratio of 5:1, and 1 ⁇ l of CD107a antibody (BD-Biosciences) and 1 ⁇ l of GolgiStop were added.
  • TM Protein Transport Inhibitor collect cells after 6 hours, and then stain for CD56 for flow cytometric detection.
  • CD19-expressing immortalized cell lines were used respectively: K562-CD19 (K562 cells genetically engineered to express CD19) and Raji (human B lymphocytes; Burkitt lymphoma, CD19 + tumor cell line, obtained from ATCC); and the CD19-negative cell line K562 (ATCC).
  • hCAR19-NK cells can be specifically activated and express IFN- ⁇ after co-culture with CD19 + tumor cells.
  • hCAR19-NK cells can be specifically activated after co-culture with CD19 + tumor cells and expose CD107a molecules to the surface of NK cells.
  • NK cells can release perforation through degranulation. cleavage of target cells by proteins and granzymes.
  • Example 5 Killing ability and cytokine release ability of hCAR19-NK cells against tumor cells
  • the hCAR19-NK cells of Example 3 were used to detect the killing ability and cytokine release ability of hCAR19-NK cells against tumor cells through the following experiments.
  • Fluorescence killing experiment compare effector cells (Mock control cells or hCAR19-NK cells) with luciferase-expressing tumor cells at different effective-to-target ratios (E:T) 10:1, 5:1, 2.5:1, 1:1.
  • E:T effective-to-target ratio
  • CD19 + tumor cells Raji-GL or CD19 - tumor cells K562-GL (the GL represents "luciferase") were co-cultured for 4 hours, D-luciferin potassium was added to a final concentration of 150 ⁇ g/mL, and Xenogen IVIS was used
  • the imaging system takes fluorescence photos and calculates the killing rate based on the fluorescence value of the tumor cells in the well.
  • killing rate [100-(fluorescence value of co-culture of effector cells and target cells/fluorescence value of target cells) x 100]%.
  • hCAR19-NK cells kill Raji-GL more strongly, while there is no significant difference in the killing of K562-GL between Mock control cells and hCAR19-NK cells.
  • Effector cells (Mock control cells and hCAR19-NK cells) and tumor cells (K562, Raji and K562-CD19) were co-cultured for 24 hours at an effect-to-target ratio of 5:1, and the supernatant was collected and centrifuged. The secretion amount of IFN- ⁇ was detected using ELISA standard method.
  • the IFN- ⁇ release caused by hCAR19-NK cells (1252 ⁇ 77) was significantly higher than the IFN- ⁇ release caused by Mock control cells (586.1 ⁇ 51.8); in In co-cultures where the target cells were K562-CD19 cells, the IFN- ⁇ release caused by hCAR19-NK control cells (4145 ⁇ 94.7) was also significantly higher than the IFN- ⁇ release caused by Mock control cells (790.2 ⁇ 53.3). Statistical significance (P ⁇ 0.001).
  • Example 6 Expansion times of NK cells by engineered feeder cells
  • This example detects the expansion fold of NK cells by the engineered feeder cells prepared in Example 2.
  • Peripheral blood samples were collected from multiple healthy volunteers. Peripheral blood mononuclear cells were isolated by centrifugation on a Lymphoprep density gradient (Nycomed, Oslo, Norway) and washed twice in RPMI-1640. To purify primary NK cells from peripheral blood mononuclear cells, CD56 + NK cells were enriched using a human NK cell isolation kit (Miltenyi Biotec).
  • K562-CD19-4-1BBL-mIL21 cells irradiated with 100Gy gamma rays were placed in a 24-well plate as feeders and mixed with enriched CD56 + NK cells at a ratio of 1:1.
  • the amplified NK cells were transfected with the hCAR19-encoding retrovirus or Mock control retrovirus prepared in Example 1 to obtain hCAR19-NK cells or Mock control cells.
  • the cell culture medium was replaced with RPMI-1640 complete medium containing IL-2 (400IU/ml), and K562-CD19-4-1BBL-mIL21 cells prepared in Example 2 irradiated with 100Gy gamma rays were used.
  • As feeder cells hCAR19-NK cells or Mock control cells were cultured.
  • the expansion folds of NK cells were calculated on days 6, 14 and 21 respectively.
  • the calculation method of "amplification fold” is as follows: divide the absolute number of NK cells on day 14 or 21 of culture by the corresponding number of NK cells on day 6.
