Cell-based therapeutic agents that target CD70

Abstract

The present invention provides natural killer (NK) cells engineered to express a chimeric antigen receptor (CAR), where the CAR comprises the extracellular domain of CD27 or a CD70-binding portion thereof, as well as compositions comprising the engineered NK cells, methods of producing the engineered NK cells, and therapeutic applications of the engineered NK cells, such as for the treatment of neoplastic disease.

Description

[Technical Field]

[0001] field The present invention is broadly applicable to the pharmaceutical field and, more specifically, relates to an immune cell-based therapeutic agent useful for treating neoplastic diseases. [Background technology]

[0002] background The CD70 (differentiation antigen group 70) protein, a tumor necrosis factor (TNF)-related molecule also known as CD27 ligand (CD27L), is normally expressed transiently only in activated T and B cells and mature dendritic cells. However, constitutive expression of CD70 has been described in malignant cells of certain solid and hematological malignancies. CD70 expression on malignant cells via its receptor, CD27, can promote immune system evasion, particularly through T cell apoptosis induction, T cell exhaustion, and an increase in suppressive regulatory T cells (Tregs). Given that CD70 is expressed in highly activated lymphocytes such as those in T and B cell lymphomas, in contrast to the short, transient CD70 expression in healthy lymphocytes, anti-CD70 antibodies have been proposed as a potential treatment for CD70-positive malignancies.

[0003] More recently, the inventors characterized CD70 expression on cancer-associated fibroblasts (CAFs) (Jacobs et al. Oncoimmunology. 2018, vol. 7(7), e1440167). CAFs are a dominant component of the tumor microenvironment (TME) and have been proposed to play a role in tumor proliferative and invasive behavior as well as immune evasion. The authors reported that the number of CD70-positive CAFs increased with the stage (T1-T4) of colorectal cancer (CRC) specimens (see Figure 2 here), and that CD70 expression acted as a significant negative predictor of overall survival and progression-free survival in CRC patients.

[0004] WO2016 / 093878 relates to an anti-CD70 chimeric antigen receptor (CAR). WO2019 / 213610 relates to natural killer (NK) cells engineered to express a CAR with immune checkpoint blockade. SUMMARY OF THE INVENTION

[0005] Summary The present invention focuses on the feasibility and therapeutic utility of targeting CD70-positive cancer cells such as cancer-associated fibroblasts (CAFs) and / or CD70-positive tumor microenvironment cells, based on the inventors' innovative insights and experimental evaluations using natural killer (NK) cells engineered to express a chimeric antigen receptor (CAR) that includes at least in part the extracellular domain of CD27 or its CD70-binding portion.

[0006] In one aspect, the present invention provides natural killer (NK) cells engineered to express a chimeric antigen receptor (CAR), wherein the CAR includes the extracellular domain of CD27 or its CD70-binding portion.

[0007] A further aspect provides a pharmaceutical composition comprising the engineered NK cells and a pharmaceutically acceptable carrier.

[0008] Another aspect provides a method of producing engineered NK cells, comprising introducing into a starting population of NK cells a nucleic acid encoding the CAR in an expressible form. Optionally and advantageously, NK cells containing the nucleic acid and capable of expressing the CAR can then be selected and / or expanded.

[0009] One aspect provides engineered NK cells or a pharmaceutical composition for use in therapy. A further aspect provides engineered NK cells or a pharmaceutical composition for use in a method of treating a neoplastic disease. A related aspect provides a method of treating a subject in need of treatment, particularly a subject having a neoplastic disease, the method comprising administering to the subject a therapeutically effective amount of the engineered NK cells or the pharmaceutical composition.

[0010] The present invention relates to an improved immune cell-based therapeutic agent using a specially designed chimeric antigen receptor (CAR) with antigen specificity for CD70, which has the ability to efficiently eliminate CD70-positive cells that may include cancerous and / or tumor microenvironment (TME) cells such as CAFs. Whether the tumor contains CD70-positive cancerous cells or not, the eradication of CD70-positive TME cells, particularly the latter such as CD70-positive CAFs, may play a significant role in the immunosuppressive characteristics of the tumor microenvironment, and their eradication may therefore offer considerable therapeutic benefits as it can make the tumor susceptible to elimination by the patient's own immune system or other (immuno)therapeutic agents.

[0011] These and further aspects and preferred embodiments of the present invention are described in the following sections and in the appended claims. The main matters of the appended claims are thus specifically incorporated herein. [Brief explanation of the drawing]

[0012] [Figure 1] The molecular mechanisms underlying or contributing to the therapeutic effects induced by an immune cell-based agent according to one embodiment of the present invention are described. Natural killer cells engineered to express a chimeric antigen receptor (CAR) (1), wherein the CAR comprises the extracellular domain of CD27 or its CD70 binding portion (2), specifically binds to the CD70 molecule (3), and is exposed on the surface of cancer-associated fibroblasts (CAFs) (4) (upper panel) or cancerous cells in a tumor (5) (lower panel), thereby driving or promoting the elimination of these CD70-positive cells and tumors.

[0013] [Figure 2]This graph reproduces the data reported by Jacobs et al. (see above), which quantifies CD70 expression on fibroblasts in normal (N), adenoma (A), carcinoma in situ (Tis), and T1-T4 colorectal cancer (CRC) specimens (shown as %CD70-positive fibroblasts, y-axis). The expression is graded as - (<1%), + (1-10%), ++ (11-50%), and +++ (>50% of CAFs expressing CD70).

[0014] [Figure 3] (A, B) Overlays of representative histograms of CD27 expression on NK-92 cells 24 hours after electroporation in the absence of CAR mRNA (MOCK), with CAR mRNA accompanied by the IL-15 cytokine cassette (CD27-CAR), and without the IL-15 cytokine cassette (CD27-CAR w / o IL-15). Further optimization and improvement of the electroporation protocol will cause the histogram to shift more to the right, indicating increased CAR expression (B).

[0015] [Figure 4] Characterization of CD70-targeted CAR-NK-92 cells is shown. (A-B) Graphs showing the percentage overtone (A) and delta-mean fluorescence intensity (△MFI) (B) of CD27 expression on NK-92 cells 24 hours after electroporation with CAR mRNA in the absence of CAR mRNA (MOCK), without the IL-15 cytokine cassette (CD27-CAR), and with the IL-15 cytokine cassette (CD27-CAR with IL-15). (C) IL-15 concentration produced in the supernatant of NK-92 cells after electroporation with MOCK, CD27-CAR, and CD27-CAR with IL-15. The dotted line indicates the detection limit of the assay. Experiments were performed in at least triplicates. Error bars indicate the standard deviation. *P≦0.05, △MFI = specific CD27 mAb staining - isotype control.

[0016] [Figure 5]This study demonstrates the in vitro cytotoxicity of CD70-targeting CAR-NK-92s against CD70+ target cell lines. Graphs show the percentage of cell death after 4 hours of co-culture of NK-92 cells electroporated with CAR mRNA in the absence of CAR mRNA (MOCK), without the IL-15 cytokine cassette (CD27-CAR), and with the IL-15 cytokine cassette (CD27-CAR with IL-15), and various CD70+ target cell lines (Raji, PANC-1, LIM2099, and RLT-PSC) 24 hours after electroporation. Target cell death percentages are identified as Annexin V+ and / or 7-AAD+ cells by flow cytometry. Experiments were performed in at least triplicates. Error bars indicate standard deviation. *P ≤ 0.05.

[0017] [Figure 6] This graph shows blockade of the antigen-recognition domain of CAR, indicating CAR-specific cell death. It shows the percentage of viable cells 24 hours after electroporation and 4 hours after co-culture for NK-92 cells electroporated with CAR mRNA in the absence of CD70+ Raji target cell line alone (Raji baseline), in the absence of CAR mRNA (MOCK), without the IL-15 cytokine cassette (CD27-CAR), and with the IL-15 cytokine cassette (CD27-CAR with IL-15), respectively. Effector cells were incubated overnight in co-culture with neutralizing anti-CD27 monoclonal antibody or IgG1 isotype control at various concentrations (10 μg / mL, 50 μg / mL, or 100 μg / mL). The percentage of viable target cells was identified as Annexin V- and 7-AAD- cells by flow cytometry. Experiments were performed in at least triplicates. Error bars indicate standard deviation. *P ≤ 0.05.

[0018] [Figure 7]This study demonstrates that stimulation of CD70-targeting CAR-NK-92s with IL-15 or IL-21 improves their cytotoxic performance. The graph shows the percentage of viable cells after electroporation with CAR mRNA in the absence of CAR mRNA (MOCK), without the IL-15 cytokine cassette (CD27-CAR), and with the IL-15 cytokine cassette (CD27-CAR with IL-15) from various CD70+ target cell lines (Raji, PANC-1, LIM2099, and RLT-PSC), 24 hours after electroporation, and 4 hours after co-culture. CD27-CAR NK-92 cells were stimulated overnight with exogenous IL-12, IL-15, and IL-21 cytokines at effector doses of 50 (ED50). The percentage of viable target cells is identified as Annexin V- and 7-AAD- cells by flow cytometry. Baseline conditions represent the viability of CD70+ target cells in the absence of effector cells. Experiments were performed in at least triplicates. Error bars indicate standard deviation. *P ≤ 0.05. [Modes for carrying out the invention]

[0019] Description of the Embodiment The singular expressions used here include both singular and plural subjects unless the context clearly requires a different interpretation.

[0020] The terms “including,” “inclusion,” and “containing” as used herein are synonymous with “encompassing,” “inclusion,” or “containing,” and are comprehensive or open-ended, and do not exclude additional, undescribed members, elements, or process steps. The terms also include “consisting of” and “essentially consisting of,” which have well-established meanings in patent terminology.

[0021] Numerical ranges indicated by endpoints include all numerical values ​​and fractions contained within each range, as well as the endpoints indicated. This applies regardless of whether the numerical range is introduced by the expression "~", the expression "between... and...", or other expressions.

[0022] When referring to measurable values ​​such as parameters, quantities, or periods, the terms "about" or "approximately" as used herein mean to include the specified value and its variation therefrom, for example, a variation of ±10%, preferably ±5%, more preferably ±1%, and even more preferably ±0.1% or less from the specified value, insofar as such variation is appropriate for the implementation of the invention disclosed. The value to which the modifying phrase "about" or "approximately" refers should be understood to also be specifically and preferably disclosed in itself.

[0023] The terms “one or more” or “at least one,” such as one or more members of a group of members or at least one member, are self-evident, and as a means of further illustration, the terms encompass, in particular, any one of the members or any two or more of the members, and even all of the members, such as any ≥3, ≥4, ≥5, ≥6, or ≥7 of the members. In other examples, “one or more” or “at least one” may mean 1, 2, 3, 4, 5, 6, 7, or more.

[0024] The background description herein includes a description of the circumstances of the present invention. This should not be construed as acknowledging that any of the materials described herein were publicly disclosed, known, or part of common technical knowledge in any country at the priority date of any of the claims herein.

[0025] Throughout this specification, various publications, patents, and patent gazettes are referenced by specific quotation marks. All references made herein are incorporated herein in whole by citation. In particular, any teachings or sections of such references specifically made herein are incorporated by citation.

[0026] Unless otherwise specified, all terms used in the disclosure of this invention, including technical and scientific terms, have meanings that are generally understood by those skilled in the art to which this invention pertains. By means of further guidance, definitions of terms are included for good understanding of the teachings of this invention. When specific terms are defined in relation to a particular aspect or embodiment of this invention, such implications or meanings are intended to apply throughout this specification, i.e., in other contexts of other aspects or embodiments of this invention, unless otherwise specified.

[0027] The following text further defines various aspects or embodiments of the present invention. Each of the aspects or embodiments thus defined may be combined with any other aspect or embodiment unless it is clearly shown to be contrary to this. In particular, any characteristic indicated as preferred or advantageous may be combined with any one or more other characteristics indicated as preferred or advantageous.

[0028] Throughout this specification, any reference to “one embodiment” or “a certain embodiment” means that a particular characteristic, structure, or feature described in relation to that embodiment is encompassed in at least one embodiment of the present invention. Therefore, the terms “one embodiment” or “a certain embodiment” found in various places in this specification do not necessarily refer to the same embodiment, but they may. Furthermore, a particular characteristic, structure, or feature can be combined in any suitable manner in one or more embodiments, as will be apparent to those skilled in the art. Moreover, while one embodiment described herein may include some characteristics included in other embodiments and not others, combinations of characteristics from various embodiments are intended to be within the scope of the present invention and, as will be understood to those skilled in the art, form different embodiments. For example, in the appended claims, any of the claimed embodiments may be used in any combination.

[0029] As supported by the experimental section illustrating a representative embodiment of the present invention, the inventors have demonstrated the feasibility and therapeutic utility of targeting CD70-positive cancer cells and / or CD70-positive tumor microenvironment cells, such as cancer-associated fibroblasts (CAFs), using natural killer (NK) cells engineered to express a chimeric antigen receptor (CAR) containing the extracellular domain of CD27 or its CD70-binding moiety. Efficient eradication of CD70-positive cancer cells and / or CAFs provides a valuable therapeutic means for treating proliferative disorders.

[0030] The present invention therefore includes embodiments as shown in the summary section, and in particular NK cells engineered to express CAR, wherein CAR comprises the extracellular domain of CD27 or its CD70 binding portion; and pharmaceutical compositions comprising such engineered NK cells, methods for producing engineered NK cells, and therapeutic uses and methods using engineered NK cells or pharmaceutical compositions, in particular for the treatment of neoplasms such as cancer.

[0031] The term “chimeric antigen receptor” or “CAR” refers to a recombinant polypeptide or set of polypeptides that, when expressed by immune effector cells, confers specificity to a target antigen on the surface of a target cell and intracellular signaling to that cell. In one embodiment, a CAR includes at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to here as the “intracellular signaling domain” or “intracellular activation domain”) that includes a functional signaling domain derived from a stimulating and / or co-stimulating molecule. The term “signaling domain” refers to a functional portion of a protein that acts by transmitting information within a cell to regulate cellular activity via a defined signaling pathway, either by producing a second messenger or by functioning as an effector in response to such a messenger. Typically, a CAR may include a chimeric fusion protein such that, for example, the antigen-binding domain and the intracellular signaling domain are contained within the same polypeptide chain. In another embodiment, a CAR may be formed from a set of non-adjacent polypeptides such that, for example, the antigen-binding domain and the intracellular signaling domain can be provided to another polypeptide chain that is designed to heterodimerize to form the CAR. As an example, the antigen-binding domain and the intracellular signaling domain may each be provided with a dimerization switch that allows polypeptides containing the domain to be bound to each other in the presence of a dimerizing molecule.

[0032] The term CAR constitutes a suitable, convenient, and well-established form to mean a receptor as intended herein; however, without using this term, such a receptor could be described, for example, as a polypeptide or set of polypeptides comprising an extracellular domain of CD27 or its CD70-binding portion and an intracellular activation domain, where the binding of the extracellular domain to CD70 on the target cell surface induces, induces, or triggers intracellular signaling via the intracellular activation domain.

[0033] Other CAR constructs can typically be characterized as belonging to multiple successive generations. In the first generation, the CAR intracellular signaling domain includes or is essentially derived from a zeta chain associated with the T cell receptor complex (CD3ζ) or the γ subunit of the immunoglobulin Fc receptor (FcRγ). The cytoplasmic signaling domain of the second-generation CAR further includes an intracellular co-stimulatory domain, i.e., a functional signaling domain derived from at least one co-stimulatory molecule such as CD28, 4-1BB (CD137), DAP10, ICOS, or OX40 (CD134), and the third-generation CAR includes a combination of two or more such co-stimulatory endodomains.

[0034] In one embodiment, CAR comprises a chimeric fusion protein comprising the extracellular domain of CD27 or its CD70 binding portion, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulating molecule. In another embodiment, CAR comprises a chimeric fusion protein comprising the extracellular domain of CD27 or its CD70 binding portion, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a co-stimulator and a functional signaling domain derived from a stimulator. In yet another embodiment, CAR comprises a chimeric fusion protein comprising the extracellular domain of CD27 or its CD70 binding portion, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more co-stimulators and a functional signaling domain derived from a stimulator.

[0035] In one embodiment, the intracellular portion of the CAR includes at least one intracellular activation domain. In one embodiment, the at least one intracellular activation domain is selected from the group consisting of a CD3ζ activation domain, an FcRγ activation domain, and combinations thereof. In one preferred embodiment, the CAR includes a CD3ζ intracellular activation domain such that the intracellular activation domain of the CAR includes, essentially consists of, or consists of a CD3ζ intracellular activation domain. In one embodiment, the intracellular portion of the CAR includes at least one intracellular co-stimulatory domain. In one embodiment, the at least one intracellular co-stimulatory domain is selected from the group consisting of a CD28 co-stimulatory domain, a 4-1BB co-stimulatory domain, a DAP10 co-stimulatory domain, an OX40 co-stimulatory domain, an ICOS co-stimulatory domain, and combinations thereof. In one preferred embodiment, the CAR includes a 4-1BB intracellular co-stimulatory domain and optionally one or more additional co-stimulatory domains. In one preferred embodiment, the CAR includes a 4-1BB intracellular co-stimulatory domain but does not include other co-stimulatory domains.

[0036] As described, CAR contains the extracellular domain of CD27 or its CD70 binding portion. The differentiation antigen group 27 (CD27) molecule, or simply CD27 or CD27 antigen, is also known as the CD27L receptor or tumor necrosis factor receptor superfamily member 7 (TNFRSF7 protein), and is a 29kDa one-pass transmembrane type I membrane glycoprotein. Human CD27 precursors are annotated under Gene ID no. 939 in the National Center for Biotechnology Information (NCBI) Genebank (http: / / www.ncbi.nlm.nih.gov / ). The human wild-type CD27 amino acid sequence is the NP_001233.2 sequence reproduced below, which may be annotated under Genbank accession number NP_001233.2 or Swissprot / Uniprot (http: / / www.uniprot.org / ) accession number P26842-1 (entry version 195, October 7, 2020; sequence version 2, November 24, 2009) (the signal or leader sequence, N-terminal extracellular domain, transmembrane or intramembrane domain and C-terminal intracellular domain of the annotated CD27 molecule in the above database entry are shown consecutively in standard, bold, and italics, respectively). [ka]

[0037] The CD27 referred to herein is specifically human CD27. In relation to the CD27 protein, the modifying term "human" used herein specifically refers to the amino acid sequence of the CD27 protein. For example, a CD27 protein having the amino acid sequence as found in humans can also be obtained by technical means, e.g., recombinant expression, cell-free translation, or non-biological peptide synthesis. Those skilled in the art will understand that the amino acid sequences of some native proteins, such as the CD27 protein, may differ between or within different individuals of the same species due to normal genetic diversity within the species (allelic variation, polymorphism) and / or differences in post-transcriptional or post-translational modifications. Any such variant or isoform of a native protein is encompassed by the reference or designation of the protein.

[0038] The extracellular domain of the human CD27 protein, annotated under Genbank accession number NP_001233.2, is reproduced below. ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIR(Sequence ID 3)

[0039] In one embodiment, the extracellular domain of CD27 is essentially composed of or a polypeptide comprising an amino acid sequence that is at least 80% identical to, for example, at least 85% identical to, preferably at least 90% identical to, for example, at least 95% identical to, preferably at least 96%, at least 97%, at least 98%, or at least 99% identical to, SEQ ID NO: 3. In a particularly preferred embodiment, the extracellular domain of CD27 is essentially composed of or a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3.

[0040] The term "sequence identity" in relation to amino acid sequences refers to the degree of overall sequence identity (i.e., including the complete or whole amino acid sequence of the amino acid sequence cited for comparison), expressed as a percentage, between amino acid sequences read from the N-terminus to the C-terminus; and, in relation to nucleic acid sequences, it refers to the degree of overall sequence identity (i.e., including the complete or whole nucleic acid sequence of the amino acid sequence cited for comparison), expressed as a percentage, between nucleic acid sequences read from the 5' end to the 3' end (complementary sequences may be used for comparison if desired). Sequence identity can be performed using known sequence alignment procedures and appropriate algorithms for sequence identity determination. For example, non-restrictive algorithms may use, for instance, the publicly available default settings or other suitable settings (e.g., for the BLASTN algorithm: gap start penalty = 5, gap extension penalty = 2, mismatch penalty = -2, match reward = 1, gap x_dropoff = 50, expected value = 10.0, word size = 28; or for the BLASTP algorithm: matrix = Blosum62 (Henikoff et al., 1992, Proc. Natl. Acad. Sci., 89:10915-10919), gap start penalty = 11, gap extension penalty = 1, expected value = 10.0, word size = 3, etc.), based on the Basic Local Alignment Search Tool (BLAST) first described by Altschul et al. 1990 (J Mol Biol 215: 403-10), for example, Tatusova and Madden 1999 (FEMS Microbiol Lett 174: Includes the "Blast 2 sequence" algorithm described in 247-250).

[0041] An exemplary procedure for determining the percentage identity between a specific amino acid sequence and a query amino acid sequence uses the Blast 2 sequences (Bl2seq) algorithm, available as a web application (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=blasttab) or a standalone executable program (BLAST version 2.11.0+) from the NCBI website (https: / / ftp.ncbi.nlm.nih.gov / blast / executables / blast+ / LATEST / ), each necessarily involving the alignment of two amino acid sequences read from the N-terminus to the C-terminus. An example of appropriate algorithm parameters includes: matrix=Blosum62, gap start penalty=11, gap extension penalty=1, expected value=10.0, word size=3). If the two compared sequences share identity, the output presents the region of identity of the aligned sequences. If the two compared sequences do not share identity, the output will not present an aligned sequence. Once aligned, the number of matches is determined by counting the number of positions where identical amino acid residues exist in both sequences. Percent identity is determined by dividing the number of matches by the query sequence length and then multiplying the resulting value by 100. The percentage identity value may, but is not necessarily, be rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 may be rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 may be rounded up to 78.2. It should also be noted that the detailed images for each segment of the alignment output by Bl2seq already conveniently include the identity percentage.

[0042] When an amino acid sequence differs from, is altered from, or is different from another amino acid sequence—for example, when the former amino acid sequence can be said to have some degree or percentage sequence identity with the latter amino acid sequence, or when the former amino acid sequence can be said to have a certain number of amino acids different from the latter amino acid sequence—such sequence diversity can consist of the addition of one or more amino acids (e.g., a stretch of one amino acid or two or more adjacent amino acids may be independently added to one location in the amino acid sequence or to two or more locations in the amino acid sequence), deletion (e.g., a stretch of one amino acid or two or more adjacent amino acids may be independently deleted to one location in the amino acid sequence or to two or more locations in the amino acid sequence), and / or substitution (e.g., a stretch of one amino acid or two or more adjacent amino acids may be independently substituted to one amino acid or two or more adjacent amino acid stretches at one location in the amino acid sequence or to two or more locations in the amino acid sequence).

[0043] Preferably, one or more amino acid substitutions, particularly one or more monoamino acid substitutions, may be conservative amino acid substitutions. A conservative amino acid substitution is a substitution of one amino acid for another having similar characteristics. Conservative amino acid substitutions include substitutions within the following groups: valine, alanine, and glycine; leucine, valine, and isoleucine; aspartic acid and glutamic acid; asparagine and glutamine; serine, cysteine, and threonine; lysine and arginine; and phenylalanine and tyrosine. Nonpolar hydrophobic amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (i.e., basic) amino acids include arginine, lysine, and histidine. Charged (i.e., acidic) amino acids include aspartic acid and glutamic acid. Any substitution of one member of the polar, basic, or acidic groups by another member of the same group can be considered a conserved substitution. In contrast, a non-conservative substitution is the substitution of one amino acid with another that has dissimilar characteristics.

[0044] This specification describes a molecule that may be a peptide, polypeptide, or protein, such as the extracellular domain or intracellular activation domain of CD27. The term “protein” generally encompasses macromolecules containing one or more polypeptide chains. The term “polypeptide” generally encompasses linear polymer chains of amino acid residues linked by peptide bonds. A “peptide bond,” “peptide linkage,” or “amide bond” is a covalent bond formed between two amino acids when the carboxyl group of one amino acid reacts with the amino group of another amino acid, thereby releasing a water molecule. The terms “protein” and “polypeptide” may be used interchangeably to refer to such proteins, especially when a protein can consist of only a single polypeptide chain. These terms are not limited by any minimum length of polypeptide chain. Polypeptide chains that essentially consist of or comprise 50 or fewer (≤50) amino acids, e.g., ≤45, ≤40, ≤35, ≤30, ≤25, ≤20, ≤15, ≤10, or ≤5 amino acids are generally referred to as “peptides.” In the context of proteins, polypeptides, or peptides, “sequence” refers to the order of amino acids in a chain from the amino terminus to the carboxyl terminus, where adjacent residues within the sequence are adjacent in the primary structure of the protein, polypeptide, or peptide. This term may encompass proteins, polypeptides, or peptides produced by natural, recombinant, semi-synthetic, or synthetic means. Thus, for example, proteins, polypeptides, or peptides may be naturally occurring or isolated, e.g., naturally or endogenously acidified or expressed by cells or tissues and isolated therefrom if desired; or proteins, polypeptides, or peptides may be recombinant, i.e., produced by recombinant DNA technology and / or partially or entirely chemically or biochemically synthesized.Proteins, polypeptides, or peptides may be produced by recombinant production by a suitable host or host cell expression system and, if desired, isolated therefrom (e.g., a suitable bacterial, yeast, fungal, plant, or animal host or host cell expression system), or by recombinant production by cell-free translation or cell-free transcription and translation, or by non-biological peptide, polypeptide, or protein synthesis. The term also includes, but is not limited to, proteins, polypeptides, or peptides that carry one or more simultaneous or post-expression modifications of polypeptide chains, such as glucosylation, lipidation, acetylation, amidation, phosphorylation, sulfonation, methylation, pegylation (typically covalent bonding of polyethylene glycol to the N-terminus or one or more Lys residues in the side chain), ubiquitination, smomination, cysteination, glutathioneation, oxidation of methionine to methionine sulfoxide or methionine sulfone, signal peptide removal, N-terminal Met removal, or conversion to an active form of an enzyme precursor or hormone precursor. Such co-expression or post-expression modifications can be introduced in vivo by host cells expressing a protein, polypeptide, or peptide (the co-translation or post-translational protein modification mechanism may be naturally occurring in the host cell and / or may be genetically engineered to include one or more (additional) co-translation or post-translational protein modification functionalities in the host cell) or in vitro by chemical (e.g., pegylation) and / or biochemical (e.g., enzymatic) modification of the isolated protein, polypeptide, or peptide.

[0045] The term "amino acid" includes naturally occurring amino acids, naturally encoded amino acids, naturally unencoded amino acids, naturally uncommon amino acids, amino acid analogs, and amino acid mimes, all of which function in a manner similar to naturally occurring amino acids, and all are stereoisomers if their structure allows for D- and L-stereoisomerization. Amino acids are here described by their nomenclature as recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or by their commonly known three-letter or one-letter symbols. "Naturally encoded amino acids" refer to the 20 common amino acids or one of the amino acids that is pyrrolicin, pyrroline-carboxy-lysine, or selenocysteine. The 20 common amino acids are alanine (A or Ala), cysteine ​​(C or Cys), aspartic acid (D or Asp), glutamic acid (E or Glu), phenylalanine (F or Phe), glycine (G or Gly), histidine (H or His), isoleucine (I or Ile), lysine (K or Lys), leucine (L or Leu), methionine (M or Met), asparagine (N or Asn), proline (P or Pro), glutamine (Q or Gln), arginine (R or Arg), serine (S or Ser), threonine (T or Thr), valine (V or Val), tryptophan (W or Trp), and tyrosine (Y or Tyr). "Naturally unencoded amino acids" refer to any amino acid that is not one of the 20 common amino acids or one of pyrrolicin, pyrroline-carboxy-lysine, or selenocysteine. This term includes, but is not limited to, amino acids resulting from modifications (e.g., post-translational modifications) of naturally encoded amino acids, but which are not naturally incorporated into the growing polypeptide chain by translational complexes, as exemplified by N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine.Further examples of naturally unencoded, unnatural, or modified amino acids include 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, beta-aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, piperidine acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,4-diaminobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, homoserine, homocysteine, hydroxylysine, allohydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, alloisoleucine, N-methylglycine, N-methylisoleucine, 6-N-methyllysine, N-methylvaline, norvaline, norleucine, ornithine. This also includes amino acid analogs in which one or more individual atoms are replaced by different atoms, isotopes of the same atom, or different functional groups. This also includes unnatural amino acids and amino acid analogs described in Ellman et al. Methods Enzymol. 1991, vol. 202, 301-36. The incorporation of unnatural amino acids into proteins, polypeptides, or peptides can be advantageous in several different ways. For example, D-amino acid-containing proteins, polypeptides, or peptides exhibit increased stability in vitro or in vivo compared to L-amino acid-containing counterparts. More specifically, D-amino acid-containing proteins, polypeptides, or peptides are resistant to endogenous peptidases and proteases, which may provide improved bioavailability and extended lifespan of the molecule in vivo.