  • K562-CD19-4-1BBL-mIL21 cells as feeder cells can expand hCAR19-NK cells or Mock control cells approximately 2000 times on day 21, and there is no significant difference in the expansion folds of these two cells. .
  • Example 7 Study on the anti-tumor effect of NK cells in vivo
  • the Raji cell line is used as the mother cell, and the green fluorescent protein (GFP) and firefly luciferase (LUC) genes are introduced into the Raji cells using a lentiviral infection method to obtain stable expression of green fluorescent protein and firefly fluorescence.
  • the NOD mice were randomly divided into 4 groups.
  • Each group is the vehicle group injected with PBS, the 21st day Mock control cell group obtained in Example 6 without transduction of CD19 CAR, the 21st day hCAR19-NK cell group obtained in Example 6, and the transduction Example 1.
  • the "FMC63 CAR" NK cell group (the preparation and expansion are the same as in Example 6, and the FMC63 CAR-NK cells on day 21 are harvested), each group has 5 mice.
  • Figure 9 shows the therapeutic effect of each NK cell on tumors in immunodeficient tumor-bearing NOD mice inoculated with human Raji tumor cells.
  • the results showed that compared with the vehicle group, each NK cell group induced significant anti-tumor effects, among which the hCAR19-NK cell group showed the largest anti-tumor effect, and was statistically significant with the FMC63 CAR-NK cell group. significance (P ⁇ 0.01).

Abstract

提供表达CD19和至少一种刺激免疫细胞活化的分子的基因工程化饲养细胞、制备所述基因工程化饲养细胞的方法和所述基因工程化饲养细胞用于扩增NK细胞的用途。还提供制备和扩增人源化抗CD19 CAR-NK细胞的方法以及通过所述方法获得的通用型人源化CD19 CAR-NK细胞和其用于临床治疗表达CD19的肿瘤的用途。

Description

用于制备和扩增通用型人源化抗CD19 CAR-NK细胞的方法及其用途 发明领域
本发明涉及基因工程领域。特别地，本发明涉及表达CD19和至少一种刺激免疫细胞活化的分子的基因工程化饲养细胞以及所述基因工程化饲养细胞用于扩增NK细胞的用途。本发明还涉及经工程化以表达人源化抗CD19 CAR的NK细胞、以及制备和扩增它们以达到临床治疗用数量级别的方法。本发明的经工程化的NK细胞为非免疫原性的，能够施与任何受试者而不被宿主免疫系统排斥(它们是“通用型”的)。
背景技术
随着肿瘤免疫学理论和临床技术的发展，嵌合抗原受体(Chimeric antigen receptor，CAR)T细胞疗法成为目前最有发展前景的肿瘤免疫疗法之一。
CD19分子是治疗B淋巴细胞系肿瘤潜在的靶点，也是CAR研究中的热点。在治疗复发性或难治性B细胞恶性肿瘤方面，靶向CD19的CAR-T细胞治疗淋巴瘤的总体有效率在50％-82％之间(Castaneda-Puglianini O等人，Assessing and Management of Neurotoxicity After CAR-T Therapy in Diffuse Large B-Cell Lymphoma[J].J Blood Med,2021,12:775-783.DOI:10.2147/JBM.S281247)。
但是，CAR-T细胞疗法由于其引起的一些毒副作用而使用受限，其中最为常见的毒副作用包括细胞因子释放综合征(cytokine release syndrome，CRS)和神经毒性等。CRS是一种可致命的急性全身性炎症反应，由活化的免疫细胞大量分泌促炎性细胞因子所引起。在靶向CD19的CAR-T细胞治疗的I期和II期临床试验中，有58-92％的患者发生了不同级别的CRS(Daher M等人，CAR-NK cells:the next wave of cellular therapy for cancer[J].Clin Transl Immunology,2021,10(4):e1274.DOI:10.1002/cti2.1274)。CAR-T细胞疗法在实体瘤的治疗中效果不佳。另外，由于存在HLA限制，异体CAR-T细胞还可能导致移植物抗宿主病(GVHD)，而需要采用自体T细胞来制备自体CAR-T细胞使得许多正在进行临床入组评估的患者由于临床上可行的制成品的产量不足而最终无法进行。引起临床上可行的制成品的产量不足的原因包括：不能从患者收获充足和足够的淋巴细 胞和有限的体外细胞扩增。因此，需要进行技术开发来克服CAR-T细胞疗法中存在的这些问题。
自然杀伤细胞(natural killer，NK)是一类重要的先天免疫细胞，它通过立即识别和消除病毒、细菌和寄生虫感染以及异常自体细胞(尤其是癌细胞)来执行身体的初级防御(先天免疫)功能。与通过表达抗原特异性受体来识别靶细胞的T细胞不同，NK细胞感知靶细胞(尤其是癌细胞)的异常变化并具有接触依赖性细胞毒性，NK细胞无需抗原特异性也无需人白细胞抗原(HLA)匹配，所述靶细胞(尤其是癌细胞)的异常变化例如抑制或激活受体的平衡，即杀伤性免疫球蛋白受体(KIR)、天然细胞毒性受体(NCR)、DNAM-1(DNAX辅助分子-1)和NKG2D(NK组2成员D)，表面MHC(主要组织相容性复合体)I类抗原缺失。
由于NK细胞作为天然免疫的效应细胞具有很强的抗肿瘤能力，且不会引起较强的CRS，也不会造成移植物抗宿主病，因此，将癌症抗原特异性CAR基因引入NK细胞来开发CAR-NK细胞治疗具有优越性。NK细胞可以取自与患者无关的健康供体，还可以通过NK细胞天然的肿瘤细胞杀伤机制克服由于抗原逃逸引起的肿瘤复发(Siegler E L等人，Off-the-Shelf CAR-NK Cells for Cancer Immunotherapy[J].Cell Stem Cell,2018,23(2):160-161.DOI:10.1016/j.stem.2018.07.007)。
但是，CAR-NK细胞疗法的广泛临床成功在一定程度上受到制造大剂量CAR-NK细胞的挑战的限制以及表达的CAR分子中鼠源scFv的免疫原性可能会造成CAR-NK细胞在体内被排斥或引起过敏反应，导致细胞CAR-NK细胞无法持续存活的风险。
因此，本领域仍有开发表达通用型人源化抗CD19 CAR的NK细胞和对其进行大量扩增以用于临床的迫切需要。
发明概述
本发明人通过研究，首次构建了一种包含人源化抗CD19 scFv的第二代CD19 CAR，并且在体外制备了表达人源化抗CD19 CAR的NK细胞(在本发明中，也简称为hCAR19-NK细胞)，并通过基因工程化饲养细胞对其进行了体外大量扩增。
本发明具体地涉及以下方面。在第一方面，本发明提供了一种基因工程化饲养细胞，其是通过基因修饰永生化细胞以表达CD19和至少一种刺激免疫细胞活化的分子而获得 的，所述基因工程化饲养细胞不表达主要组织相容性复合物(MHC)I分子，并且其中所述基因工程化饲养细胞与NK细胞的共培养导致NK细胞的活化和扩增。
在一些实施方案中，本发明提供了一种基因工程化饲养细胞，其是通过基因修饰例如K562髓性白血病细胞、HL-60人早幼粒白血病细胞或OCI-AML3人急性髓细胞性白血病细胞以表达CD19和至少一种刺激免疫细胞活化的分子而获得的，所述至少一种刺激免疫细胞活化的分子例如选自4-1BBL、膜结合的IL-12、膜结合的IL-7、膜结合的IL-15(mIL15)、膜结合的IL-18(mIL18)、膜结合的IL-21(mIL-21)、膜结合的IL-22(mIL-22)。
在一些实施方案中，本发明的基因工程化饲养细胞是经遗传修饰以表达CD19、4-1BBL和膜结合的IL-21(mIL-21)的K562细胞。
在一些实施方案中，对本发明的基因工程化饲养细胞采用病毒载体(例如，逆转录病毒载体)进行遗传修饰，使其表达SEQ ID NO:13或与SEQ ID NO:13具有至少90％或至少95％序列同一性的氨基酸序列的CD19、SEQ ID NO:14或与SEQ ID NO:14具有至少90％或至少95％序列同一性的氨基酸序列的4-1BBL和SEQ ID NO:15或与SEQ ID NO:15具有至少90％或至少95％序列同一性的氨基酸序列的膜结合的IL-21(mIL-21)。
在第二方面，本发明提供了制备本发明第一方面的基因工程化饲养细胞的方法，所述方法包括：
用编码CD19和至少一种刺激免疫细胞活化的分子的核酸转导或转染癌细胞，其中所述至少一种刺激免疫细胞活化的分子例如选自4-1BBL、膜结合的IL-12、膜结合的IL-7、膜结合的IL-15(mIL15)、膜结合的IL-18(mIL18)、膜结合的IL-21(mIL-21)、膜结合的IL-22(mIL-22)，所述癌细胞例如K562髓性白血病细胞、HL-60细胞、OCI-AML3细胞；
分离表达所述CD19和至少一种刺激免疫细胞活化的分子的细胞；以及
辐照所分离的细胞，从而产生基因工程化饲养细胞。
在第三方面，本发明提供了本发明第一方面的基因工程化饲养细胞的用途，用于扩增NK细胞，其中所述NK细胞可以是天然存在的NK细胞，也可以是经遗传修饰的NK细胞。在一些实施方案中，待扩增的天然存在的NK细胞分离自或者包含在外周血液、骨髓、淋巴结组织、脐带血、胸腺组织、感染部位组织、腹水、胸腔积液、脾组织、或肿瘤中。在一些实施方案中，待扩增的经遗传修饰的NK细胞是CAR-NK细胞，例如，人 源化抗CD19 CAR-NK细胞。
在第四方面，本发明提供了用于制备和扩增人源化抗CD19 CAR-NK细胞的方法，包括：
a)将经辐照(例如，伽马射线照射)的本发明第一方面的基因工程化饲养细胞与天然存在的NK细胞共培养；例如，共培养2-10天，例如，共培养6天，例如，所述天然存在的NK细胞分离自或者包含在外周血液、骨髓、淋巴结组织、脐带血、胸腺组织、感染部位组织、腹水、胸腔积液、脾组织、或肿瘤中；
b)将编码人源化抗CD19 CAR的核酸序列导入NK细胞中，获得人源化抗CD19 CAR-NK细胞(即，hCAR19-NK细胞)，例如，通过逆转录病毒将编码人源化抗CD19 CAR的核酸序列导入NK细胞中；以及
c)将人源化抗CD19 CAR-NK细胞与经辐照(例如，伽马射线照射)的本发明第一方面的基因工程化饲养细胞和任选地IL-2共培养足以扩增人源化抗CD19 CAR-NK细胞的一段时间(例如，7-42天，例如，14-28天)，从而扩增人源化抗CD19 CAR-NK细胞；例如，所述人源化抗CD19 CAR-NK细胞扩增至少1500至5000倍。
在一些实施方案中，hCAR19-NK细胞表达的人源化抗CD19 CAR包含：
(1)人源化抗CD19 scFv序列，其中所述scFv序列特异性结合CD19且包含：
(i)轻链可变区，其包含SEQ ID NO:1的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，和
(ii)重链可变区，其包含SEQ ID NO:2的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；
例如，(i)轻链可变区，其包含SEQ ID NO:1的序列，和
(ii)重链可变区，其包含SEQ ID NO:2的序列；
(2)铰链区/间隔区，其选自CD8a铰链区(SEQ ID NO 5)，或其具有至少90％、至少95％的序列同一性的CD8铰链区；
(3)跨膜区(TM)，其选自
(i)SEQ ID NO:6所示的CD4跨膜结构域或其具有1-5个氨基酸修饰的变体；
(ii)SEQ ID NO:7所示的CD8跨膜结构域或其具有1-5个氨基酸修饰的变体；
(iii)SEQ ID NO:8所示的CD28跨膜结构域或其具有1-5个氨基酸修饰的变体；
(4)共刺激信号结构域(CSD)，其是：
(i)SEQ ID NO:9所示的CD28共刺激结构域或其具有1-5个氨基酸修饰的变体；和、或
(ii)SEQ ID NO:10所示的4-1BB共刺激结构域或其具有1-5个氨基酸修饰的变体；
(5)刺激信号结构域(SSD)，为SEQ ID NO:11所示的CD3ζ信号传导结构域或其具有1-5个氨基酸修饰的变体；
其中所述氨基酸修饰是氨基酸的添加、缺失或取代。
在一些具体实施方案中，hCAR19-NK细胞表达的人源化抗CD19 CAR包含：
(1)人源化抗CD19 scFv序列，其包含：SEQ ID NO:4的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，
(2)铰链区/间隔区，为SEQ ID NO 5所示的CD8a铰链区或其具有1个氨基酸修饰的变体；
(3)跨膜区(TM)，为SEQ ID NO:8所示的CD28跨膜结构域或其具有1个氨基酸修饰的变体；
(4)共刺激信号结构域(CSD)，为SEQ ID NO:9所示的CD28共刺激结构域或其具有1个氨基酸修饰的变体；
(5)刺激信号结构域(SSD)，为SEQ ID NO:11所示的CD3ζ信号传导结构域或其具有1个氨基酸修饰的变体；
其中所述氨基酸修饰是氨基酸的添加、缺失或取代。
在第五方面，本发明提供了通过本发明第四方面的方法制备和扩增的人源化抗CD19CAR-NK细胞，其与表达CD19的肿瘤细胞接触后，表达IFN-γ、发生脱颗粒，并杀伤表达CD19的肿瘤细胞。
在第六方面，本发明提供了本发明第五方面的人源化抗CD19 CAR-NK细胞的用途，用于制备治疗表达CD19的肿瘤的药物。本发明的人源化抗CD19 CAR-NK细胞能够与肿瘤细胞上的CD19结合，并且破坏所述肿瘤细胞。
在一些实施方案中，表达CD19的肿瘤选自多发性骨髓瘤、白血病、淋巴瘤或实体瘤，例如，所述白血病为急性淋巴细胞白血病(ALL)或急性髓系白血病(AML)，所述淋巴瘤为非霍奇金淋巴瘤，所述实体瘤为脑部、前列腺、乳腺、肺部、结肠、子宫、皮肤、肝脏、 骨头、胰脏、卵巢、睾丸、膀胱、肾脏、头部、颈部、胃部、子宫颈、直肠、喉部、或食道的肿瘤。
附图简述
结合以下附图一起阅读时，将更好地理解以下详细描述的本发明的优选实施方案。出于说明本发明的目的，图中显示了目前优选的实施方案。然而，应当理解本发明不限于图中所示实施方案的精确安排和手段。
图1显示了“人源化抗CD19 scFv-28Z”CAR(在本文中也称为“hCAR19”)的组成结构域的示意图。图中，5’LTR和3’LTR分别表示5’端和3’端的长末端重复序列；CD8a代表CD8a铰链区；CD28代表CD28跨膜结构域和CD28共刺激结构域；ζchain代表CD3ζ信号传导结构域。
图2显示了使用包含hCAR19编码序列的逆转录病毒转染NK细胞后，流式细胞术检测hCAR19的表达结果。图中，“Mock”表示使用不包含hCAR19编码序列的逆转录病毒转染NK细胞后获得的Mock对照细胞。
图3显示了使用包含hCAR19编码序列的逆转录病毒转染NK细胞后，用抗CD3ζmAb通过蛋白质印迹检测经转染细胞的外源性CD3ζ的表达结果。图中从左到右分别为泳道1：分子量标志物；泳道2：Mock对照细胞；泳道3：hCAR19-NK细胞。
图4显示了Mock对照细胞和hCAR19-NK细胞与K562、Raji或K562-CD19细胞共培养6小时后，通过流式细胞术检测IFN-γ的产生。
图5显示了Mock对照细胞和hCAR19-NK细胞与K562、Raji或K562-CD19细胞共培养6小时后，通过流式细胞术检测NK细胞表面的CD107a分子。
图6显示了Mock对照细胞或hCAR19-NK细胞与CD19 +细胞系(Raji-GL)和CD19 -细胞系(K562-GL)以不同E:T比共培养4小时后，对靶细胞的细胞毒性。结果显示为来自三个独立实验的平均值±SD(**P<0.001，Mock对照细胞vs hCAR19-NK细胞，n＝3)。
图7显示了Mock对照细胞或hCAR19-NK细胞与CD19 +细胞系(Raji和K562-CD19)或CD19 -细胞系(K562)以E:T比5:1共培养24小时后，检测Mock对照细胞和hCAR19-NK细胞的IFN-γ产生。结果显示为来自三个独立实验的平均值±SD(**P<0.001，Mock对照细胞vs hCAR19-NK细胞，n＝3)。
图8显示了基因工程化饲养细胞体外扩增hCAR19-NK细胞和Mock对照细胞。培养至第21天，hCAR19-NK细胞和Mock对照细胞的扩增倍数没有显著性差异。结果显示为三个独立实验的平均值±SD(P>0.05，Mock对照细胞vs hCAR19-NK细胞，n＝3)。
图9显示了Raji-GFP-LUC肿瘤细胞接种小鼠后，载剂组(黑色线)、Mock对照细胞组(棕黄色线)、hCAR19-NK细胞组(红色线)、FMC63 CAR-NK细胞组(蓝色线)的小鼠的存活百分数，由此显示各NK细胞对肿瘤的治疗效应。
发明详述
除非另外限定，否则本文中所用的全部技术与科学术语具有如本发明所属领域的普通技术人员通常理解的相同含义。本文所提及的全部出版物、专利申请、专利和其他参考文献通过引用的方式完整地并入。此外，本文中所述的材料、方法和例子仅是说明性的并且不意在是限制性的。本发明的其他特征、目的和优点将从本说明书及附图并且从后附的权利要求书中显而易见。
I.定义
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值。
如本文中所用，术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。
术语“CD19”是指分化簇19蛋白质，其是在白血病前体细胞上可检测的抗原决定簇。人和鼠氨基酸和核酸序列可以在诸如GenBank、UniProt和Swiss-Prot的公共数据库中找到。例如，人CD19的氨基酸序列可以以UniProt/Swiss-Prot登录号P15391(全长为556个氨基酸)找到，编码人CD19的核苷酸序列可以以登录号NM_001178098找到。CD19在大多数B谱系癌症上表达，包括例如急性淋巴细胞性白血病、慢性淋巴细胞白血病和非霍奇金淋巴瘤。
术语“嵌合抗原受体(CAR)”是指一种具有细胞外结构域的嵌合融合蛋白，所述细胞外结构域通过跨膜结构域与能够激活细胞的细胞内信号转导结构域融合。CAR分子的细胞内信号转导结构域包括两个不同的细胞质信号转导结构域。例如，一个信号转导结构域可以是细胞质效应功能信号转导结构域，第二个信号转导结构域可以是细胞质共刺激信 号转导结构域。接头可将结构域彼此连接。