[0046] In one embodiment, CAR may comprise the CD70 binding portion or fragment of the extracellular domain of CD27. The terms “fragment” or “part” of a protein, polypeptide, or peptide generally encompass forms of a full-length protein, polypeptide, or peptide in which the N-terminus and / or C-terminus are deleted or cleaved. The terms include, but are not limited to, fragments resulting from any mechanism such as the expression of a cleaved form of a full-length protein, polypeptide, or peptide or the physical, chemical, or enzymatic proteolysis of a full-length protein, polypeptide, or peptide in vivo or in vitro. A fragment of a protein, polypeptide, or peptide may mean at least about 50% (in terms of the number of amino acids) of the adjacent amino acid sequence of the full-length protein, polypeptide, or peptide, for example, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. For example, a fragment or portion of the 172-amino acid-length extracellular domain of CD27 in the example shown in SEQ ID NO: 3 may include a sequence of at least about 90, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, or at least about 170 consecutive amino acids in SEQ ID NO: 3.

[0047] Originally, CD27 is a cognate receptor for the CD70 molecule expressed on the cell surface. Thus, the extracellular domain of CD27 included in the CARs taught herein has the intrinsic ability to specifically bind to the CD70 protein on the cell surface in physiologically and therapeutically relevant settings. As used herein, the term "specifically bind" means that an agent binds to one or more desired molecules or analytes, substantially excluding other molecules that are random or irrelevant, and optionally substantially excluding other molecules that are structurally related. In other words, an agent specifically binds to a target molecule when it preferentially recognizes the target molecule in a complex mixture of proteins and / or macromolecules.

[0048] When the CAR includes a CD70-binding portion or fragment of the extracellular domain of CD27, the ability of such a CD27 portion to bind to the CD70 protein is significantly similar or equivalent to the therapeutic effect achieved by cells expressing a CAR that includes the CD70-binding portion of the CD27 extracellular domain, and is significantly similar or equivalent to the ability of the full-length CD27 extracellular domain to bind to CD70, such that the therapeutic effect shown by the CAR-expressing cells containing the CD70-binding portion of the CD27 extracellular domain is significantly similar or equivalent to the therapeutic effect achieved by cells expressing the same CAR but containing the full-length CD27 extracellular domain.

[0049] Typically, a target-binding protein (e.g., the extracellular domain of CD27 or its CD70-binding fragment) binds to a cognate target protein (e.g., CD70) at 1×10 -5 ~1×10 -12 moles per liter (M) or less, and preferably 1×10 -7 ~1×10 -12 M or less, and more preferably 1×10 -8 ~1×10 -12 M or less, and even more preferably 1×10 -9 ~1×10 -12 M or less, e.g., 1×10 -9 ~1×10 -10 M or 1×10 -10 ~1×10 -11M can bind with a dissociation constant (KD), where KD = [TBP][TG] / [TBP-TG], where TBP is the target-binding protein, TP is the target protein, and TBP-TG is the complex of these two. 10 -4 A KD value exceeding M is typically considered an indicator of nonspecific binding. Therefore, in one embodiment, the CD70 binding portion of the CD27 extracellular domain is 10 -4 M or less, preferably 10 -5 M or less, more preferably 10 -6 M or less, more preferably 10 -7 M or less, more preferably 10 -8 M or less, for example, 10 -9 M or less, 10 -10 M or less or 10 -11 CD70 can be bound with a KD of M or less. In one embodiment, the CD70-binding portion of the extracellular domain of CD27 can bind to CD70 with a KD up to two orders of magnitude higher, preferably up to one order of magnitude higher, and more preferably the same order of magnitude or more lower, than the KD that typically represents the binding of a full-length extracellular domain of CD27 to CD70 under otherwise substantially identical conditions. Specific binding of a target-binding protein to its target can be determined by any suitable method known to the art, including, for example, scatchard plot analysis and / or competitive binding assays such as radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competition assays and various variants thereof known to the art.

[0050] In one embodiment, the suitability of a portion or fragment of the CD27 extracellular domain in the therapeutic context of the present invention can be evaluated by in vitro cell death experiments as described in the examples. For example, if, under otherwise substantially identical conditions, NK cells expressing a CAR containing a portion of the CD27 extracellular domain exhibit 10% of the efficiency of NK cells expressing the otherwise identical CAR containing the full-length CD27 extracellular domain in CD70-positive cell death, then that portion of the CD27 extracellular domain can be considered the CD70 binding intended herein. Preferably, the death efficiency of NK cells expressing a CAR containing the CD70 binding portion of the CD27 extracellular domain may be at least 20%, for example at least 30% or at least 40%, more preferably at least 50%, for example at least 60% or at least 70%, even more preferably at least 80%, for example at least 90%, or substantially equivalent to or higher than the death efficiency of NK cells expressing a CAR containing the full-length CD27 extracellular domain.

[0051] In one embodiment, the CAR may further include an intramembrane domain of CD27. The intramembrane or transmembrane domains of the human CD27 protein, annotated under Genbank accession number NP_001233.2, are reproduced below. ILVIFSGMFLVFTLAGALFLH (Sequence ID 4)

[0052] In one embodiment, the intramembrane domain of CD27 is essentially composed of or a polypeptide comprising an amino acid sequence that is at least 80% identical to, for example, at least 85% identical to, for example, at least 90% identical to, for example, at least 95% identical to, for example, at least 96%, at least 97%, at least 98%, or at least 99% identical to, for example,5%. In a particularly preferred embodiment, the intramembrane domain of CD27 is essentially composed of or a polypeptide comprising the amino acid sequence shown in, for example, at least 95%.

[0053] The polypeptide consisting of the extracellular and intramembrane domains of the human CD27 protein, annotated under Genbank accession number NP_001233.2, is reproduced below. ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFLH(Sequence ID 10)

[0054] In one embodiment, the extracellular and intramembrane domains of CD27 are essentially composed of or comprise a polypeptide comprising an amino acid sequence that is at least 80% identical to, for example, at least 85% identical to, preferably at least 90% identical to, for example, at least 95% identical to, preferably at least 96%, at least 97%, at least 98%, or at least 99% identical to, SEQ ID NO: 10. In a particularly preferred embodiment, the extracellular and intramembrane domains of CD27 are essentially composed of or comprise a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 10.

[0055] In some other embodiments, the CAR may include intramembrane domains derived from transmembrane proteins other than CD27, preferably single-pass type I transmembrane proteins. For example, and not limited to, many CAR constructs in the art use the CD8α transmembrane domain, optionally together with the CD8α hinge domain, which can also be employed in the present CAR molecule. In yet another embodiment, the CAR may include non-naturally occurring or synthetic intramembrane domains scanned based on biophysical criteria known to be applicable to this type of transmembrane domain, such as high hydrophobicity and alpha-helical primary structure.

[0056] In one embodiment, CAR lacks all or part of the intracellular domain of CD27. The intracellular or cytoplasmic domains of the human CD27 protein, annotated under Genbank accession number NP_001233.2, are reproduced below. QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP (Sequence ID 5)

[0057] In a preferred embodiment, the CAR lacks the entire intracellular domain of CD27, i.e., the CAR lacks the entire intracellular domain of CD27, such as the amino acid sequence shown in SEQ ID NO: 5. In another embodiment, the CAR may include a modified CD27 intracellular domain that lacks at least one, e.g., at least two, at least five, at least ten, at least 20, at least 30, or at least 40 adjacent or non-adjacent amino acids of the full-length CD27 intracellular domain. In a specific embodiment, the CD27 intracellular domain may be modified such that normal CD27 signaling by the domain is impossible. Such modifications may include any amino acid sequence changes, such as the deletion and / or substitution of one or more amino acids in the CD27 intracellular domain.

[0058] Therefore, in one embodiment, CAR comprises, essentially or consists of, the extracellular and intramembrane domains and intracellular activation domain of CD27. In another embodiment, CAR comprises, essentially or consists of, the extracellular and intramembrane domains, intracellular activation domain and intracellular costimulatory domain of CD27. In a further embodiment, CAR comprises, essentially or consists of, the extracellular and intramembrane domains, intracellular activation domain and two or more intracellular costimulatory domains, such as exactly two. In one embodiment, CAR comprises, essentially or consists of, the extracellular and intramembrane domains, CD3ζ intracellular activation domain and intracellular costimulatory domain of CD27. In one embodiment, CAR comprises, essentially or consists of, the extracellular and intramembrane domains, CD3ζ intracellular activation domain and two or more intracellular costimulatory domains, such as exactly two. In a particularly preferred embodiment, CAR comprises, essentially or consists of, the extracellular and intramembrane domains, CD3ζ intracellular activation domain and 4-1BB intracellular costimulatory domain of CD27.

[0059] A specific example of the human CD3ζ intracellular activation domain is reproduced below. RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(Sequence ID 6)

[0060] In one embodiment, the CD3ζ intracellular activation domain is essentially composed of or a polypeptide comprising an amino acid sequence that is at least 80% identical to, for example, at least 85% identical to, preferably at least 90% identical to, for example, at least 95% identical to, preferably at least 96%, at least 97%, at least 98%, or at least 99% identical to, SEQ ID NO: 6. In a particularly preferred embodiment, the CD3ζ intracellular activation domain is essentially composed of or a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 6.

[0061] A specific example of the human 4-1BB intracellular co-stimulatory domain is reproduced below. KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (Sequence ID 7)

[0062] In one embodiment, the 4-1BB intracellular costimulatory domain is essentially composed of or a polypeptide comprising an amino acid sequence that is at least 80% identical to, for example, at least 85% identical to, for example, at least 90% identical to, for example, at least 95% identical to, for example, at least 96%, at least 97%, at least 98%, or at least 99% identical to, for example, the amino acid sequence of

[0063] In one particularly preferred embodiment, CAR is essentially composed of or comprises the amino acids shown in Sequence ID No. 1 below, where the extracellular and intramembrane domains of CD27 are in standard letters, the intracellular costimulatory domain of 4-1BB is in italics, and the intracellular activation domain of CD3ζ is underlined. [ka]

[0064] In one embodiment, CAR essentially consists of or comprises an amino acid sequence that is at least 80% identical, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95% identical, for example, particularly preferably at least 96%, at least 97%, at least 98%, or at least 99% or 100% identical to SEQ ID NO: 1.

[0065] In one embodiment, the CAR essentially consists of or comprises (i) extracellular and intramembrane domains of CD27, (ii) an intracellular costimulatory domain of 4-1BB, and (iii) an intracellular activation domain of CD3ζ, where domains (i) to (iii) each independently exhibit at least 80% identity, preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, for example, particularly preferably at least 96%, at least 97%, at least 98%, or at least 99% or 100% identity with (i) amino acid positions 1 to 193, (ii) amino acid positions 194 to 235, and (iii) 236 to 348 of SEQ ID NO: 1.

[0066] Those skilled in the art will recognize that the CAR molecules described herein are transmembrane proteins and typically require the inclusion of an appropriate signal or leader sequence during expression to achieve cell membrane localization of the protein. Such signal sequences are typically short (3-60 amino acid length) N-terminally positioned peptide chains that are optionally and advantageously removed by cleavage or processing with a signal peptidase after the protein has been transported, for example, to produce a mature protein. Signal sequences are widely known in the art and can be applied to the expression of the CARs taught herein. In some embodiments, the signal sequence may be derived from a protein other than CD27. In some preferred embodiments, the signal sequence may be derived from CD27. The native signal sequence of the human CD27 protein, annotated under Genbank accession number NP_001233.2, is reproduced below. MARPHPWWLCVLGTLVGLS(Sequence ID 8)

[0067] In one embodiment, the signal sequence contained in the CAR molecule consists of or comprises essentially the same amino acid sequence as SEQ ID NO: 8, comprising at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% the same amino acid sequence as SEQ ID NO: 8. In one embodiment, such a signal sequence may be fused to the N-terminus of any of the individualized CAR molecules above.

[0068] Therefore, as an example, in one particularly preferred embodiment, the precursor form of CAR is essentially composed of or comprises the amino acids shown in Sequence ID No. 9 below, where the signal sequence is in bold, the extracellular and intramembrane domains of CD27 are in standard letters, the 4-1BB intracellular costimulatory domain is in italics, and the CD3ζ intracellular activation domain is underlined. [ka]

[0069] Aspects of the present invention also relate to any of the CAR molecules described herein. Further aspects of the present invention relate to nucleic acids encoding the CAR molecules described herein. "Encoding" means that a nucleic acid sequence or part thereof corresponds to a specific amino acid sequence, e.g., the amino acid sequence of one or more desired proteins or polypeptides, or to other nucleic acid sequences in a template-transcript (e.g., RNA or RNA analog) relationship, by the genetic code of the organism in question.

[0070] As used herein, the term “nucleic acid” refers to polymers of any length (preferably linear polymers) that typically consist of nucleoside units. Nucleoside units generally include heterocyclic bases and sugar groups. Heterocyclic bases include, among others, purine and pyrimidine bases such as adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U), which are widely present in naturally occurring nucleic acids, other naturally occurring bases (e.g., xanthine, inosine, hypoxanthine), and chemically or biochemically modified (e.g., methylated), unnatural, or derivatized bases. Examples of modified nucleic acid bases include, but are not limited to, 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. In particular, 5-methylcytosine substitution has been shown to increase the double-strand stability of nucleic acids. The sugar groups may include, in particular, pentose (pentofuranose) groups, for example, ribose and / or 2-deoxyribose or arabinose, 2-deoxyarabinose, threose or hexose sugar groups, which are common in naturally occurring nucleic acids, as well as modified or substituted sugar groups (for example, but not limited to, 2'-O-alkylation, e.g., 2'-O-methylation or 2'-O-ethylated sugars, e.g., ribose; 2'-O-alkyloxyalkylation, e.g., 2'-O-methoxyethylated sugars, e.g., ribose; or 2'-O,4'-C-alkylene linkage, e.g., 2'-O,4'-C-methylene linkage or 2'-O,4'-C-ethylene linkage sugars, e.g., ribose; 2'-fluoro-arabinose, etc.).Nucleoside units can be linked to one another by any of the many known internucleoside bonds, including phosphodiester bonds common to naturally occurring nucleic acids and further modified phosphate- or phosphonate-based bonds, such as phosphorothioates, alkylphosphorothioates, such as methylphosphorothioates, phosphorodithioates, alkylphosphonates, such as methylphosphonates, alkylphosphonothioates, phosphotriesters, such as alkylphosphotriesters, phosphoramidates, phosphoropiperadates, phosphoromolholides, cross-linked phosphoramidates, cross-linked methylenephosphonates, cross-linked phosphorothioates; and further, siloxanes, carbonates, sulfamates, carboalkoxys, acetamidates, carbamates, such as 3'-N-carbamates, morpholino, borano, thioethers, 3'-thioacetal, and sulfone internucleoside bonds. Preferably, the nucleoside bond may be a modified phosphate-based bond, for example, a phosphate-based bond comprising a phosphodiester, phosphorothioate, or phosphorodithioate bond or a combination thereof. The term “nucleic acid” also encompasses nucleic acid mimes, including any other nucleic acid bases including polymers, for example, but not limited to peptide nucleic acids (PNA), peptide nucleic acids with phosphate groups (PHONA), locked nucleic acids (LNA), morpholinophosphodiamide backbone nucleic acids (PMO), cyclohexene nucleic acids (CeNA), tricyclo-DNA (tcDNA), and nucleic acids having a backbone section with an alkyl linker or amino linker (see, e.g., Kurreck 2003 (Eur J Biochem 270: 1628-1644)). The term "alkyl" as used herein particularly includes lower hydrocarbon moieties, such as C1-C4 straight-chain or branched-chain, saturated or unsaturated hydrocarbons, such as methyl, ethyl, ethenyl, propyl, 1-propenyl, 2-propenyl, and isopropyl.

[0071] The nucleic acids as intended herein may include naturally occurring nucleosides, modified nucleosides, or mixtures thereof. Modified nucleosides may include modified heterocyclic bases, modified sugar moieties, modified nucleoside bonds, or combinations thereof. The term “nucleic acid” more preferably encompasses DNA, RNA, and DNA / RNA hybrid molecules, particularly hnRNA, premRNA, mRNA, cDNA, genomic DNA, amplification products, oligonucleotides, and synthetic (e.g., chemosynthetic) DNA, RNA, or DNA / RNA hybrids. Nucleic acids may be naturally occurring, e.g., present in or isolated from natural products, recombinant, i.e., produced by recombinant DNA technology, and / or partially or entirely chemically or biochemically synthesized. “Nucleic acid” may be double-stranded, partially double-stranded, or single-stranded. When single-stranded, the nucleic acid may be either a sense strand or an antisense strand. Furthermore, the nucleic acid may be cyclic or linear.

[0072] In one embodiment, the extracellular and intramembrane domains of CD27, particularly those shown in SEQ ID NO: 10, are represented by SEQ ID NO: 11 (Sequence ID 11) A nucleic acid sequence comprising, essentially consisting of, or encoding a nucleic acid comprising the nucleic acid sequence shown in (SEQ ID NO: 11) may optionally be replaced, hereby any codon in SEQ ID NO: 11 with other codons that independently encode the same amino acid, preferably, hereby the nucleic acid sequence is codon-optimized for expression in cells of a desired species, such as human cells, according to codon optimization principles known in the art, such that, for example, the extracellular and intramembrane domains of CD27 are encoded, essentially consisting of, or encoding a nucleic acid comprising, a nucleic acid sequence identical to, SEQ ID NO: 11 by at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%.

[0073] In one embodiment, the human CD3ζ intracellular activation domain, particularly as shown in SEQ ID NO. 6, is represented by SEQ ID NO. 12 CGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCC TGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG (SEQ ID NO: 12) A nucleic acid sequence comprising, essentially consisting of, or encoded by, the nucleic acid sequence shown in, wherein any codon in SEQ ID NO: 12 may be replaced, respectively, with other codons that independently encode the same amino acid, preferably, wherein the nucleic acid sequence is codon-optimized for expression in cells of a desired species, such as human cells, according to codon optimization principles known in the art, such that, for example, the human CD3ζ intracellular activation domain comprises, essentially consisting of, or encoded by, the nucleic acid sequence that is identical to, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of, SEQ ID NO: 12.

[0074] In one embodiment, the human 4-1BB intracellular co-stimulatory domain, particularly as shown in SEQ ID NO: 7, is represented by SEQ ID NO: 13 AAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTG (SEQ ID NO: 13) A nucleic acid sequence comprising, essentially consisting of, or encoded by, the nucleic acid sequence shown in, wherein any codon in SEQ ID NO: 13 may be replaced, respectively, with other codons that independently encode the same amino acid, preferably, wherein the nucleic acid sequence is codon-optimized for expression in cells of a desired species, such as human cells, according to codon optimization principles known in the art, such that, for example, the human 4-1BB intracellular costimulatory domain comprises, essentially consisting of, or encoded by, the nucleic acid sequence that is identical to, SEQ ID NO: 13 by at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%.

[0075] In one embodiment, a precursor form of CAR taught herein, such as that shown in SEQ ID NO: 9, is shown in SEQ ID NO: 14. A nucleic acid sequence comprising, essentially consisting of, or encoded by, a nucleic acid comprising the nucleic acid sequence shown in (SEQ ID NO: 14), wherein any codon in SEQ ID NO: 14 may be replaced, respectively, with other codons that independently encode the same amino acid, preferably wherein the nucleic acid sequence is codon-optimized for expression in cells of a desired species, such as human cells, according to codon optimization principles known in the art, such that, for example, CAR comprises, essentially consisting of, or encoded by, a nucleic acid comprising, a nucleic acid sequence identical to, SEQ ID NO: 14 by at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%.

[0076] For the expression of nucleic acids encoding CARs, which are driven by the genetic information contained in the nucleic acid to produce CAR proteins in a suitable system such as a host cell, nucleic acids can be provided in an expressible form, such as being inserted as a component of an expression cassette or vector, as is common in the art, or otherwise prepared. Accordingly, aspects of the present invention also relate to nucleic acids encoding the CAR molecules disclosed herein in an expressible form. One embodiment relates to an expression cassette or expression vector containing a CAR-encoding nucleic acid.

[0077] The term “expression cassette” refers to a nucleic acid molecule, typically DNA, into which the coding sequences of one or more proteins of interest can be inserted for expression, wherein the nucleic acid molecule includes one or more nucleic acid sequences (regulatory sequences) that are manipulatively linked to the coding sequence and control its expression, and non-limiting examples include promoter sequences and transcription terminators. “Manipulative linkage” is a linkage in which a regulatory sequence and a sequence to be expressed are connected in a manner that enables such expression. Sequences such as a promoter and the coding sequence of the protein of interest can be considered manipulatively linked unless the nature of the linkage between the sequences (1) results in the introduction of a frameshift, (2) interferes with the promoter’s ability to direct the transcription of the coding sequence, or (3) interferes with the coding sequence’s ability to be transcribed from the promoter sequence. Thus, “manipulative linkage” can be incorporated into a genetic construct so that an expression regulatory sequence, such as a promoter, efficiently controls the transcription / expression of the sequence of interest. The exact properties of the transcriptional and translational regulatory sequences or elements required for expression may vary depending on the expression environment, but typically, transcriptional regulatory sequences include promoters, and optionally enhancers and transcriptional terminators.

[0078] The term “promoter” should be interpreted in its broadest sense, encompassing the transcriptional regulatory sequences necessary for precise transcription initiation and, where applicable, precise spatial and / or temporal control of gene expression or its response to, for example, internal or external (e.g., exogenous) stimuli. More specifically, “promoter” can represent a region of a nucleic acid molecule, preferably a DNA molecule, to which RNA polymerase binds and initiates transcription. The promoter is preferably, but not necessarily, located upstream of the transcription-regulating sequence, i.e., at the 5' end. Typically, in prokaryotes, the promoter region includes both the promoter itself and a sequence (e.g., the Shine-Dalgano sequence) that, when transcribed into RNA, transmits the signal for the initiation of protein synthesis. The promoter sequence may also include “enhancer regions,” which are one or more regions of DNA (i.e., trans-acting factors) that can bind to proteins to enhance the transcriptional level of a gene in a gene cluster. Enhancers are typically at the 5' end of the coding region, but may be detached from the promoter sequence, for example, within an intron region of the gene or 3' relative to the coding region of the gene.

[0079] In some embodiments, the promoter may be constitutive or inductive. A constitutive promoter is understood as a promoter whose expression is constant under standard culture conditions. An inductive promoter is a promoter that is responsive to one or more inducers. For example, an inductive promoter may be chemically controlled (e.g., a promoter whose transcriptional activity is controlled by the presence or absence of a chemical inducer such as alcohol, tetracycline, steroid, metal, or other small molecules) or physically controlled (e.g., a promoter whose transcriptional activity is controlled by the presence or absence of a physical inducer such as light or high or low temperature). Inductive promoters may also be indirectly controlled by one or more transcription factors, or directly controlled by chemical or physical factors. Non-limiting examples of promoters include the T7, U6, H1, retrovirus Roussarcoma virus (RSV) LTR promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, phosphoglycerol kinase (PGK) promoter, and EF1α promoter.

[0080] The term "terminator" or "transcriptional terminator" generally refers to a sequence element at the end of a transcription unit that transmits a transcription termination signal. For example, a terminator is usually located downstream of the coding sequence encoding the polypeptide of interest, i.e., at 3'. For example, when a recombinant nucleic acid contains two or more coding sequences, for example, consecutively aligned and collectively forming a polycistronic transcription unit, the transcriptional terminator is advantageously located relative to the furthest downstream coding sequence.

[0081] As used herein, the term “expression vector” or “vector” refers to a nucleic acid molecule, typically DNA, into which a nucleic acid fragment can be inserted and cloned, i.e., replicated. Therefore, a vector typically contains one or more specific restriction sites and is autonomously reproducible in a defined cell or medium such that the clonal sequence is reproducible. A vector may also preferably include selection markers, such as antibiotic resistance genes, which allow for the selection of recipient cells containing the vector. A vector may appropriately include, but is not limited to, plasmids, phagemids, bacteriophages, bacteriophage-derived vectors, PACs, BACs, linear nucleic acids, such as linear DNA, transposons, and viral vectors (see, e.g., Sambrook et al., 1989; Ausubel 1992). Viral vectors may include, among others, retroviral vectors, lentiviral vectors, adenovirus vectors, or adeno-associated virus vectors, such as vectors based on HIV, SV40, EBV, HSV, or BPV. Expression vectors are generally configured to enable and / or act on the expression of a desired expression system, such as an introduced nucleic acid or open reading frame in vitro, in cells, organs, and / or organisms. For example, an expression vector may contain advantageously suitable regulatory sequences.

[0082] Important factors in the selection of a particular vector include, among other things, the selection of recipient cells that are easily recognizable and can be sorted from recipient cells that do not contain the vector; the desired copy number of the vector in the particular recipient cells; whether it is desired that the vector be integrated into the chromosome or remain extrachromosomal in the recipient cells; and whether it is desired that the vector can be "shuttle" between recipient cells of different species.

[0083] Expression vectors can be autonomous or integrative. Nucleic acids can be introduced into cells in the form of expression vectors such as plasmids, phages, transposons, cosmids, or viral particles. Recombinant nucleic acids can remain extrachromosomal or be integrated into cellular chromosome DNA. Expression vectors may contain selection marker genes encoding proteins necessary for cell viability under selection conditions (e.g., URA3, an enzyme required for uracil biosynthesis, LEU2, an enzyme required for leucine biosynthesis, or TRP1, an enzyme required for tryptophan biosynthesis) to enable detection and / or selection of cells transformed with the desired nucleic acid. Expression vectors may also contain autonomous reproducible sequences (ARS). ARS may contain a centromere (CEN) and a reproducible origin (ORI). For example, an ARS may be ARS18 or ARS68.

[0084] An integrated vector typically contains a sequence of at least a first insertable DNA fragment, a selectable marker gene, and a second insertable DNA fragment arranged in sequence. Each of the first and second insertable DNA fragments is approximately 200 nucleotides long (e.g., approximately 250, 300, 350, 400, 450, 500, or 1000 or more) and contains a nucleotide sequence homologous to a portion of the genomic DNA of the cell type to be transformed. The nucleotide sequence containing the nucleic acid of interest for expression is inserted into the vector either between the first and second insertable DNA fragments, or before or after the marker gene. The integrated vector may be linearized before transformation to facilitate the integration of the nucleotide sequence of interest into the cell genome.

[0085] Before introducing the vector into target cells, the vector may be grown (e.g., amplified) in bacterial cells such as Escherichia coli. The vector DNA can be isolated from the bacterial cells by any method known in the art that results in the purification of vector DNA from the bacterial environment. The purified vector DNA is then thoroughly extracted with phenol, chloroform, and ether to ensure that E. coli proteins are not present in the plasmid DNA preparation, as these proteins may be toxic to mammalian cells.

[0086] In one embodiment, a nucleic acid encoding CAR in an expressible form may be a DNA molecule configured to drive CAR protein expression (transcription of the DNA CAR coding sequence into a corresponding CAR messenger RNA molecule and translation of the latter into a protein by a cellular translation mechanism) when the DNA molecule is introduced into mammalian cells, particularly animal cells such as human NK cells. In one embodiment, a nucleic acid encoding CAR in an expressible form may be an expression cassette or expression vector containing the coding sequence of the CAR protein. In one embodiment, this may be a DNA expression cassette or expression vector containing the DNA coding sequence of the CAR protein. In another embodiment, a nucleic acid encoding CAR in an expressible form may be an RNA molecule such as mRNA configured to drive CAR protein expression (translation of the coding sequence contained in the RNA molecule into a protein by a cellular translation mechanism) when the messenger RNA (mRNA) molecule is introduced into mammalian cells, particularly animal cells such as human NK cells. Such mRNA may be produced by any suitable means available in the art, such as in vitro transcription from an expression cassette or vector containing the CAR coding sequence.