术语“饲养细胞”是指在离体或体外培养中向另一细胞类型提供支持的细胞。饲养细胞可提供用饲养细胞培养的细胞的存活、生长和/或分化所需的一种或多种因子。通常，辐照或者另外处理饲养细胞以防止其在培养物中增殖。在本文所述的一些实施方案中，用饲养细胞如辐照的经修饰的K562细胞培养NK细胞。
术语“扩增”是指来自初始细胞群的特征细胞类型的数量的提高。
如本文中所用，免疫细胞的“活化”或“激活”是指免疫细胞响应并在可测量的水平上表现出本领域技术人员已知的相应细胞的免疫功能的能力。测量免疫细胞活性的方法也是本领域技术人员已知的。
术语“自然杀伤(NK)细胞”是指免疫系统的一种细胞，其在不存在特定抗原刺激物并且不受根据MHC类别限制的情况下杀伤靶细胞。靶细胞可以是肿瘤细胞或携带病毒的细胞。NK细胞的特征在于存在CD56表面标志物以及不存在CD3表面标志物。NK细胞通常约占正常外周血单个核细胞的10％至15％。认为NK细胞提供针对病毒和肿瘤的“后备”保护机制，这些病毒和肿瘤可能通过下调MHC I类呈递分子而逃避细胞毒性T淋巴细胞(CTL)反应。除了参与直接细胞毒性杀伤外，NK细胞通过产生细胞因子发挥作用。
可以通过“转染”、“转化”或“转导”将编码本发明的CAR的核酸序列引入细胞。本文使用的术语“转染”、“转化”或“转导”指通过利用物理或化学方法将一种或多种外源多核苷酸引入宿主细胞。许多转染技术在本领域是已知的，并且其包括，例如磷酸钙DNA共沉淀(参见，例如Murray E.J.(编著)，Methods in Molecular Biology，第7卷，Gene Transfer and Expression Protocols,Humana Press(1991))；DEAE-葡聚糖；电穿孔；阳离子脂质体介导的转染；钨颗粒促进的微粒轰击(Johnston,Nature,346:776-777(1990))；以及磷酸锶DNA共沉淀(Brash等人，Mol.Cell Biol.,7:2031-2034(1987))。在感染性颗粒于合适的包装细胞(它们中的许多可商购获得)中生长之后，可将噬菌体载体或病毒载体引入宿主细胞。
术语“CAR-NK细胞”是指用编码CAR的异源核酸转导的NK细胞。
术语“scFv”指一种融合蛋白，其包含至少一个包含轻链可变区的抗体片段和至少一个包含重链可变区的抗体片段，其中轻链可变区和重链可变区任选地借助柔性短多肽接头连续地连接，并且能够表达为单链多肽，并且其中scFv保留衍生它的完整抗体的特异性。 除非另外指出，否则如本文所用，scFv可以具有按任何顺序(例如，相对于多肽的N末端和C末端)的VL可变区和VH可变区，scFv可以包含VL-接头-VH或可以包含VH-接头-VL。
术语“CD3ζ”定义为GenBan登录号BAG36664.1提供的蛋白质或其等同物，并且“CD3ζ胞内信号传导结构域”定义为来自CD3ζ链胞质结构域的氨基酸残基，所述氨基酸残基足以在功能上传播细胞活化必需的初始信号。在一个实施方案中，CD3ζ的胞质结构域包含GenBank登录号BAG36664.1的残基52至残基164或作为其功能直向同源物的来自非人类物种(例如，小鼠、啮齿类、猴、猿等)的等同残基。在一个实施方案中，“CD3ζ刺激信号结构域”是在SEQ ID NO:11中提供的序列或其变体。
术语“共刺激分子”是指细胞上的与共刺激配体特异性结合从而介导细胞的共刺激反应(例如但不限于增殖)的相应结合配偶体。共刺激分子是除抗原受体或其配体之外的有助于有效免疫应答的细胞表面分子。共刺激分子包括但不限于MHC I类分子、TNF受体蛋白、免疫球蛋白样蛋白、细胞因子受体、整联蛋白、信号传导淋巴细胞活化分子(SLAM蛋白)、激活NK细胞受体、OX40、CD40、GITR、4-1BB(即CD137)、CD27和CD28。在一些实施方案中，“共刺激分子”是4-1BB(即CD137)或CD28。共刺激信号结构域是指共刺激分子的胞内部分。
术语“信号传导途径”指在从细胞一个部分传播信号至细胞的另一个部分中发挥作用的多种信号传导分子之间的生物化学关系。
术语“细胞因子”是由一种细胞群释放，作为细胞间介质作用于另一细胞的蛋白质的通称。此类细胞因子的例子有淋巴因子、单核因子、白介素(IL)，诸如IL-1，IL-1α，IL-2，IL-3，IL-4，IL-5，IL-6，IL-7，IL-8，IL-9，IL-11，IL-12，IL-15；肿瘤坏死因子，诸如TNF-α或TNF-β；及其它多肽因子，包括γ-干扰素。
本文所用的术语“同源性”或“同一性”指两个给定氨基酸序列或核苷酸序列之间的关联度，并且可以表示为百分比。术语同源性和同一性通常可以互换使用。
为确定两个氨基酸序列或两个核酸序列的同一性百分数，将所述序列出于最佳比较目的比对(例如，可以为了最佳比对而在第一和第二氨基酸序列或核酸序列之一或二者中引入空位或可以为比较目的而抛弃非同源序列)。在一个优选实施方案中，为比较目的，所比对的参考序列的长度是至少30％、优选地至少40％、更优选地至少50％、60％和甚至 更优选地至少70％、80％、90％、100％的参考序列长度。随后比较在对应氨基酸位置或核苷酸位置处的氨基酸残基或核苷酸。当第一序列中的位置由第二序列中对应位置处的相同氨基酸残基或核苷酸占据时，则所述分子在这个位置处是相同的。
可以利用数学算法实现两个序列间的序列比较和同一性百分数的计算。在一个优选实施方案中，使用已经集成至GCG软件包的GAP程序中的Needlema和Wunsch((1970)J.Mol.Biol.48:444-453)算法(在http://www.gcg.com可获得)，使用Blossum 62矩阵或PAM250矩阵和空位权重16、14、12、10、8、6或4和长度权重1、2、3、4、5或6，确定两个氨基酸序列之间的同一性百分数。在又一个优选的实施方案中，使用GCG软件包中的GAP程序(在http://www.gcg.com可获得)，使用NWSgapdna.CMP矩阵和空位权重40、50、60、70或80和长度权重1、2、3、4、5或6，确定两个核苷酸序列之间的同一性百分数。特别优选的参数集合(和除非另外说明否则应当使用的一个参数集合)是采用空位罚分12、空位延伸罚分4和移码空位罚分5的Blossum 62评分矩阵。
还可以使用PAM120加权余数表、空位长度罚分12，空位罚分4)，利用已经并入ALIGN程序(2.0版)的E.Meyers和W.Miller算法,((1989)CABIOS,4:11-17)确定两个氨基酸序列或核苷酸序列之间的同一性百分数。
额外地或备选地，可以进一步使用本文所述的核酸序列和蛋白质序列作为“查询序列”以针对公共数据库执行检索，以例如鉴定其他家族成员序列或相关序列。
术语“氨基酸变化”和“氨基酸修饰”可互换地使用，是指氨基酸的添加、缺失、取代和其他修饰。可以进行氨基酸的添加、缺失、取代和其他修饰的任意组合，条件是最终的多肽序列具有所需的特性。氨基酸取代包括用非天然存在的氨基酸或二十种标准氨基酸的天然存在的氨基酸衍生物(例如、4-羟基脯氨酸、3-甲基组氨酸、鸟氨酸、高丝氨酸、5-羟基赖氨酸)的取代。可以使用本领域公知的遗传或化学方法产生氨基酸变化。遗传方法可包括定点诱变、PCR、基因合成等。通过除基因工程化之外的方法(如化学修饰)改变氨基酸侧链基团的方法可能是有用的。
术语“保守序列修饰”、“保守序列变化”指未显著影响或改变含有氨基酸序列的抗体或抗体片段的结合特征的氨基酸修饰或变化。这类种保守修饰包括氨基酸取代、添加和缺失。可以通过本领域已知的标准技术，如位点定向诱变和PCR介导的诱变向本发明的抗体或抗体片段引入修饰。保守性取代是氨基酸残基由具有相似侧链的氨基酸残基替换的 氨基酸取代。已经在本领域中定义了具有相似侧链的氨基酸残基的家族。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸、组氨酸)、酸性侧链(例如，天冬氨酸、谷氨酸)、不带电荷极性侧链(例如，甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如，丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、蛋氨酸)、β-侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因而，可以将本发明CAR内部的一个或多个氨基酸残基替换为来自相同侧链家族的其他氨基酸残基，并且可以使用本文所述的功能测定法测试改变的CAR。
术语“自体的”指这样的任何物质，所述物质从稍后将向个体再次引入所述物质的相同个体衍生。
术语“同种异体的”指这样的任何物质，所述物质从与引入所述物质的个体相同的物种的不同动物衍生。当一个或多个基因座处的基因不相同时，两位或更多位个体据称彼此是同种异体的。在一些方面，来自相同物种的个体的同种异体物质可以在遗传上足够地不相似以发生抗原性相互作用。
术语“异种的”指从不同物种的动物衍生的移植物。
II.本发明的基因工程化饲养细胞
本发明提供了用于扩增和活化NK细胞的基因工程化饲养细胞。例如，在一些实施方案中，基因工程化饲养细胞不表达主要组织相容性复合物(major histocompatibility complex，MHC)I分子，其与NK细胞的共培养导致NK细胞的活化和扩增。
在一些实施方案中，基因工程化饲养细胞来源于永生的细胞系。例如，在培养中表现出永生细胞的一种或更多种特征的细胞系。
在一些实施方案中，基因工程化饲养细胞来源于癌细胞，其被修饰以表达促进和/或增强NK细胞活化和/或扩增的一种或更多种分子。
在一些实施方案中，基因工程化饲养细胞表达膜结合白细胞介素-21(mbIL21)。在另外一些实施方案中，除了mbIL21或代替mbIL21，基因工程化饲养细胞被修饰以表达膜结合4-1BB配体(4-1BBL)。在另一些实施方案中，除了或代替mbIL21、4-1BBL和/或其 他活化/扩增促进因子，基因工程化饲养细胞被修饰以表达至少一种刺激NK细胞活化的另外的膜结合白细胞介素。