[0087] Various well-known methods exist for introducing nucleic acids into animal cells, and any of them will be used here. Most simply, nucleic acids can be directly injected into target cells. Other methods include the fusion of recipient cells with nucleic acid-containing bacterial protoplasts, the use of compositions such as calcium chloride, rubidium chloride, lithium chloride, calcium phosphate, DEAE dextran, cationic lipids, or liposomes, or receptor-mediated endocytosis, microparticle guns ("gene gun" methods), infection with viral vectors (i.e., derived from lentiviruses, adeno-associated viruses (AAV), adenoviruses, retroviruses, or antiviruses), and electroporation. Other techniques or methods suitable for the delivery of nucleic acid molecules to target cells include the continuous delivery of NA molecules from poly(lactic acid-coglycolic acid) polymer microspheres or direct injection into a micropump to deliver the product of protected (stabilized) NA molecules. Another possibility is the use of implantable drug-releasing biodegradable microspheres. Also conceivable are the encapsulation of NA into various types of liposomes (immunoliposomes, pegylated (immuno)liposomes), cationic lipids and polymers, nanoparticles or dendrimers, poly(lactic acid-coglycolic acid) polymer microspheres, implantable drug-releasing biodegradable microspheres, etc., and the co-injection of NA with protective agents such as the nuclease inhibitor aurintricarboxylic acid. It is also clear that various combinations of the above delivery modes or methods may be used.

[0088] In addition to the above explanation and guidance, the term “cells engineered to express” the protein of interest means, in particular, cells that have been artificially modified or engineered to contain the exogenous nucleic acid encoding the protein of interest, such as cells into which the exogenous nucleic acid encoding the protein of interest has been introduced or inserted by available technological means.

[0089] As previously stated, the present invention aims to genetically modify natural killer (NK) cells to express the CAR proteins described herein. The expression level of CAR, which is the amount of CAR protein produced by NK cells, can vary within an acceptable range, typically determined by the priority or necessity, at the lower limit, that the expression level is sufficient to make the cells specific to CD70 on target cells, associated with the production of meaningfully strong intracellular signals within the NK cells, such as inducing cytotoxic effects (e.g., damage or death of CD70-positive target cells), and at the upper limit, the priority or necessity, to avoid a decrease in the viability or functionality of NK cells due to excessive overexpression of exogenous CAR proteins. Such quantitative considerations can be applied by those skilled in the art.

[0090] The modifying phrase “isolated” does not need to be explicitly stated in relation to the NK cells intended herein, but since the invention relates to the manipulation of NK cells in vitro, it may be convenient to include in vitro or ex vivo and subsequent therapeutic uses of such manipulated NK cells. The term “isolated” referring to a specific component generally means that such component exists apart from one or more other components of its natural environment—for example, it is separated or prepared and / or maintained apart. More specifically, the term “isolated” as used herein in relation to cells or cell populations means that such cells or cell populations are not part of an animal or human body, for example, that cells may be cultured, sorted or stored in vitro or ex vivo.

[0091] Native NK cells are a distinct population of lymphocytes in terms of both phenotypic and functional characteristics. NK cells possess a macrogranular lymphocyte morphology, express characteristic NK cell surface receptors, and lack both T cell receptor (TCR) rearrangements and T cell, B cell, monocyte, and / or macrophage cell surface markers. The cells are killed by small cytoplasmic granule proteins (perforin and granzyme), which kill target cells by apoptosis. NK cells have a mechanism to distinguish potential "target" cells from healthy cells via numerous inhibitory and activating receptors that bind to MHC class I molecules, MHC class I-like molecules, and MHC-independent molecules. Inhibitory NK cell receptors include HLA-E (CD94 / NKG2A); HLA-C (group 1 or 2), KIR2DL; KIR3DL (HLA-B Bw4), and HLA-A3 or A4+ peptides. Activated NK cell receptors include HLA-E (CD94 / NKG2C); KIR2DS (HLA-C) and KIR3DS (HLA-Bw4). Other receptors include low-affinity receptors for NK cell receptor protein-1 and the IgGFc moiety (FcyRIII; CD16). "Activating" and "inhibitory" surface receptors regulate the cytotoxic activity of native NK cells.

[0092] NK cells, along with the absence of CD3 expression, can be detected in humans by specific surface markers such as CD16, CD56, and CD8.

[0093] In some embodiments, NK cells may be isolated from a subject such as a human subject. In some embodiments, NK cells may be isolated from the subject's umbilical cord blood or peripheral blood. In some embodiments, NK cells may be differentiated in vitro from hematopoietic stem cells or induced pluripotent stem (iPS) cells. In some embodiments, NK cells may originate from human peripheral blood mononuclear cells (PBMCs), unstimulated leukocyte depletion products (PBSCs), human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), bone marrow, or umbilical cord blood. Methods for isolating or deriving NK cells from such sources are well known in the art. In specific embodiments, NK cells may be isolated from umbilical cord blood (CB), peripheral blood (PB), bone marrow, or stem cells. In specific embodiments, NK cells may be isolated from stored CBs. CBs may be stored from 2, 3, 4, 5, 6, 7, 8, 10, 20 or more units.

[0094] In one embodiment, the starting population of NK cells may be obtained by isolating mononuclear cells using Ficol density gradient centrifugation. Cell culture may be depleted of any cells expressing CD3, CD14 and / or CD19 cells and may be characterized for determining the percentage of CD56+ / CD3 cells (NK cells).

[0095] In one embodiment, the NK cells may be autologous or allogeneic to the target being administered. In one preferred embodiment, the NK cells may be allogeneic to the target being administered, so that the NK cells can be applied in an allogeneic setting without promoting graft-versus-host disease. In one embodiment, the NK cells may be haplotype-matched to the target being administered.

[0096] In one embodiment, NK cells may be clonal NK cell lines. NK cell lines are typically derived from NK lymphoma / leukemia, which are well known in this field. Non-limiting examples of NK cell lines include YTS, NK92 (NK-92), NK3.3, NKL, and NK101 cell lines. In one preferred embodiment, the NK cells are NK-92 cells. NK-92 cells are well-characterized and are often used in preclinical and clinical settings.

[0097] Primary NK cells can be isolated from patients, from healthy donors, or purchased from public cell collections. For example, but not limited to, primary human CD56+ NK cells isolated from blood are available from the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 20110, USA, under catalog number PCS-800-019; NK cell lines, particularly the NK-92 cell line, are available from ATCC under catalog numbers CRL-2407 and CRL-2408 (the latter derived from CRL-2407 and being interleukin-2 independent); and from the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) under catalog number # ACC 488.

[0098] In one embodiment, NK cells can be cultured in vitro. The culture, proliferation, and expansion of NK cells, as well as the modification of their properties in vitro, including ex vivo, are well characterized. Without limiting, the examples section shows the culture of NK cells in standard α-MEM medium supplemented with one or more suitable serums (e.g., fetal bovine serum and / or equine serum), an antibiotic mixture, L-glutamine, and interleukin 2 (IL-2). Further methods for culturing NK cells, such as the use of cytokine interleukins including IL-2, 12, 15, 18, and / or 21, co-culture of auto-attached non-NK cells, or addition of proliferating inactivated feeder cells, are described, for example, in Granzin et al. (Front Immunol. 2017, 8:458).

[0099] In one embodiment, the expression of the CAR and the optional one or more immunostimulatory cytokines may be manipulated to further express one or more immunostimulatory interleukins (ILs), particularly one or more human cytokines or interleukins. Examples of suitable interleukins include IL-15, IL-12 and / or IL-21, particularly human IL-15, IL-12 and / or IL-21. Preferably, the immunostimulatory interleukin is IL-15 or IL-21 or both. Preferably, the immunostimulatory interleukin is human IL-15 or human IL-21 or both. Particularly preferably, the immunostimulatory interleukin is human IL-15. In one embodiment, the expression of the CAR and the optional one or more immunostimulatory cytokines may be independently constitutive or inducible.

[0100] As a means of guidance, and not limited to that, the human interleukin-15 isoform 1 preproprotein and the human interleukin-15 isoform 2 preproprotein are annotated under Genbank accession numbers NP_000576.1 and NP_751915.1, respectively.

[0101] A specific example of the human IL-15 isoform 1 preprotein is reproduced below. MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(Sequence ID 15).

[0102] In one embodiment, IL-15 means a polypeptide consisting essentially of or comprising an amino acid sequence that is at least 80% identical to, for example, at least 85% identical to, preferably at least 90% identical to, for example, at least 95% identical to, preferably at least 96%, at least 97%, at least 98%, or at least 99% identical to, SEQ ID NO: 15. In a particularly preferred embodiment, IL-15 consists essentially of or comprises an amino acid sequence that is shown in SEQ ID NO: 15.

[0103] In one embodiment, human IL-15, such as that shown in SEQ ID NO: 15, is used in SEQ ID NO: 16 ATGAGAATCAGCAAGCCCCACCTGAGATCCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTTCTGACAGAGGCCGGCATCCACGTGTTCATCCTGGGCTGTTTTTCTGCC GGCCTGCCTAAGACCGAGGCCAACTGGGTTAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACACTGTACACCGAGAGCGACGTGCACCCTAGCT GTAAAGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAAGTGATCAGCCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAAAACCTGATCATCCTGGCCAACAACAGCCTGAGCA GCAACGGCAATGTGACCGAGTCCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAATATCAAAGAGTTCCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGC (SEQ ID NO: 16) A nucleic acid sequence comprising, essentially consisting of, or encoded by, a nucleic acid comprising the nucleic acid sequence shown in (SEQ ID NO: 16), wherein any codon in SEQ ID NO: 16 may be replaced, respectively, with other codons that independently encode the same amino acid, preferably wherein the nucleic acid sequence is codon-optimized for expression in cells of a desired species, such as human cells, according to codon optimization principles known in the art, such that, for example, human IL-15 comprises, essentially consisting of, or encoded by, a nucleic acid comprising the nucleic acid sequence, which is at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to SEQ ID NO: 16.

[0104] Human interleukin-21 isoform 1 precursor and human IL-21 isoform 2 precursor are annotated under Genbank accession numbers NP_068575.1 and NP_001193935.1, respectively, as means of guidance and without limitation.

[0105] A specific example of the human IL-21 isoform 1 precursor (NP_068575.1) is reproduced below. MRSSPGNMERIVICLMVIFLGTLVHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (Sequence ID 23).

[0106] A specific example of the human IL-21 isoform 2 precursor (NP_001193935.1) is reproduced below. MRSSPGNMERIVICLMVIFLGTLVHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKVSTLSFI (Sequence ID 24).

[0107] In one embodiment, IL-21 means a polypeptide consisting essentially of or comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 23 or 24, preferably SEQ ID NO: 23; for example, SEQ ID NO: 23 or 24, preferably at least 85% identical to SEQ ID NO: 23; preferably SEQ ID NO: 23 or 24, preferably at least 90% identical to SEQ ID NO: 23; for example, SEQ ID NO: 23 or 24, preferably at least 95% identical to SEQ ID NO: 23; more preferably at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 23 or 24. In a particularly preferred embodiment, IL-21 consists essentially of or comprises an amino acid sequence that is at least 99% identical to the amino acid sequence shown in SEQ ID NO: 23 or 24, preferably SEQ ID NO: 23.

[0108] In one embodiment, human IL-21, such as those shown in particular sequence numbers 23 or 24, is annotated in Genbank under accession numbers NM_021803.4 or NM_001207006.2, respectively, or sequence number 25 ATGAGATCCAGTCCTGGCAACATGGAGAGGATTGTCATCTGTCTGATGGTCATCTTCTTGGGGACACTGGTCCACAAATCAAGCTCCCAAGGTCAAGATCGCCACATGATTAGAATGCGTCAA CTTATAGATATTGTTGATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGCTTTCAGAAGGCCC AACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATCAATTAAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGCATCTGTCCTCTAGAACACACGGAAGTGAAGATTCC (SEQ ID NO: 25) A nucleic acid sequence comprising, essentially consisting of, or encoded by, a nucleic acid comprising the nucleic acid sequence shown, wherein any codon in NM_021803.4, NM_001207006.2, or SEQ ID NO: 25 may be optionally replaced with other codons that independently encode the same amino acid, preferably wherein the nucleic acid sequence is codon-optimized for expression in cells of a desired species, such as human cells, according to codon optimization principles known in the art, wherein human IL-21 comprises, essentially consisting of, or encoded by, a nucleic acid sequence that is at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to NM_021803.4, NM_001207006.2, or SEQ ID NO: 25.

[0109] As a means of guidance, and not limited thereto, human interleukin-12 subunit alpha (IL-12A) precursors are annotated under Genbank accession numbers NP_000873.2 (isoform 1), NP_001341511.1 (isoform 2), NP_001341512.1 (isoform 3), and NP_001384921.1 (isoform 4). As a means of guidance, and not limited thereto, human interleukin-12 subunit beta (IL-12B) precursors are annotated under Genbank accession number NP_002178. Collectively, IL-12A(p35) and IL-12B(p40) form a heterodimeric active cytokine referred to as p70.

[0110] A specific example of human IL-12A, i.e., isoform 1 precursor (NP_000873.2), is reproduced below. MWPPGSASQPPPSPAAATGLHPAARPVSLQCRLSMCPARSLLLVATLVLLDHLSLARNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (Sequence ID 26).

[0111] A specific example of a human IL-12A subunit co-expressed with CAR is reproduced below. MCPARSLLLVATLVLLDHLSLARNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (SEQ ID NO: 27).

[0112] A specific example of the human IL-12B precursor (NP_002178.2) is reproduced below. MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNK eye YSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS (SEQ ID NO: 28).

[0113] In one embodiment, IL-12A and IL-12B refer to each polypeptide essentially consisting of or comprising the amino acid sequence of SEQ ID NOs. 26-28, which is at least 80% identical, for example, at least 85% identical, preferably at least 90% identical, for example at least 95% identical, more preferably at least 96%, at least 97%, at least 98%, or at least 99% identical. In a particularly preferred embodiment, IL-12A essentially consists of or comprises the amino acid sequence shown in SEQ ID NOs. 26 or 27. In a particularly preferred embodiment, IL-12B essentially consists of or comprises the amino acid sequence shown in SEQ ID NOs. 28.