在一些实施方案中，基因工程化饲养细胞的群体至少包含表达mbIL21的多个第一细胞和表达4-1BBL的多个第二细胞，使得所述基因工程化饲养细胞的群体整体表达mbIL21和4-1BBL二者。在另一些实施方案中，基因工程化饲养细胞的群体包含表达mbIL21和4-1BBL二者的多个细胞。在另一些实施方案中，基因工程化饲养细胞包含表达mbIL21的一些细胞，表达4-1BBL的一些细胞，以及表达这二者的一些细胞。在一些实施方案中，除了或代替mbIL21和/或4-1BBL，可另外地表达其他配体和/或活化因子。
在一些实施方案中，由基因工程化饲养细胞表达的mbIL21具有如SEQ ID NO.15所示的氨基酸序列。在一些实施方案中，由基因工程化饲养细胞表达的4-1BBL具有如SEQ ID NO.14所示的氨基酸序列。
在一些实施方案中，基因工程化饲养细胞来源于选自以下的细胞系：K562细胞、HL-60细胞、OCI-AML3细胞。
在一些实施方案中，基因工程化饲养细胞还表达其他白细胞介素分子，例如，膜结合的IL-12、膜结合的IL-7、膜结合的IL-15(mIL15)、膜结合的IL-18(mIL18)、膜结合的IL-21(mIL-21)、膜结合的IL-22(mIL-22)。可以以多种组合表达各种白细胞介素的组合。
在一些实施方案中，基因工程化饲养细胞还表达CD19，其中所述表达的CD19与本发明的hCAR19-NK细胞接触后，通过与hCAR19所编码的人源化抗CD19 scFv特异性结合而特异性活化和/或扩增hCAR19-NK细胞。在一些实施方案中，基因工程化饲养细胞表达的CD19CD19具有如SEQ ID NO.13所示的氨基酸序列。
本发明还提供了制备本发明的基因工程化饲养细胞的方法。通过用至少一种异源核酸(如编码CD19、4-1BBL、膜结合的IL-12、膜结合的IL-7、膜结合的IL-15(mIL15)、膜结合的IL-18(mIL18)、膜结合的IL-21(mIL-21)、膜结合的IL-22(mIL-22)中的一或多种核酸)转导或转染饲养细胞(例如，K562细胞)，产生基因工程化或重组的饲养细胞。在具体的非限制性实例中，基因工程化饲养细胞包含编码mIL-21的异源核酸、编码CD19的异源核酸、编码4-1BBL的异源核酸。
在一些实施方案中，用包含至少一种异源核酸的载体(如慢病毒或逆转录病毒载体)转导或转化饲养细胞(例如，K562细胞)。例如，用至少一种、至少2种、至少3种、至 少4种或更多种选自编码CD19、4-1BBL、膜结合的IL-12、膜结合的IL-7、膜结合的IL-15(mIL15)、膜结合的IL-18(mIL18)、膜结合的IL-21(mIL-21)、膜结合的IL-22(mIL-22)的异源核酸转导或转染饲养细胞(例如，K562细胞)。
可以使用任意转导或转染方法，如病毒转导(例如使用逆转录病毒如MoMLV或慢病毒)或非病毒转导、mRNA转染。
在一些实施方案中，用病毒载体将至少一种异源核酸递送至饲养细胞(例如，K562细胞)中。适宜的病毒载体的实例包括逆转录病毒(例如MoMLV或慢病毒)载体、腺病毒载体、腺相关病毒载体和痘苗病毒载体。在具体的实例中，使用逆转录病毒系统将一种或多种异源核酸引入饲养细胞(例如，K562细胞)中。在一些实例中，可以使用MoMLV载体，如SFG逆转录病毒载体。SFG载体来源于鼠白血病病毒(MLV)骨架。这种类型的基于鼠白血病病毒(MLV)的逆转录病毒载体是常用的基因递送运载体并且已经广泛用于临床试验。当前的SFG载体针对淋巴细胞遗传修饰、蛋白质表达和病毒滴度的基因表达进行了全面优化。
在一些实施方案中，用编码mbIL21的第一构建体转导细胞，从而产生第一经转导的细胞群并扩增该第一经转导的细胞群；用编码4-1BBL的第二构建体转导第一经转导的细胞群，从而产生第二经转导的细胞群；用编码CD19的第三构建体转导第二经转导的细胞群，从而产生第三经转导的细胞群，以及扩增第三经转导的细胞群。在一些实施方案中，通过用编码mbIL21的第一构建体、编码4-1BBL的第二构建体和编码CD19的第三构建体同时转导细胞群来制备本发明的基因工程化饲养细胞。
在一些实施方案中，基因工程化饲养细胞可通过包括用编码mbIL21、4-1BB、CD19，以及膜结合的IL-12、膜结合的IL-7、膜结合的IL-15(mIL15)、膜结合的IL-18(mIL18)、膜结合的IL-22(mIL-22)中的一种或更多种的构建体转导细胞的方法来制备。
本发明还提供了通过用包含编码CD19、膜结合的IL-21(mIL-21)和4-1BBL的cDNA序列的逆转录病毒载体转导K562细胞来产生基因工程化饲养细胞。
通过流式细胞术分析CD19、膜结合的IL-21(mIL-21)和4-1BBL的表面表达。通过流式细胞术富集表达高水平转基因的亚克隆并将其用于刺激NK细胞扩增。
III.本发明的hCAR19-NK细胞
NK细胞作为天然杀伤细胞，具有利用其自身优势克服CAR-T细胞的缺点。首先，异基因NK细胞输注既不会产生GVHD，也不会产生明显的毒副作用，相较于CAR-T会更安全，也不会受限于自体细胞。其次，除了通过CAR依赖途径抑制肿瘤细胞之外，CAR-NK细胞还可以通过NK细胞受体依赖途径更有效地杀伤残留的肿瘤细胞，减少复发率。最后，NK细胞来源多样化，可以从外周血(PB)、脐带血(UCB)、人胚胎干细胞(HESC)以及诱导多能干细胞(IPSC)，甚至NK-92细胞系中产生(Marofi F等人，CAR-engineered NK cells；a promising therapeutic option for treatment of hematological malignancies[J].Stem Cell Res Ther,2021,12(1):374.DOI:10.1186/s13287-021-02462-y)，因此，构建CAR-NK细胞可为肿瘤免疫治疗开辟更广阔的前景。
CAR-NK细胞免疫治疗的主要局限性之一是在没有细胞因子的支持下，输注的细胞缺乏体内持久性，这将会影响CAR-NK细胞治疗的疗效。有研究表明鼠源scFv的免疫原性可能会造成CAR-T细胞在体内产生排斥或引起过敏反应，导致CAR-T细胞无法持续存活(Turtle C J等人，CD19 CAR-T cells of defined CD4+:CD8+composition in adult B cell ALL patients[J].J Clin Invest,2016,126(6):2123-2138.DOI:10.1172/JCI85309)，影响治疗效果。因此，本发明构建了人源化抗CD19 scFv来降低人体对CAR产生的免疫应答，同时在体外活化和/或扩增hCAR19-NK细胞，以提高hCAR19-NK细胞在体内的持久性，从而提高疗效。
本发明成功地从外周血分离纯化出NK细胞，并且在辐照后(例如，100Gy辐照后)的基因工程化饲养细胞(例如，K562-CD19-4-1BBL-mIL21细胞)刺激下体外扩增。本发明还构建了包含靶向CD19的人源化抗CD19 scFv的CAR，经过逆转录病毒包装所述CAR、转导入NK细胞中，并扩增hCAR19-NK细胞，获得了足够数量的hCAR19-NK细胞，体外实验证明了这种hCAR19-NK细胞可以有效杀伤CD19+肿瘤细胞并分泌大量IFN-γ，并且CD19+肿瘤细胞可以特异性刺激hCAR19-NK细胞表达IFN-γ和脱颗粒，为hCAR19-NK细胞在体内的功能验证奠定了基础。
本发明采用外周血分离的NK细胞首次构建了靶向CD19的人源化CAR-NK细胞，并验证了其可以特异性杀伤淋巴瘤细胞。
具体地，本发明的人源化抗CD19 CAR包含
(1)人源化抗CD19 scFv序列，其中所述scFv序列特异性结合CD19且包含：
(i)轻链可变区，其包含SEQ ID NO:1的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，和
(ii)重链可变区，其包含SEQ ID NO:2的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；
(2)铰链区/间隔区，其选自CD8a铰链区(SEQ ID NO 5)，或其具有至少90％、至少95％的序列同一性的CD8铰链区；
(3)跨膜区I，其选自
(i)SEQ ID NO:6所示的CD4跨膜结构域或其具有1-5个氨基酸修饰的变体；
(ii)SEQ ID NO:7所示的CD8跨膜结构域或其具有1-5个氨基酸修饰的变体；
(iii)SEQ ID NO:8所示的CD28跨膜结构域或其具有1-5个氨基酸修饰的变体；
(4)共刺激信号结构域(CSD)，其是：
(i)SEQ ID NO:9所示的CD28共刺激结构域或其具有1-5个氨基酸修饰的变体；和、或
(ii)SEQ ID NO:10所示的4-1BB共刺激结构域或其具有1-5个氨基酸修饰的变体；
(5)刺激信号结构域(SSD)，为SEQ ID NO:11所示的CD3ζ信号传导结构域或其具有1-5个氨基酸修饰的变体；
其中所述氨基酸修饰是氨基酸的添加、缺失或取代；
在一个具体实施方案中，本发明的人源化抗CD19 CAR包含
(1)SEQ ID NO:4所示的人源化抗CD19 scFv序列；
(2)SEQ ID NO 5所示的CD8a铰链区；
(3SEQ ID NO:8所示的CD28跨膜结构域；
(4)SEQ ID NO:9所示的CD28共刺激结构域；
(5)SEQ ID NO:11所示的CD3ζ信号传导结构域。
本领域普通技术人员将理解，本发明的CAR多肽还可以进行修饰，从而在氨基酸序列上变动，但是在所需的活性方面不变动。例如，可以对CAR多肽进行导致“非必需”氨基酸残基处氨基酸置换的额外核苷酸置换。例如，可以将分子中的非必需氨基酸残基替换为来自相同侧链家族的另一个氨基酸残基。在另一个实施方案中，可以将一个氨基酸片 段替换为结构上相似的片段，所述的片段在侧链家族成员的顺序和组成方面中不同，例如，可以进行保守性置换，其中将氨基酸残基替换为具有相似侧链的氨基酸残基。