[0114] In one embodiment, the human IL-12A precursor is annotated in Genbank under accession numbers NM_000882.3 (isoform 1), NM_001354582.2 (isoform 2), NM_001354583.2 (isoform 3), NM_001397992.1 (isoform 4), or SEQ ID NO: 29: ATGTGTCCAGCGCGCAGCCTCCTCCTTGTGGCTACCCTGGTCCTCCTGGACCACCTCAGTTTGGCCAGAAACCTCCCCGTGGCCACTCCAGACCCAGGAATGTTCCCATGCCTTCACCACTCCCAAAACCTGCTGAGGGCCGTCAGCAACATGCTCCAGAAGGCCAGACAAACTCTAGAATTTTACCCTTGCACTTCTGAAGAGATTGATCATGAAGATATCACAAAAGATAAAACCAGCACAGTGGAGGCCTGTTTACCATTGGAATTAACCAAGAATGAGAGTTGCCTAAATTCCAGAGAGACCTCTTTCATAACTAATGGGAGTTGCCTGGCCTCCAGAAAGACCTCTTTTATGATGGCCCTGTGCCTTAGTAGTATTTATGAAGACTTGAAGATGTACCAGGTGGAGTTCAAGACCATGAATGCAAAGCTTCTGATGGATCCTAAGAGGCAGATCTTTCTAGATCAAAACATGCTGGCAGTTATTGATGAGCTGATGCAGGCCCTGAATTTCAACAGTGAGACTGTGCCACAAAAATCCTCCCTTGAAGAACCGGATTTTTATAAAACTAAAATCAAGCTCTGCATACTTCTTCATGCTTTCAGAATTCGGGCAGTGACTATTGATAGAGTGATGAGCTATCTGAATGCTTCC(SEQ ID NO: 29) A nucleic acid sequence comprising, essentially consisting of, or encoding a nucleic acid comprising the nucleic acid sequence shown, wherein any codon in NM_000882.3, NM_001354582.2, NM_001354583.2, NM_001397992.1, or SEQ ID NO: 29 may be replaced, each independently, with another codon encoding the same amino acid, preferably wherein the nucleic acid sequence is codon-optimized for expression in cells of a desired species, such as human cells, according to codon optimization principles known in the art, wherein human IL-12A comprises, essentially consisting of, or encoding a nucleic acid comprising the nucleic acid sequence, which is at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to NM_000882.3, NM_001354582.2, NM_001354583.2, NM_001397992.1, or SEQ ID NO: 29.

[0115] In one embodiment, the human IL-12B precursor is annotated in Genbank under accession number NM_002187.2 or SEQ ID NO: 30: ATGTGTCACCAGCAGTTGGTCATCTCTTGGTTTTCCCTGGTTTTTCTGGCATCTCCCCTCGTGGCCATATGGGAACTGAAGAAAGATGTTTATGTCGTAGAATTGGATTGGTATCCGGATGCCCCTGGAGAAATGGTGGTCCTCACCTGTGACACCCCTGAAGAAGATGGTATCACCTGGACCTTGGACCAGAGCAGTGAGGTCTTAGGCTCTGGCAAAACCCTGACCATCCAAGTCAAAGAGTTTGGAGATGCTGGCCAGTACACCTGTCACAAAGGAGGCGAGGTTCTAAGCCATTCGCTCCTGCTGCTTCACAAAAAGGAAGATGGAATTTGGTCCACTGATATTTTAAAGGACCAGAAAGAACCCAAAAATAAGACCTTTCTAAGATGCGAGGCCAAGAATTATTCTGGACGTTTCACCTGCTGGTGGCTGACGACAATCAGTACTGATTTGACATTCAGTGTCAAAAGCAGCAGAGGCTCTTCTGACCCCCAAGGGGTGACGTGCGGAGCTGCTACACTCTCTGCAGAGAGAGTCAGAGGGGACAACAAGGAGTATGAGTACTCAGTGGAGTGCCAGGAGGACAGTGCCTGCCCAGCTGCTGAGGAGAGTCTGCCCATTGAGGTCATGGTGGATGCCGTTCACAAGCTCAAGTATGAAAACTACACCAGCAGCTTCTTCATCAGGGACATCATCAAACCTGACCCACCCAAGAACTTGCAGCTGAAGCCATTAAAGAATTCTCGGCAGGTGGAGGTCAGCTGGGAGTACCCTGACACCTGGAGTACTCCACATTCCTACTTCTCCCTGACATTCTGCGTTCAGGTCCAGGGCAAGAGCAAGAGAGAAAAGAAAGATAGAGTCTTCACGGACAAGACCTCAGCCACGGTCATCTGCCGCAAAAATGCCAGCATTAGCGTGCGGGCCCAGGACCGCTACTATAGCTCATCTTGGAGCGAATGGGCATCTGTGCCCTGCAGT(SEQ ID NO: 3) A nucleic acid sequence comprising, essentially consisting of, or encoded by, a nucleic acid comprising the nucleic acid sequence shown, wherein any codon in NM_002187.2 or SEQ ID NO: 30 may be optionally replaced with other codons that independently encode the same amino acid, preferably wherein the nucleic acid sequence is codon-optimized for expression in cells of a desired species, such as human cells, according to codon optimization principles known in the art, wherein human IL-12B comprises, essentially consisting of, or encoded by, a nucleic acid sequence that is at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to NM_002187.2 or SEQ ID NO: 30.

[0116] In one embodiment, CARs and immunostimulatory cytokines, such as IL-15, IL-12, and / or IL-21, for example, IL-15 and / or IL-21, for example, more specifically IL-15, may be encoded by two separate open reading frames (ORFs). In one embodiment, the two separate ORFs may be contained in the same polynucleotide or in two separate polynucleotides. In one embodiment, the two separate ORFs may be contained in a single transcription unit or in two separate transcription units. In one embodiment, the two separate ORFs may be contained in a single mRNA molecule or in two separate mRNA molecules.

[0117] In one embodiment, a CAR and an immunostimulatory cytokine, such as IL-15, IL-12, and / or IL-21, particularly IL-15 and / or IL-21, and more specifically IL-15, may be included in a single ORF along with a peptide sequence sensitive to ribosome skipping or spontaneous proteolysis, positioned between the CAR and cytokine amino acid sequences. For example, a sequence encoding a 2A self-cleaving peptide may be inserted between the sequence encoding the CAR and the sequence encoding the cytokine. Examples of 2A peptides include T2A (EGRGSLLTCGDVEENPGP, SEQ ID NO: 17), P2A (ATNFSLLKQAGDVEENPGP, SEQ ID NO: 18), E2A (QCTNYALLKLAGDVESNPGP, SEQ ID NO: 19), and F2A (VKQTLNFDLLKLAGDVESNPGP, SEQ ID NO: 20). An optional GSG tripeptide may be introduced at the N-terminus to increase efficiency. Therefore, in one embodiment, the CAR and cytokine are encoded within the same ORF as the sequence encoding the polypeptide containing the 2A peptide inserted between them.

[0118] Accordingly, as an example, in one particularly preferred embodiment, the CAR-P2A-IL15 precursor is essentially composed of or comprises the amino acids shown in Sequence ID No. 21 below, where the signal sequence is in bold, the extracellular and intramembrane domains of CD27 are in standard letters, the 4-1BB intracellular costimulatory domain is in italics, the CD3ζ intracellular activation domain is underlined, the GSG-P2A autocleavage peptide is in bold italics, and IL-15 is in underlined bold. [ka]

[0119] In one embodiment, the CAR-P2A-IL15 precursor comprises or consists of essentially the same amino acid sequence as SEQ ID NO: 21, comprising at least 80%, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95%, for example, particularly preferably at least 96%, at least 97%, at least 98%, or at least 99% or 100% identical.

[0120] In one embodiment, the CAR-P2A-IL15 precursor forms taught herein, particularly those shown in SEQ ID NO: 21, are as shown in SEQ ID NO: 22 or 31. [ka] [ka] A nucleic acid sequence comprising, essentially consisting of, or encoding a nucleic acid comprising, the nucleic acid sequence shown in (1) below, wherein the ATG start and TAA stop codons of the CAR-P2A-IL15 coding sequence are bold, the SpeI restriction site is underlined, the GCCACC Kozak sequence is italicized, the XhoI restriction site is double underlined, and the EcoRI restriction site adjacent to the IL-15 cassette is bold and italicized (obviously, other restriction sites may or may not be included for experimental convenience), and optionally, here, any codon in SEQ ID NO: 22 or 31 may be replaced with other codons that independently code for the same amino acid, preferably, here, the nucleic acid sequence is codon-optimized for expression in cells of a desired species, such as human cells, according to codon optimization principles known in the art, and here, the CAR-P2A-IL15 precursor comprises, essentially consisting of, or encoding a nucleic acid comprising, the nucleic acid sequence that is at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to SEQ ID NO: 22 or 31.

[0121] Furthermore, as an example, in one particularly preferred embodiment, the CAR-P2A-IL21 precursor is essentially composed of or comprises the amino acids shown in Sequence ID No. 32 below, where the signal sequence is in bold, the extracellular and intramembrane domains of CD27 are in standard letters, the 4-1BB intracellular costimulatory domain is in italics, the CD3ζ intracellular activation domain is underlined, the GSG-P2A autocleavage peptide is in bold italics, and IL-21 is in underlined bold. [ka]

[0122] In one embodiment, the CAR-P2A-IL21 precursor comprises or consists of essentially the same amino acid sequence as SEQ ID NO: 32, comprising at least 80% identical, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95%, for example, particularly preferably at least 96%, at least 97%, at least 98%, or at least 99% or 100% identical.

[0123] In one embodiment, particularly as shown in Sequence ID No. 32, the CAR-P2A-IL21 precursor form taught herein is shown in Sequence ID No. 33 [ka] A nucleic acid sequence comprising, essentially consisting of, or encoding a nucleic acid comprising, the nucleic acid sequence shown in, where the ATG start and TAA stop codons of the CAR-P2A-IL21 coding sequence are bold, the SpeI restriction site is underlined, the GCCACC Kozak sequence is italicized, the XhoI restriction site is double underlined, and the EcoRI restriction site adjacent to the IL-21 cassette is bold and italicized (obviously, other restriction sites may or may not be included for experimental convenience), and optionally, here any codon in SEQ ID NO: 33 may be replaced with other codons that independently code for the same amino acid, preferably here the nucleic acid sequence is codon-optimized for expression in cells of a desired species, such as human cells, according to codon optimization principles known in the art, where the CAR-P2A-IL21 precursor comprises, essentially consisting of, or encoding a nucleic acid comprising, the nucleic acid sequence that is at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to SEQ ID NO: 33.

[0124] Furthermore, as an example, in one particularly preferred embodiment, the CAR-P2A-IL12 precursor is essentially composed of or comprises the amino acids shown in Sequence ID No. 34 below, where the signal sequence is in bold, the extracellular and intramembrane domains of CD27 are in standard letters, the 4-1BB intracellular costimulatory domain is in italics, the CD3ζ intracellular activation domain is underlined, the GSG-P2A autocleavage peptide is in bold italics, and IL-12A and 12B (in that order and separated by the GSG-P2A peptide) are in underlined bold. [ka]

[0125] In one embodiment, the CAR-P2A-IL12 precursor comprises or consists of essentially the same as SEQ ID NO: 34, comprising an amino acid sequence that is at least 80% identical, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95%, particularly preferably at least 96%, at least 97%, at least 98%, or at least 99% or 100% identical.

[0126] In one embodiment, the CAR-P2A-IL12 precursor form taught herein, particularly as shown in SEQ ID NO: 34, is also shown in SEQ ID NO: 35 [ka] [ka] A nucleic acid sequence comprising, essentially consisting of, or encoding by a nucleic acid consisting of, the nucleic acid sequence shown below, where the ATG start and TAA stop codons of the CAR-P2A-IL12A-P2A-IL12B coding sequence are in bold, the SpeI restriction site is underlined, the GCCACC Kozak sequence is in italics, the XhoI restriction site is double underlined, and the EcoRI restriction site adjacent to the IL-12 cassette is in bold italics (obviously, other restriction sites may or may not be included for experimental convenience), and optionally, where any of the sequences of Sequence ID No. 35 The codons may be replaced by other codons that independently encode the same amino acid, preferably therein the nucleic acid sequence is codon-optimized for expression in cells of a desired species, such as human cells, according to codon optimization principles known in the art, wherein the CAR-P2A-IL21 precursor contains a nucleic acid sequence that is at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to SEQ ID NO: 35, and is essentially composed of or encoded by a nucleic acid composed of the same.

[0127] Further aspects of the present invention provide a method for producing the NK cells taught herein, comprising introducing a nucleic acid encoding a CAR as defined herein in an expressible form and optionally a nucleic acid encoding one or more immunostimulatory cytokines in an expressible form into a starting population of NK cells.

[0128] In one embodiment, the nucleic acid introduced into the cells may be a DNA expression cassette or vector, in which mRNA encoding a CAR and optionally a cytokine can be transcribed using the cell's transcription mechanism, as described above, optionally within a single ORF. In another embodiment, the nucleic acid introduced into the cells may be mRNA encoding a CAR and optionally a cytokine, as described above, which can be translated by the cell's protein translation mechanism. In one preferred embodiment, nucleic acids such as mRNA may be introduced into a starting population of NK cells by electroporation.

[0129] The method may, if desired but not necessarily, further include the selection and / or expansion of NK cells capable of expressing one or more nucleic acids, CARs, and optionally one or more immunostimulatory cytokines. Methods for selecting transfect or transduced cells are routine in the art, and established positive selection markers such as genes conferring resistance to blastosidine, genethecin, hygromycin B, puromycin, or zeosin may be used.

[0130] In one particularly preferred embodiment, the method may involve transient transfection of NK cells with a DNA construct or mRNA molecule, preferably an mRNA molecule. In one embodiment, transient transfection may not require any selection of transfected cells. In one embodiment, the method may involve stable transfected NK cells, in which case selection of manipulated cells is desirable.

[0131] Further embodiments provide pharmaceutical compositions comprising engineered NK cells and a pharmaceutically acceptable carrier as taught herein.

[0132] The term "pharmaceutically acceptable" as used herein means that it is consistent with the technology, compatible with other components of the pharmaceutical composition, and not harmful to its recipient.

[0133] The “carriers” or “additives” used herein include any and all solvents, diluents, buffers (e.g., neutral buffered saline or phosphate-buffered saline), solubilizers, colloids, dispersion media, media, fillers, chelating agents (e.g., EDTA or glutathione), amino acids (e.g., glycine), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavoring agents, fragrances, thickeners, agents for achieving depot effects, coatings, antifungal agents, preservatives, stabilizers, antioxidants, tonicity regulators, absorption retarders, etc. The use of such media and agents for pharmaceutically active substances is well known in the art. Such substances are non-toxic and should not interfere with cellular activity.

[0134] The exact properties of the carrier, additive, or other substance depend on the route of administration. For example, the composition may be in the form of a non-enterally acceptable aqueous solution that is pyrogen-free and has appropriate pH, isotonicity, and stability. For general principles in pharmaceutical formulations, readers should refer to *Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy*, by G. Morstyn & W. Sheridan eds., Cambridge University Press, 1996; and *Hematopoietic Stem Cell Therapy*, ED Ball, J. Lister & P. ​​Law, Churchill Livingstone, 2000.

[0135] Liquid pharmaceutical compositions may generally contain a liquid carrier such as water or a pharmaceutically acceptable aqueous solution. For example, they may contain physiological saline solution, tissue or cell culture medium, dextrose or other sugar solutions, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol.

[0136] The composition may contain one or more cytoprotective molecules, cell regeneration molecules, growth factors, anti-apoptotic factors, or factors that regulate gene expression in cells. Such substances may make cells independent of their environment.

[0137] Such pharmaceutical compositions may contain further components to ensure cell viability. For example, the composition may include a suitable buffer system (e.g., a phosphate or carbonate buffer system) to achieve a desired pH, more generally a nearly neutral pH, and may include sufficient salts to ensure isotonic conditions for the cells to prevent osmotic stress. For example, suitable solutions for these purposes may be phosphate-buffered saline (PBS), sodium chloride solution, Ringer's injection, or lactated Ringer's injection, which are known in the art. Furthermore, the composition may include a carrier protein, such as albumin (e.g., bovine or human albumin), which may enhance cell viability.

[0138] More preferably pharmaceutically acceptable carriers or additives are well known to those skilled in the art and can be selected from, for example, proteins such as collagen or gelatin, carbohydrates such as starch, polysaccharides, sugars (dextrose, glucose and sucrose), cellulose derivatives such as sodium or calcium carboxymethylcellulose, hydroxypropylcellulose or hydroxypropyl methylcellulose, pregelatinized starch, pectin agar, carrageenan, clay, hydrophilic rubbers (acacia gum, guar gum, acacia gum and xanthan gum), alginic acid, alginates, hyaluronic acid, polyglycolic acid and polylactic acid, dextran, pectin, synthetic polymers such as water-soluble acrylic acid polymers or polyvinylpyrrolidone, proteoglycans, calcium phosphate, and the like.

[0139] In one embodiment, the pharmaceutical cell preparation defined above may be administered in the form of a liquid composition. In one embodiment, cells or a pharmaceutical composition containing them may be administered systemically, topically, intra-organ, to a site of organ dysfunction or lesion, or to a site of tissue lesion.

[0140] Preferably, the pharmaceutical composition may contain a therapeutically effective amount of the desired cells. The term “therapeutic amount” means an amount that can induce a biological or pharmacokinetic response in a tissue, system, animal or human, particularly one or more local or systemic symptoms or characteristics of the disease or condition being treated, as sought by researchers, veterinarians, physicians or other clinicians. An appropriate therapeutically effective amount may be determined by a qualified physician, taking into account the properties of the desired cells, the disease state and severity, as well as the age, size and condition of the subject.

[0141] Also provided is a method for producing the pharmaceutical composition by mixing the cells of the present invention with one or more of the above-mentioned additional components and one or more of the above-mentioned pharmaceutical additives.

[0142] Disclosed are arrangements or kits of parts that include surgical devices or apparatus for administering the cells or pharmaceutical compositions as defined herein to a subject, for example, by injection, for example, systemically, and further include the cells or pharmaceutical compositions as defined herein.

[0143] In one embodiment, the pharmaceutical composition defined above may be administered in the form of a liquid composition.

[0144] The amount of cells to be administered varies depending on the target of treatment. In one embodiment, the amount of cells to be administered is 10 2 ~10 10 or 10 2 ~10 9 or 10 3 ~10 10 or 10 3 ~10 9 or 10 4 ~10 10 or 10 4~10 9 For example, 10 4 ~10 8 or 10 5 ~10 7 For example, approximately 1 × 10 5 , about 5×10 5 , about 1×10 6 , about 5×10 6 , about 1×10 7 , about 5×10 7 , about 1×10 8 , about 5×10 8 , about 1×10 9 , about 5×10 9 Or approximately 1 x 10 10 Cells can be administered to human subjects. For example, such administration may be preferably administered over a period of one day or more (e.g., over one, two, three, four, or five days or more), and may be divided into one or more doses (e.g., two, three, four, five, six, seven, eight, nine, or ten or more doses). However, the precise determination of the therapeutically effective dose can be readily determined by those skilled in the art from this disclosure and knowledge of the art, based on individual factors of each patient, including physique, age, and tissue damage. Preferably, but not limited to, in the composition to be administered, the cells are approximately 10 4 / ml~about 10 9 / ml, preferably about 10 5 / ml~about 10 8 / ml, more preferably about 1 × 10⁻⁶ 6 / ml ~ approx. 1×10 8 It can exist at a concentration of / ml.

[0145] Further embodiments provide engineered NK cells or pharmaceutical compositions taught herein for use in therapeutic purposes. Particularly applicable are engineered NK cells or pharmaceutical compositions taught herein for use in methods of treating neoplastic diseases. Particularly applicable are engineered NK cells or pharmaceutical compositions taught herein for use in methods of treating cancer.

[0146] The term “treatment” or “procedure” broadly encompasses both curative and preventive measures, and may particularly refer to the alleviation or measurable reduction of one or more symptoms or measurable markers of a pathological condition such as a disease or disorder. The term encompasses primary treatment as well as neoadjuvant treatment, adjuvant treatment, and adjuvant therapy. Measurable reduction includes a statistically significant decrease in a measurable marker or symptom. Generally, the term encompasses both curative treatment and treatment intended to reduce the symptoms and / or delay the progression of a disease. The term encompasses both therapeutic treatment and preventive or protective measures for already established pathological conditions, where the objective is to prevent or reduce the opportunity for the onset of the condition. In some embodiments, the term may refer to therapeutic treatment. In other embodiments, the term may refer to preventive treatment. Treatment of chronic conditions during remission may also be considered to constitute therapeutic treatment. The term may include ex vivo or in vivo treatment, insofar as it is appropriate in the configuration of the present invention.

[0147] The terms “subject,” “individual,” or “patient” are used interchangeably throughout this specification and typically and preferably mean human, but may also include non-human animals, preferably warm-blooded animals, and even more preferably non-human mammals. Human subjects, including both sexes and all age classifications, are particularly preferred. In other embodiments, subjects are experimental animals or surrogate animals used as disease models. The terms do not imply any particular age or sex. Thus, adult and neonatal subjects, as well as fetuses, whether male or female, are intended to be covered. The term subject is further intended to include transgenic non-human species.

[0148] The term "subjects requiring treatment" or similar terms used herein refers to subjects diagnosed with or possessing any of the diseases described herein, and / or subjects for whom prevention of such diseases is necessary.

[0149] The term "neoplastic disease" generally refers to any disease or disorder characterized by neoplastic cell growth and proliferation, whether benign (not invading surrounding normal tissue, not forming metastases), premalignant (precancerous), or malignant (capable of invading adjacent tissues and producing metastases). The term neoplastic disease generally includes all transformed cells and tissues, as well as all cancerous cells and tissues. Neoplastic diseases or disorders include, but are not limited to, abnormal cell proliferation, benign tumors, premalignant or precancerous lesions, malignant tumors, and cancer. Examples of neoplastic diseases or disorders are benign, premalignant, or malignant neoplasms located in any tissue or organ, such as the prostate, colon, abdomen, bones, breasts, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal glands, parathyroid glands, pituitary gland, testes, ovaries, thymus, thyroid gland), eyes, head and neck, nerves (central and peripheral), lymphatic system, pelvic cavity, skin, soft tissues, spleen, thoracic cavity, or urogenital tract.

[0150] The terms “tumor” or “tumor tissue” as used herein refer to an abnormal mass of tissue resulting from excessive cell division. Tumors or tumor tissue include tumor cells, which are neoplastic cells that have abnormal proliferative properties and no useful bodily function. Tumors, tumor tissue, and tumor cells may be benign, premalignant, or malignant and may present with no potential for cancer. Tumors or tumor tissue may also include tumor-associated non-tumor cells, such as vascular cells that form blood vessels supplying the tumor or tumor tissue. Non-tumor cells may be regenerated and promoted by tumor cells, for example, through the induction of angiogenesis in the tumor or tumor tissue.

[0151] As used herein, the term "cancer" refers to a malignant neoplasm characterized by uncontrolled or unregulated cell proliferation. The term "cancer" includes primary malignant cells or tumors (e.g., cells that have not migrated to a site other than the original malignant tumor or tumor site in the body in question) and secondary malignant cells or tumors (e.g., malignant cells or tumor cells resulting from metastasis or migration to a secondary site different from the original tumor site). The term "metastatic" or "metastasis" generally refers to the spread of cancer from one organ or tissue to another non-adjacent organ or tissue. The appearance of a neoplastic disease in another non-adjacent organ or tissue is called metastasis.

[0152] Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemia or lymphoid malignancies. Further specific examples of such cancers include, but are not limited to, squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma and large cell lung cancer, peritoneal cancer, hepatocellular carcinoma, gastric cancer including gastrointestinal cancer, pancreatic cancer, glioma, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, as well as CNS cancers, melanoma, head and neck cancer, bone cancer, bone marrow cancer, duodenal cancer, esophageal cancer, thyroid cancer or hematological cancer.

[0153] Other non-limiting examples of cancer or malignant tumors include acute childhood lymphoblastic leukemia, acute lymphoblastic leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, adult (primary) hepatocellular carcinoma, adult (primary) liver cancer, adult acute lymphoblastic leukemia, adult acute myeloid leukemia, adult Hodgkin's disease, adult Hodgkin's lymphoma, adult lymphoblastic leukemia, adult non-Hodgkin's lymphoma, adult primary liver cancer, adult soft tissue sarcoma, AIDS-associated lymphoma, AIDS-associated malignant tumors, anal cancer, astrocytoma, cholangiocarcinoma, bladder cancer, bone cancer, brainstem glioma, brain tumor, breast cancer, cancer of the renal pelvis and urethra, and central nervous system cancer. (Primary) Lymphoma, Central Nervous System Lymphoma, Cerebellar Astrocytoma, Brain Astrocytoma, Cervical Cancer, Pediatric (Primary) Hepatocellular Carcinoma, Pediatric (Primary) Liver Cancer, Pediatric Acute Lymphoblastic Leukemia, Pediatric Acute Myeloid Leukemia, Pediatric Brainstem Glioma, Glioblastoma, Pediatric Cerebellar Astrocytoma, Pediatric Brain Astrocytoma, Pediatric Extracranial Germ Cell Tumor, Pediatric Hodgkin's Disease, Pediatric Hodgkin's Lymphoma, Pediatric Hypothalamic and Visual Tract Glioma, Pediatric Lymphoblastic Leukemia, Pediatric Medulloblastoma, Pediatric Non-Hodgkin's Lymphoma, Pediatric Pineal and Supratentorial Primitive Neuroectodermal Tumor, Pediatric Primary Liver Cancer, Pediatric Rhabdomyosarcoma, Pediatric Soft Tissue Sarcoma, Pediatric Visual Pathological and hypothalamic glioma, chronic lymphocytic leukemia, chronic myeloid leukemia, colon cancer, cutaneous T-cell lymphoma, endocrine pancreatic islet cell carcinoma, endometrial cancer, ependymoma, epithelial carcinoma, esophageal cancer, Ewing's sarcoma and related tumors, exocrine pancreatic carcinoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic cholangiocarcinoma, eye cancer, female breast cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal tumor, germ cell tumor, pregnancy trophoblast tumor, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, Hodgkin's disease, Hodgkin's lymphoma, hypergammaglobulinemia, hypopharyngeal cancer, intestinal cancer, intraocular melanoma, islet cell carcinoma, islet cell pancreatic cancer, capillary cancer Di sarcoma, kidney cancer, laryngeal cancer, lip and oral cancer, liver cancer, lung cancer, lymphoproliferative disorders, macroglobulinemia, male breast cancer, malignant mesothelioma, malignant thymoma, medulloblastoma, melanoma, mesothelioma, metastatic or potentially metastatic primary squamous cell carcinoma of the neck, metastatic primary squamous cell carcinoma of the neck, metastatic squamous cell carcinoma of the neck, multiple myeloma, multiple myeloma / plasmacytic neoplasm, myelodysplastic syndrome, myeloid leukemia, myeloid leukemia, myeloproliferative disorders, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma during pregnancy, non-melanoma skin cancer, non-small cell lung cancer, potentially primary metastatic squamous cell carcinoma of the neck, oropharyngeal cancer, bone / malignant fibrous sarcoma,Osteosarcoma / malignant fibrous histiocytoma, osteosarcoma / malignant fibrous histiocytoma of bone, ovarian epithelial carcinoma, ovarian germ cell tumor, low-grade ovarian tumor, pancreatic cancer, paraproteinemia, purpura, parathyroid cancer, penile cancer, pheochromocytoma, pituitary tumor, plasma cell neoplasm / multiple myeloma, primary central nervous system lymphoma, primary liver cancer, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and urethral cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoidosis sarcoma, Sézary syndrome, skin cancer, small-scale cancers. This includes, but is not limited to, alveolar lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma of the neck, gastric cancer, supratentorial cerebellar primitive neuroectodermal tumors and pineal gland tumors, T-cell lymphoma, testicular cancer, thymoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and urethra, transitional renal pelvis and urethral cancer, trophoblastic cell carcinoma, urethral and pyelocyte carcinoma, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, optic tract and hypothalamic glioma, vulvar cancer, Waldenström macroglobulinemia, or Wilms' tumor.

[0154] In one embodiment, the cancer is a hematological malignancy. In one embodiment, the cancer is Burkitt lymphoma. In one embodiment, the cancer is a solid malignancy (solid tumor). In one embodiment, the cancer is colorectal cancer or pancreatic cancer. In one embodiment, when the cancer is a hematological malignancy, NK cells may be engineered to further express one or more immunostimulatory cytokines, although this is not essential. In one embodiment, when the cancer is a solid tumor, NK cells are engineered to further express one or more immunostimulatory cytokines, preferably one or more immunostimulatory interleukins, such as IL-15 and / or IL-21, for example, at least IL-15 or IL-15 alone, for example. We have shown that the co-expression of immunostimulatory interleukins and CARs in NK cells greatly improves the ability of NK cells to target solid tumor cells, including cancer cells and cells in the tumor environment such as CAFs.

[0155] In one embodiment, a neoplastic disease such as cancer includes CD70-positive cancer cells. Cells such as tumor cells or cells of the tumor microenvironment described herein may be described in the context herein as "expressing" or "not expressing" one or more markers, such as one or more genes, polypeptides or proteins, such as CD70, or as "positive" (+) or "negative" (-) for one or more markers, such as one or more genes, polypeptides or proteins, such as CD70.