通过将编码本发明的人源化抗CD19 CAR的核酸序列导入NK细胞中，能够获得人源化抗CD19 CAR-NK细胞。
本领域已经开发了众多基于病毒的系统用于转移基因至哺乳动物细胞中。例如，逆转录病毒提供了用于基因递送系统的便利平台。可以使用本领域已知的技术，将选择的基因插入载体并且包装在逆转录病毒粒子中。随后可以分离重组病毒并将其在体内或离体递送至受试者的细胞。众多逆转录病毒系统是本领域已知的。
衍生自逆转录病毒的载体是实现长期基因转移的合适工具，因为它们允许转基因的长期、稳定整合和其在子代细胞中增殖。逆转录病毒载体可以例如是γ逆转录病毒载体。Γ逆转录病毒载体可以例如包含启动子、包装信号(ψ)、引物结合位点(PBS)、一个或多个(例如，两个)长末端重复序列(LTR)和目的转基因，例如，编码CAR的基因。Γ逆转录病毒载体可以缺少病毒结构性基因如gag、pol和env。
在一些实施方案中，本发明提供了在哺乳动物NK细胞中表达本发明的CAR的方法和由此产生的hCAR19-NK细胞。
在一些实施方案中，本发明构建了人源化的第二代CD19 CAR的逆转录病毒载体；使用经辐照的K562-CD19-4-1BBL-mIL21细胞刺激外周血来源的NK细胞，然后通过逆转录病毒转导NK细胞获得hCAR19-NK细胞。本发明通过流式细胞术和Western blot检测了CAR对NK细胞的转导效率，表明了本发明构建的CAR可以成功转导外周血来源的NK细胞。
由于产生足够数量的hCAR19-NK细胞仍然是患者过继性免疫治疗所需要的。因此，本发明进一步提供了体外扩增hCAR19-NK细胞的方法。
将本发明的基因工程化饲养细胞(例如，K562-CD19-4-1BBL-mIL21细胞)进行致命照射，以确保无增殖和无存活的饲养细胞；然后与hCAR19-NK细胞共培养一段时间(例如，7-42天，例如，14-28天)，从而扩增hCAR19-NK细胞。在一些实施方案中，本发明的hCAR19-NK细胞能够体外扩增至少1500至5000倍。
采用CD107a脱颗粒实验和IFN-γ胞内染色评估了表达CD19的淋巴瘤细胞对hCAR19-NK细胞的特异性激活；以及采用荧光杀伤实验和ELISA法检测了hCAR19-NK 细胞对淋巴瘤细胞的杀伤能力和细胞因子释放水平。结果表明，hCAR19-NK与不同CD19阳性细胞株共培养后IFN-γ释放、CD107a脱颗粒，荧光杀伤实验和检测IFN-γ释放表明hCAR19-NK细胞对CD19阳性淋巴瘤细胞的杀伤能力和IFN-γ释放水平均显著高于对照组。
IV.本发明的hCAR19-NK细胞的用途
本发明提供了本发明的hCAR19-NK细胞的用途，其用于在受试者中治疗与表达CD19相关的疾病。
在一个实施方案中，本发明的hCAR19-NK细胞用于在受试者中治疗表达CD19的癌症，并且能够减轻癌症的至少一种症状或指征的严重性或抑制癌细胞生长。
本发明提供了在受试者中治疗与表达CD19相关的疾病(例如，表达CD19的癌症)的方法，其包括向有需要的个体施用治疗有效量的本发明的hCAR19-NK细胞。
本发明提供了前述本发明的hCAR19-NK细胞在制备用于治疗与表达CD19相关的疾病(例如，表达或过量表达CD19的癌症)的药物中的用途。
本发明的hCAR19-NK细胞也可以施用于已经用一种或多种先前疗法治疗癌症但随后复发或转移的个体。
在一些实施方案中，本发明的hCAR19-NK细胞用于胃肠外、经皮、腔内、动脉内、静脉内、鞘内施用，或直接注入组织或肿瘤中。
在一些实施方案中，本发明的hCAR19-NK细胞是自自体NK细胞或同种异体NK细胞制备的表达本发明CAR多肽的NK细胞。
本发明的hCAR19-NK细胞可以以合适的剂量施用于受试者。剂量方案将由主治医生和临床因素决定。如医学领域中公知的，用于任何一名患者的剂量取决于许多因素，包括患者的体重、身体表面积、年龄、待施用的特定化合物、性别、施用时间和途径、一般健康状况、和待并行施用的其他药物。
在一些实施方案中，本发明的hCAR19-NK细胞以1×10 6个细胞－1×10 12个细胞、优选地为5×10 6个细胞－1×10 11个、更优选地为1×10 7个细胞－1×10 10个免疫效应细胞、例如5×10 7个细胞、2.5×10 8个细胞、1.25×10 9个免疫效应细胞的剂量以单次或多次静脉内施用。
在一些实施方案中，向患有癌症的个体施用本发明的hCAR19-NK细胞导致肿瘤的完全消失。在一些实施方案中，向患有癌症的个体施用本发明的hCAR19-NK细胞导致肿瘤细胞或肿瘤大小减少至少85％或更多。可以通过本领域已知的任何方法测量肿瘤的减少，例如X-线、正电子发射断层扫描(PET)、计算机断层扫描(CT)、磁共振成像(MRI)、细胞学、组织学或分子遗传分析。
可通过给需要过继性细胞疗法的受试者施用本发明的hCAR19-NK细胞来缓解表达CD19的癌症，例如，血液恶性肿瘤，包括但不限于急性和慢性白血病、淋巴瘤、多发性骨髓瘤和骨髓增生异常综合征；实体瘤，包括但不限于脑部、前列腺、乳腺、肺部、结肠、子宫、皮肤、肝脏、骨头、胰脏、卵巢、睾丸、膀胱、肾脏、头部、颈部、胃部、子宫颈、直肠、喉部、或食道的肿瘤。
本发明所述的各个实施方案/技术方案以及各个实施方案/技术方案中的特征应当被理解为可以任意进行相互组合，这些相互组合得到的各个方案均包括在本发明的范围内，就如同在本文中具体地且逐一地列出了这些相互组合而得到的方案一样，除非上下文清楚地显示并非如此。
描述以下实施例以辅助对本发明的理解。不意在且不应当以任何方式将实施例解释成对本发明的保护范围的限制。
实施例
实验材料及试剂：
K562、Raji(人B淋巴细胞；伯基特淋巴瘤细胞系，CD19 +肿瘤细胞系)和PG-13细胞系购自ATCC。
K562-GL、Raji-GL、K562-CD19细胞由本实验室构建，构建方法参考Kochenderfer J N等人，Construction and preclinical evaluation of an anti-CD19 chimeric antigen receptor[J].J Immunother,2009,32(7):689-702.DOI:10.1097/CJI.0b013e3181ac6138。所有细胞系支原体检测均为阴性。
RPMI 1640培养基和DMEM培养基购自Lonza公司。淋巴细胞分离液购自美国MP公司。胎牛血清购自Thermo公司。重组人白细胞介素-2购自双鹭药业。
人NK细胞分离试剂盒购自Miltenyi公司。
所有流式细胞检测用抗体均购自美国BD公司。IFN-γELISA检测试剂盒购自美国R&D公司。D-虫荧光素钾盐(D-Luciferin potassium salt)购自桥生物。
统计学处理：
采用GraphPad Prism 5软件进行数据统计，数据以均值±标准误形式表示，每组实验至少独立重复三次，以P<0.05为差异有统计学意义。
实施例1.靶向CD19的包含人源化scFv的CAR基因合成及病毒表达载体的构建
使用本实验室之前构建的MSGV-FMC63-28Z重组逆转录病毒载体，其编码MSGV(基于小鼠干细胞病毒的splice-gag载体)逆转录病毒骨架和FMC63-28Z CAR。所述FMC63-28Z CAR由源自FMC63小鼠杂交瘤39的抗CD19 scFv；人CD28分子的部分胞外区、整个跨膜和细胞质部分；以及人TCR-ζ分子的胞内成分组成。FMC63-28Z CAR中包含的CD28分子的确切序列对应于Genbank登录号NM_006139，包括从氨基酸序列IEVMYPPY开始一直到蛋白质羧基末端的所有氨基酸(Kochenderfer J N,Feldman S A,Zhao Y,et al.Construction and preclinical evaluation of an anti-CD19 chimeric antigen receptor[J].J Immunother,2009,32(7):689-702.DOI:10.1097/CJI.0b013e3181ac6138)，将所述MSGV-FMC63-28Z重组逆转录病毒载体中的FMC63-28Z CAR(在本文中也称为“FMC63 CAR”)编码序列替换为本发明的经设计为包含人源化抗CD19 scFv的图1所示的CAR，获得MSGV-人源化抗CD19 scFv-28Z重组逆转录病毒载体，所述“人源化抗CD19 scFv-28Z”在本文中也称为“hCAR19”(图1)，其包含来源于人粒细胞-巨噬细胞集落刺激因子(GM-CSF)受体的信号序列(SEQ ID NO:12)、人源化抗CD19 scFv(SEQ ID NO:4)、修饰的CD8a铰链区(SEQ ID NO:5)、CD28跨膜结构域(SEQ ID NO:8)和CD28共刺激结构域(SEQ ID NO:9)以及CD3ζ信号传导结构域(SEQ ID NO:11)的编码核苷酸序列。
将MSGV-人源化抗CD19 scFv-28Z重组逆转录病毒载体中的“人源化抗CD19 scFv-28Z”构建体删除，获得无hCAR19构建体的空载体，在以下实验中用作阴性对照，也称为Mock对照。
为了产生逆转录病毒，利用LIPOFECTAMINE TM 2000(Life Technologies,Carlsbad,CA)，用编码hCAR19的质粒以及编码RD114包膜蛋白的质粒(Porter等人， Human Gene Therapy,7(8):913-919(1996))转染PG13包装细胞体系(Kochenderfer J N等人，Construction and preclinical evaluation of an anti-CD19 chimeric antigen receptor[J].J Immunother,2009,32(7):689-702.DOI:10.1097/CJI.0b013e3181ac6138)。将转染的细胞在37℃，于不含抗生素的D10培养基中孵育6-8小时。然后用新鲜的D10培养基替代用于转染的培养基，并将细胞再孵育36-48小时。在转染期间和转染之后，在聚-D-赖氨酸包被的平皿(BD Biosciences,San Jose,CA)上培养PG13细胞。从平皿中移出包含逆转录病毒的上清液，并离心，以移除细胞碎片。将上清液于-80℃储存。
将产生的编码hCAR19的逆转录病毒或Mock对照的逆转录病毒，用于后续转导NK细胞。
实施例2.工程化饲养细胞的制备
在本实施例中，工程化改造K562细胞(ATCC)，使其共表达如下分子：CD19、4-1BBL、膜结合的IL-21(mIL-21)，获得了工程化饲养细胞，即，K562-CD19-4-1BBL-mIL21细胞。
具体地，K562(人骨髓性白血病细胞系)获自美国典型培养物保藏中心(ATCC,Manassas,VA,USA)。将K562细胞在补充有10％热灭活胎牛血清(FBS)(Gibco,US)、100单位/mL青霉素和100μg/mL链霉素(Invitrogen,CA,USA)的RPMI 1640培养基中于37℃培养于加湿的5％CO 2培养箱中。
合成包含编码SEQ ID NO：13所示氨基酸序列的CD19、SEQ ID NO：14所示氨基酸序列的4-1BBL和SEQ ID NO：15所示氨基酸序列的膜结合IL-21(mIL-21)的cDNA序列，其中各编码核苷酸之间通过编码P2A自裂解肽(SEQ ID NO：16)的核苷酸连接。
用包含编码CD19、4-1BBL和膜结合的IL-21(mIL-21)的cDNA序列的逆转录病毒载体转导K562细胞来产生K562-CD19-4-1BBL-mIL21细胞。
与实施例1类似地，将本实验室之前构建的MSGV-FMC63-28Z重组逆转录病毒载体中的FMC63-28Z CAR编码序列替换为编码CD19、4-1BBL和膜结合的IL-21(mIL-21)的cDNA序列，获得MSGV-CD19-4-1BBL-mIL21重组逆转录病毒载体；并产生编码CD19-4-1BBL-mIL21的逆转录病毒上清液。
向培养的K562细胞连续两天添加新鲜的编码CD19-4-1BBL-mIL21的逆转录病毒上清液。然后将细胞维持在含有10％胎牛血清和抗生素的RPMI-1640中。
通过流式细胞术分析CD19、4-1BBL和mIL21的表面表达，并富集表达高水平CD19、4-1BBL和mIL21的亚克隆细胞作为工程化饲养细胞，用于刺激NK细胞扩增。
实施例3.hCAR19-NK细胞的制备
本实施例阐述了产生表达hCAR19的NK细胞的方法。
采集多位健康志愿者外周血样品。通过在Lymphoprep密度梯度(Nycomed，Oslo，Norway)上离心分离外周血单个核细胞，并在RPMI-1640中洗涤两次。为了从外周血单个核细胞中纯化原代NK细胞，使用人NK细胞分离试剂盒(Miltenyi Biotec)富集CD56 +NK细胞。
在第0天，将100Gy伽马射线照射的实施例2制备的K562-CD19-4-1BBL-mIL21细胞作为饲养细胞(feeder)置于24孔板中，与富集的CD56 +NK细胞按细胞数1:1的比例于RPMI 1640培养基(10％FBS、100U/mL青霉素、100μg/mL链霉素和4mmol/L L-谷氨酰胺)中共培养，以扩增NK细胞。
在第6天，采用实施例1制备的编码hCAR19的逆转录病毒或Mock对照的逆转录病毒转染经扩增的NK细胞，获得了hCAR19-NK细胞或Mock对照细胞。
在第7天，将细胞培养基更换为含有IL-2(400IU/ml)的RPMI-1640完全培养基，使用100Gy伽马射线照射的实施例2制备的K562-CD19-4-1BBL-mIL21细胞作为饲养细胞(feeder)，进行hCAR19-NK细胞或Mock对照细胞的培养。
在第14天，采用抗CD19Fab抗体通过流式细胞术检测hCAR19-NK细胞表面上的hCAR19的表达，并用蛋白质印迹验证经转染的hCAR19-NK细胞是否表达外源性CD3ζ。
由图2可见，包含hCAR19编码序列的逆转录病毒转染NK细胞后，高达23.8％的NK细胞表达hCAR19，而Mock对照细胞中仅为1.04％。
通过蛋白质印迹检测经转染细胞的外源性CD3ζ的表达，结果显示与Mock对照细胞相比，hCAR19-NK细胞显著地表达外源性CD3ζ(图3)。
该实施例的结果表明，通过本发明的方法，可将NK细胞工程化，以稳定表达本发明的hCAR19。
实施例4.CD19 +肿瘤细胞对hCAR19-NK细胞的特异性激活
本实施例在第15天，使用实施例3的hCAR19-NK细胞，通过如下实验检测了CD19 +肿瘤细胞对hCAR19-NK细胞的特异性激活。
IFN-γ胞内染色实验：将效应细胞(Mock对照细胞或hCAR19-NK细胞)分别与3种肿瘤细胞按照效靶比5:1共培养，加入1μl的GolgiStop TM Protein Transport Inhibitor(Fisher Scientific，BD 554724)，6h后收集细胞，按照IFN-γ胞内染色标准流程通过流式细胞术检测IFN-γ。
CD107a表面表达的脱颗粒实验：将效应细胞(Mock对照细胞或hCAR19-NK细胞)分别与3种肿瘤细胞按照效靶比5:1共培养，加入1μl CD107a抗体(BD-Biosciences)和1μl的GolgiStop TM Protein Transport Inhibitor，6h后收集细胞，再染色CD56进行流式检测。
作为3种肿瘤细胞，分别使用下述表达CD19的永生化细胞系：K562-CD19(经基因工程重组而表达CD19的K562细胞)和Raji(人B淋巴细胞；伯基特淋巴瘤，CD19 +肿瘤细胞系，获自ATCC)；以及CD19阴性细胞系K562(ATCC)。
IFN-γ胞内染色实验结果如图4所示。由图4可见，与Mock对照细胞相比较，hCAR19-NK细胞在与CD19 +肿瘤细胞共培养后，可以被特异性激活并表达IFN-γ。
CD107a脱颗粒实验结果如图5所示。由图5可见，与Mock对照细胞相比较，hCAR19-NK细胞在与CD19 +肿瘤细胞共培养后，可以被特异性激活并发生CD107a分子暴露于NK细胞表面，NK细胞能够通过脱颗粒来释放穿孔素和颗粒酶等裂解靶细胞。
实施例5.hCAR19-NK细胞对肿瘤细胞的杀伤能力和细胞因子释放能力
本实施例在第15天，使用实施例3的hCAR19-NK细胞，通过如下实验检测了hCAR19-NK细胞对肿瘤细胞的杀伤能力和细胞因子释放能力。
荧光杀伤实验：以不同效靶比(E:T)10:1、5:1、2.5:1、1:1将效应细胞(Mock对照细胞或hCAR19-NK细胞)与表达荧光素酶的肿瘤细胞(CD19 +肿瘤细胞Raji-GL或CD19 -肿瘤细胞K562-GL)(所述GL表示“荧光素酶”)共培养4小时，加入D-虫荧光素钾至150μg/mL终浓度，采用Xenogen IVIS成像系统进行荧光拍照，基于孔中的肿瘤细胞 的荧光值计算杀伤率。
计算公式为：杀伤率＝[100-(效应细胞和靶细胞共培养荧光值/靶细胞荧光值)x100]％。
hCAR19-NK细胞对肿瘤细胞的杀伤能力如图6所示。由图6可见，与Mock对照细胞相比较，hCAR19-NK细胞对Raji-GL的杀伤更强，而Mock对照细胞和hCAR19-NK细胞对K562-GL的杀伤无显著性差异。
ELISA法检测IFN-γ释放：按照效靶比5:1将效应细胞(Mock对照细胞和hCAR19-NK细胞)与肿瘤细胞(K562、Raji和K562-CD19)共培养24h，收集上清离心后，用ELISA标准方法检测IFN-γ的分泌量。
结果如图7所示。由图7可见，在靶细胞为K562细胞的共培养中，hCAR19-NK细胞所致的IFN-γ释放(1078±10.7)与Mock对照细胞所致的IFN-γ释放(1036±132.7)无显著性差异；在靶细胞为Raji细胞的共培养中，hCAR19-NK细胞所致的IFN-γ释放(1252±77)显著高于Mock对照细胞所致的IFN-γ释放(586.1±51.8)；在靶细胞为K562-CD19细胞的共培养中，hCAR19-NK对照细胞所致的IFN-γ释放(4145±94.7)也显著高于Mock对照细胞所致的IFN-γ释放(790.2±53.3)，具有统计学意义(P<0.001)。
实施例6.工程化饲养细胞对NK细胞的扩增倍数
本实施例检测了实施例2制备的工程化饲养细胞对NK细胞的扩增倍数。
采集多位健康志愿者外周血样品。通过在Lymphoprep密度梯度(Nycomed，Oslo，Norway)上离心分离外周血单个核细胞，并在RPMI-1640中洗涤两次。为了从外周血单个核细胞中纯化原代NK细胞，使用人NK细胞分离试剂盒(Miltenyi Biotec)富集CD56 +NK细胞。
在第0天，将100Gy伽马射线照射的K562-CD19-4-1BBL-mIL21细胞作为饲养细胞(feeder)置于24孔板中，与富集的CD56 +NK细胞按细胞数1:1的比例于RPMI 1640培养基(10％FBS、100U/mL青霉素、100μg/mL链霉素和4mmol/L L-谷氨酰胺)中共培养，以扩增NK细胞。
在第6天，采用实施例1制备的编码hCAR19的逆转录病毒或Mock对照的逆转录病毒转染经扩增的NK细胞，获得了hCAR19-NK细胞或Mock对照细胞。
在第7天，将细胞培养基更换为含有IL-2(400IU/ml)的RPMI-1640完全培养基， 使用100Gy伽马射线照射的实施例2制备的K562-CD19-4-1BBL-mIL21细胞作为饲养细胞(feeder)，进行hCAR19-NK细胞或Mock对照细胞的培养。
分别在第6天、第14天和第21天计算NK细胞的扩增倍数。“扩增倍数”的计算方法：将培养第14天或第21天时NK细胞的绝对数除以第6天时的相应NK细胞数。
结果如图8所示。由图8可见，K562-CD19-4-1BBL-mIL21细胞作为饲养细胞能够在第21天时使hCAR19-NK细胞或Mock对照细胞扩增约2000倍，对这两种细胞扩增的倍数无显著差异。
实施例7.NK细胞的体内抗肿瘤效应研究
按照常规方法，以Raji细胞系为母细胞，采用慢病毒感染的方法，将绿色荧光蛋白(GFP)和萤火虫荧光素酶(Fireflyluciferase，LUC)基因引入Raji细胞，获得稳定表达绿色荧光蛋白和萤火虫荧光素酶的细胞株Raji-GFP-LUC。
用1x PBS重悬Raji-GFP-LUC细胞，制备成细胞浓度为5×10 6个/mL细胞悬液。免疫缺陷型NOD小鼠尾静脉注射5×10 6个/mL的Raji-GFP-LUC细胞悬液，0.1mL/只小鼠，即接种量为5×10 5个/只小鼠。
Raji-GFP-LUC肿瘤细胞接种NOD小鼠后第2天，将NOD小鼠随机分成4组。各组分别为注射PBS的载剂组、没有转导CD19 CAR的实施例6获得的第21天Mock对照细胞组、实施例6获得的第21天hCAR19-NK细胞组、以及转导实施例1的“FMC63 CAR”的NK细胞组(其制备和扩增同实施例6，收获第21天的FMC63 CAR-NK细胞)，每组5只小鼠。
用1x PBS分别重悬Mock对照细胞组、hCAR19-NK细胞组、FMC63 CAR-NK细胞组的细胞，制备成2.5×10 7个/mL细胞悬液。在Raji-GFP-LUC肿瘤细胞接种后第2天，尾静脉注射各NK细胞悬液0.2mL/只小鼠，即尾静脉注射各NK细胞5×10 6个/只小鼠。每隔七天通过荧光定量监测肿瘤负荷大小，同时观察NOD小鼠生存质量和状态。该动物实验重复三次。小鼠生存图采用KAPLAN-MEIER生存函数(SAS 2020版)进行统计。
图9显示了在接种人Raji肿瘤细胞的免疫缺陷荷瘤NOD小鼠中各NK细胞对肿瘤的治疗效应。结果显示，在相对于载剂组，各NK细胞组均诱导了显著的抗肿瘤效应，其中hCAR19-NK细胞组显示最大的抗肿瘤效应，且与FMC63 CAR-NK细胞组之间统计学 有显著意义(P<0.01)。
以上描述了本发明的示例性实施方案，本领域技术人员应当理解的是，这些公开内容仅是示例性的，在本发明的范围内可以进行各种其它替换、适应和修改。因此，本发明不限于文中列举的具体实施方案。
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Figure PCTCN2022119604-appb-000001
Figure PCTCN2022119604-appb-000002

Claims (10)

  1. 一种基因工程化饲养细胞，其是通过基因修饰永生化细胞以表达CD19和至少一种刺激免疫细胞活化的分子而获得的，所述基因工程化饲养细胞不表达主要组织相容性复合物(MHC)I分子，并且其中所述基因工程化饲养细胞与NK细胞的共培养导致NK细胞的活化和扩增，例如，所述至少一种刺激免疫细胞活化的分子选自4-1BBL、膜结合的IL-12、膜结合的IL-7、膜结合的IL-15(mIL15)、膜结合的IL-18(mIL18)、膜结合的IL-21(mIL-21)、膜结合的IL-22(mIL-22)。
  2. 根据权利要求1所述的基因工程化饲养细胞，其中所述永生化细胞来源于癌细胞，例如K562髓性白血病细胞、HL-60细胞、OCI-AML3细胞。
  3. 根据权利要求1或2所述的基因工程化饲养细胞，其中所述基因工程化饲养细胞是经遗传修饰以表达CD19、4-1BBL和膜结合的IL-21(mIL-21)的K562细胞；例如，其中所述CD19包含SEQ ID NO:13或与SEQ ID NO:13具有至少90％或至少95％序列同一性的氨基酸序列；所述4-1BBL包含SEQ ID NO:14或与SEQ ID NO:14具有至少90％或至少95％序列同一性的氨基酸序列；所述膜结合的IL-21(mIL-21)包含SEQ ID NO:15或与SEQ ID NO:15具有至少90％或至少95％序列同一性的氨基酸序列。
  4. 根据权利要求1-3中任一项所述的基因工程化饲养细胞，其中采用病毒载体在所述饲养细胞中表达所述CD19和至少一种刺激免疫细胞活化的分子，例如，所述病毒载体为逆转录病毒载体。
  5. 制备权利要求1-4中任一项所述的基因工程化饲养细胞的方法，所述方法包括：用编码所述CD19和至少一种刺激免疫细胞活化的分子的核酸转导或转染癌细胞，例如K562髓性白血病细胞、HL-60细胞、OCI-AML3细胞；分离表达所述CD19和至少一种刺激免疫细胞活化的分子的细胞；以及辐照所分离的细胞，从而产生基因工程化饲养细胞，例如，所述至少一种刺激免疫细胞活化的分子选自4-1BBL、膜结合的IL-12、膜结合的IL-7、膜结合的IL-15(mIL15)、膜结合的IL-18(mIL18)、膜结合的IL-21(mIL-21)、膜结合的IL-22(mIL-22)。
  6. 根据权利要求1-4中任一项所述的基因工程化饲养细胞的用途，用于扩增NK细胞，其中所述NK细胞是天然存在的NK细胞或经遗传修饰的NK细胞，例如，所述天然存在的NK细胞分离自或者包含在外周血液、骨髓、淋巴结组织、脐带血、胸腺组织、感染 部位组织、腹水、胸腔积液、脾组织、或肿瘤中；例如，所述经遗传修饰的NK细胞是CAR-NK细胞，例如，人源化抗CD19 CAR-NK细胞。
  7. 用于制备和扩增人源化抗CD19 CAR-NK细胞的方法，包括：
    a)将经辐照(例如，伽马射线照射)的权利要求1-4中任一项所述的基因工程化饲养细胞与天然存在的NK细胞共培养；例如，共培养2-10天，例如，共培养6天，例如，所述天然存在的NK细胞分离自或者包含在外周血液、骨髓、淋巴结组织、脐带血、胸腺组织、感染部位组织、腹水、胸腔积液、脾组织、或肿瘤中；
    b)将编码人源化抗CD19 CAR的核酸序列导入NK细胞中，获得人源化抗CD19 CAR-NK细胞，例如，通过逆转录病毒将编码人源化抗CD19 CAR的核酸序列导入NK细胞中，
    例如，所述人源化抗CD19 CAR包含
    (1)人源化抗CD19 scFv序列，其中所述scFv序列特异性结合CD19且包含：
    (i)轻链可变区，其包含SEQ ID NO:1的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，和
    (ii)重链可变区，其包含SEQ ID NO:2的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；
    例如，(i)轻链可变区，其包含SEQ ID NO:1的序列，和
    (ii)重链可变区，其包含SEQ ID NO:2的序列；
    (2)铰链区/间隔区，其选自CD8a铰链区(SEQ ID NO 5)，或其具有至少90％、至少95％的序列同一性的CD8铰链区；
    (3)跨膜区(TM)，其选自
    (i)SEQ ID NO:6所示的CD4跨膜结构域或其具有1-5个氨基酸修饰的变体；
    (ii)SEQ ID NO:7所示的CD8跨膜结构域或其具有1-5个氨基酸修饰的变体；
    (iii)SEQ ID NO:8所示的CD28跨膜结构域或其具有1-5个氨基酸修饰的变体；
    (4)共刺激信号结构域(CSD)，其是：
    (i)SEQ ID NO:9所示的CD28共刺激结构域或其具有1-5个氨基酸修饰的变体；和、或
    (ii)SEQ ID NO:10所示的4-1BB共刺激结构域或其具有1-5个氨基酸修饰的变体；
    (5)刺激信号结构域(SSD)，为SEQ ID NO:11所示的CD3ζ信号传导结构域或其具有1-5个氨基酸修饰的变体；
    其中所述氨基酸修饰是氨基酸的添加、缺失或取代；
    以及
    c)将人源化抗CD19 CAR-NK细胞与经辐照(例如，伽马射线照射)的权利要求1-4中任一项所述的基因工程化饲养细胞和任选地IL-2共培养足以扩增人源化抗CD19 CAR-NK细胞的一段时间(例如，7-42天，例如，14-28天)，从而扩增人源化抗CD19 CAR-NK细胞；例如，所述人源化抗CD19 CAR-NK细胞扩增至少1500至5000倍，例如，2000倍、3000倍、4000倍。
  8. 根据权利要求7所述的方法，其中，所述人源化抗CD19 CAR包含
    (1)人源化抗CD19 scFv序列，其包含：SEQ ID NO:4的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，
    (2)铰链区/间隔区，为SEQ ID NO 5所示的CD8a铰链区或其具有1个氨基酸修饰的变体；
    (3)跨膜区(TM)，为SEQ ID NO:8所示的CD28跨膜结构域或其具有1个氨基酸修饰的变体；
    (4)共刺激信号结构域(CSD)，为SEQ ID NO:9所示的CD28共刺激结构域或其具有1个氨基酸修饰的变体；
    (5)刺激信号结构域(SSD)，为SEQ ID NO:11所示的CD3ζ信号传导结构域或其具有1个氨基酸修饰的变体；
    其中所述氨基酸修饰是氨基酸的添加、缺失或取代。
  9. 通过权利要求7或8所述的方法制备和扩增的人源化抗CD19 CAR-NK细胞，其与表达CD19的肿瘤细胞接触后，表达IFN-γ、发生脱颗粒，并杀伤表达CD19的肿瘤细胞。
  10. 通过权利要求7或8所述的方法制备和扩增的人源化抗CD19 CAR-NK细胞的用途，用于制备治疗表达CD19的肿瘤的药物，其中所述人源化抗CD19 CAR-NK细胞与所述肿瘤的细胞上的CD19结合，并且破坏所述肿瘤细胞，例如，所述表达CD19的肿瘤选自多发性骨髓瘤、白血病、淋巴瘤或实体瘤，例如，所述白血病为急性淋巴细胞白血病(ALL)或急性髓系白血病(AML)，所述淋巴瘤为非霍奇金淋巴瘤，所述实体瘤为脑部、前列腺、 乳腺、肺部、结肠、子宫、皮肤、肝脏、骨头、胰脏、卵巢、睾丸、膀胱、肾脏、头部、颈部、胃部、子宫颈、直肠、喉部、或食道的肿瘤。
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