[0156] Such terminology is standard and well understood by those skilled in the art when characterizing cellular phenotypes. As a means of additional guidance, when a cell is positive for, expresses, or contains expression for a certain marker, e.g., a gene, polypeptide, or protein, e.g., CD70, a person skilled in the art will conclude the presence or evidence of a clear signal for the marker when performing a measurement capable of detecting or quantifying the marker intracellularly or above. Preferably, the presence or evidence of a clear signal for a marker is concluded based on a comparison of the measurement results obtained in a cell with the results of the same measurement performed in a negative control (e.g., a cell known not to express the marker) and / or a positive control (e.g., a cell known to express the marker). If the measurement method allows for the quantitative evaluation of the marker, then the positive cell will produce a signal for the marker at least 1.5 times higher than the signal for the marker produced by the negative control cell or the average signal for the marker produced by the negative control cell population, e.g., at least 2 times, at least 4 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, or even higher. Furthermore, positive cells may produce signals for markers that are 3.0 standard deviations or more higher than the mean signal for markers produced by the negative control cell population, for example, 3.5 standard deviations or more, 4.0 standard deviations or more, 4.5 standard deviations or more, or 5.0 standard deviations or more.

[0157] The presence or absence (e.g., readout is presence vs. absence; or detectable vs. undetectable) and / or quality (e.g., readout is absolute or relative quality) of CD70-positive cells in a biological sample from a subject can be measured using any existing, available, or conventional separation, detection, and / or quantification method. Examples include, but are not limited to, standard immunological assay methods, including immunohistochemistry, immunocytochemistry, flow cytometry, mass cytometry, fluorescent cell sorting (FACS), fluorescence microscopy, fluorescence-based cell sorting using microfluidic systems, immunoaffinity adsorption-based techniques such as affinity chromatography, magnetic particle separation, magnetically activated cell sorting or bead-based cell sorting using microfluidic systems, enzyme-linked immunosorbent assay (ELISA) and ELISPOT-based techniques, radioimmunoassay (RIA), and Western blotting. Anti-CD70 antibodies suitable for such techniques are commercially available from a variety of suppliers.

[0158] As used herein, the terms “sample” or “biological sample” include any biological specimen obtained (isolated, removed) from a subject. A sample may include, but is not limited to, organ tissue (e.g., primary or metastatic tumor tissue), whole blood, plasma, serum, whole blood cells, red blood cells, white blood cells (e.g., peripheral blood mononuclear cells), saliva, urine, feces, tears, sweat, sebum, nipple aspirate, intraductal lavage, tumor exudate, synovial fluid, cerebrospinal fluid, lymph, fine-needle aspiration, amniotic fluid, any other body fluid, exudate or secretion, cell lysate, cell secretions, inflammatory fluid, semen, and vaginal secretions. Preferably, a sample may be obtained by non-invasive or minimally invasive methods such as blood collection ('liquid biopsy'), urine collection, fecal collection, tissue (e.g., tumor tissue) biopsy, or fine-needle aspiration, which allow for the supply / removal / isolation of the sample from the subject. The term "tissue" as used herein encompasses all types of cells in the human body, including not only organ cells but also blood and other bodily fluids mentioned above. Tissue may be healthy or affected by pathological changes, for example, tumor tissue. Tissue may be from a living subject or from a corpse. Particularly useful samples are those known, expected, or predicted to contain tumor cells and / or tumor microenvironment cells.

[0159] Any suitable weight or volume of sample can be taken from the subject for analysis. Liquid samples may have volumes ranging from 1 mL to 20 mL, e.g., 5 mL, 7.5 mL, 10 mL, 15 mL, or 20 mL. Solid samples may have weights ranging from 1 g to 20 g, e.g., 5 g, 7.5 g, 10 g, 15 g, or 20 g.

[0160] In one embodiment, a neoplasm such as cancer contains 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or up to 100% CD70-positive cancer cells, where the percentage of CD70-positive cancer cells represents the ratio to total cancer cells in a representative sample of the neoplasm lesion. In another embodiment, a neoplasm such as cancer contains 10% or less CD70-positive cancer cells, for example, 8% or less, 6% or less, 4% or less, 2% or less, or at least 0%, where the percentage of CD70-positive cancer cells represents the ratio to total cancer cells in a representative sample of the neoplasm lesion. Due to their ability to target CD70-positive tumor microenvironment (TME) cells such as cancer-associated fibroblasts (CAFs), NK cells manipulated according to embodiments of the present invention may also be therapeutically effective in cancers with relatively low CD70 expression levels in cancer cells.

[0161] In one embodiment, neoplasms such as cancer include CD70-positive cancer-associated fibroblasts (CAFs). The terms “cancer-associated fibroblasts,” “CAF,” “tumor-associated fibroblasts,” “oncogenicity-associated fibroblasts,” or “activated fibroblasts” refer to cell types within the tumor microenvironment that have been reported to promote oncogenic characteristics by initiating extracellular matrix remodeling or cytokine secretion. CAFs have been reported to originate from normal fibroblasts, pericytes, smooth muscle cells, fibrous cells, or mesenchymal stem cells. Based on current knowledge, CAFs may support and stimulate tumor growth by secreting growth factors such as vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF), as well as other chemokines, to stimulate angiogenesis.

[0162] In one embodiment, neoplasms such as cancer contain 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or up to 100% CD70-positive CAFs, and the percentage of CD70-positive CAFs represents the ratio to total CAFs in a representative sample of the neoplasm lesion.

[0163] In one embodiment, neoplastic diseases such as cancer include CD70-positive cancer cells and CD70-positive cancer-associated fibroblasts (CAFs).

[0164] In one embodiment, a neoplasm, such as cancer, contains less than 10% CD70-positive cancer cells, e.g., 8% or less, 6% or less, 4% or less, 2% or less, or at least 0% (the percentage of CD70-positive cancer cells represents the ratio of all cancer cells in a representative sample of the neoplasm); and 10% or more CD70-positive CAFs, e.g., 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or at most 100% (the percentage of CD70-positive CAFs represents the ratio of all CAFs in a representative sample of CD70-positive CAFs in the neoplasm).

[0165] In one embodiment, the neoplasm is colorectal cancer (CC), and more specifically in one embodiment, colorectal cancer including CD70-positive carcinomatous fibroblasts (CAFs). The inventors characterize CC as often having a small number of CD70-positive cancer cells (e.g., less than 10% or less than 5%) but simultaneously having a relatively large proportion of CD70-positive CAFs. Furthermore, the percentage of CD70-positive CAFs is thought to increase as the stage of CC progresses. Therefore, preferably, CC is stage T1, more preferably stage T2, even more preferably stage T3, and most preferably stage T4.

[0166] In one embodiment, colorectal cancer contains 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or up to 100% CD70-positive CAFs, where the percentage of CD70-positive CAFs represents the ratio to total CAFs in a representative sample of colorectal cancer lesions.

[0167] In some embodiments, the engineered NK cells taught herein may be administered as a sole agent (active pharmaceutical ingredient) or in combination with one or more other agents, provided that the combination does not cause unacceptable adverse effects. For example, NK cells may be combined with one or more known anti-cancer treatments, such as surgery, radiotherapy, chemotherapy, biological therapy, or a combination thereof. The term “chemotherapy” as used herein is understood to broadly and generally encompass treatments using chemical substances or compositions. Chemotherapy agents typically exhibit cytotoxic or cell proliferation inhibitory effects. In some embodiments, chemotherapy agents may be alkylating agents, cytotoxic compounds, antimetabolites, plant alkaloids, terpenoids, topoisomerase inhibitors, or combinations thereof. The term “biological therapy” as used herein is understood to broadly and generally encompass treatments using biomolecules such as viruses or cells, or biological substances or compositions such as biological preparations. In some embodiments, biomolecules may be peptides, polypeptides, proteins, nucleic acids or small molecules (e.g., primary metabolites, secondary metabolites or natural products), or combinations thereof. Examples of suitable biomolecules include, but are not limited to, interleukins, cytokines, anti-cytokines, tumor necrosis factor (TNF), cytokine receptors, vaccines, interferons, enzymes, therapeutic antibodies, antibody fragments, antibody-like protein scaffolds, or combinations thereof. Examples of suitable biomolecules include aldesleukin, alemtuzumab, atezolizumab, bevacizumab, blinatumomab, brentuximab, vedotin, catumakisomab, cetuximab, daratumumab, denileukin difutitox, denosumab, dinutuximab, elotuzumab, gemtuzumab, ozogamicin, 90 Y-ibritumomab tiuxetan, idarucizumab, interferon A, ipilimumab, necitumumab, nivolumab, obinutuzumab, ofatumumab, olaratumumab, panitumumab, pembrolizumab, ramucirumab, rituximab, tasonelmin, 131This includes, but is not limited to, I-tositumomab, trastuzumab, ad-trastuzumab ethansine, and combinations thereof. Suitable examples of oncolytic viruses include, but are not limited to, tarimodine raherparepbec. Further categories of anticancer treatment include, among others, hormone therapy (endocrine therapy), immunotherapy, and stem cell therapy, which are generally considered to fall within the realm of biological therapy. Suitable hormone therapies include, but are not limited to, tamoxifen; aromatase inhibitors, e.g., anastrozole, exemestane, letrozole, and combinations thereof; luteinizing hormone blockers, e.g., goserelin, leuprorelin, triptorelin, and combinations thereof; antiandrogens, e.g., bicalutamide, cyproterone acetate, flutamide, and combinations thereof; gonadotropin-releasing hormone blockers, e.g., degarelix; progesterone treatments, e.g., medroxyprogesterone acetate, megestrol, and combinations thereof; and combinations thereof. The term “immunotherapy” broadly encompasses any treatment that modulates the immune system of a target. In particular, the term includes any treatment that modulates an immune response, such as humoral immune response, cell-mediated immune response, or both. Immunotherapy includes cell-based immunotherapy in which immune cells, such as T cells and / or dendritic cells, are transferred to a patient. The term also includes the administration of substances or compositions that modulate the immune system of a target, such as chemical compounds and / or biomolecules (e.g., antibodies, antigens, interleukins, cytokines, or combinations thereof). Examples of cancer immunotherapy include, but are not limited to, treatments using monoclonal antibodies against proteins expressed by tumor cells, such as Fc-modified monoclonal antibodies, immune checkpoint inhibitors, prophylactic or therapeutic cancer vaccines, adoptive cell therapy, and combinations thereof.Examples of immune checkpoints targeted for inhibition include, but are not limited to, PD-1 (examples of PD-1 inhibitors include, but are not limited to, pembrolizumab, nivolumab, and combinations thereof), CTLA-4 (examples of CTLA-4 inhibitors include, but are not limited to, ipilimumab, tremelimumab, and combinations thereof), PD-L1 (examples of PD-L1 inhibitors include, but are not limited to, atezolizumab), LAG3, B7-H3 (CD276), B7-H4, TIM-3, BTLA, A2aR, killer cell immunoglobulin-like receptors (KIRs), IDO, and combinations thereof. Other approaches for therapeutic anti-cancer vaccination include dendritic cell vaccines. This term broadly encompasses vaccines containing dendritic cells loaded with antigens to which an immune response is desired. Adoptive cell therapy (ACT) can be defined as the transfer of cells, most commonly immune-derived cells, such as cytotoxic T cells (CTLs), to the same patient or a new recipient host, with the aim of transmitting immunological functionality and characteristics to a new host. Where possible, the use of autologous cells facilitates minimizing recipient tissue rejection and graft-versus-host disease problems. Various strategies may include, for example, the use of genetically modified T cells by altering the specificity of the T cell receptor (TCR), such as by introducing novel TCR α and β chains with selective peptide specificity. Alternatively, chimeric antigen receptors (CARs) can be used to produce immune-responsive cells, such as T cells, that are specific to a selective target, such as malignant cells, and a wide variety of receptor chimeric constructs have been described. Stem cell therapy in cancer generally aims to replace bone marrow stem cells destroyed by radiotherapy and / or chemotherapy, and includes, but is not limited to, autologous, syngeneic, or allogeneic stem cell transplantation. Stem cells, particularly hematopoietic stem cells, are typically obtained from bone marrow, peripheral blood, or umbilical cord blood. Details regarding the administration routes, dosages, and treatment regimens of anticancer drugs are well known in the field, as can be found, for example, in “Cancer Clinical Pharmacology” (2005) ed. By Jan HM Schellens, Howard L. McLeod, and David R. Newell, Oxford University Press.The active ingredients of any combination therapy may be mixed or physically separated, and may be administered simultaneously or sequentially in any order.

[0168] The present invention also provides the embodiments and aspects described below: Description 1. Natural killer (NK) cells engineered to express a chimeric antigen receptor (CAR), wherein the CAR contains the extracellular domain of CD27 or its CD70 binding portion.

[0169] Description 2. NK cells of Description 1, in which the CAR further contains the intramembrane domain of CD27.

[0170] Description 3. NK cells of Description 1 or 2, in which the CAR lacks all or part of the intracellular domain of CD27.

[0171] Description 4. NK cells of any of descriptions 1-3, in which the CAR lacks all intracellular domains of CD27.

[0172] Description 5. Any NK cell described in Descriptions 1-4, wherein the intracellular portion of the CAR contains at least one intracellular activation domain.

[0173] Description 6. The NK cells of Description 5, wherein at least one intracellular activation domain is selected from the group consisting of a CD3ζ activation domain, an FcRγ activation domain, and combinations thereof.

[0174] Description 7. Any NK cell described in Descriptions 1-6, wherein the intracellular portion of the CAR contains at least one intracellular costimulatory domain.

[0175] Description 8. NK cells as described in Description 7, wherein at least one intracellular co-stimulatory domain is selected from the group consisting of the CD28 co-stimulatory domain, the 4-1BB co-stimulatory domain, the DAP10 co-stimulatory domain, the OX40 co-stimulatory domain, the ICOS co-stimulatory domain, and combinations thereof.

[0176] Description 9. NK cells from any of descriptions 5-8, in which the CAR contains the CD3ζ intracellular activation domain.

[0177] Description 10. NK cells from any of descriptions 5-9, in which the CAR contains the 4-1BB intracellular costimulatory domain.

[0178] Description 11. CAR is: It includes the extracellular and intramembrane domains of CD27, the CD3ζ intracellular activation domain, and the 4-1BB intracellular costimulatory domain; Essentially consisting of the extracellular and intramembrane domains of CD27, the intracellular activation domain of CD3ζ, and the intracellular costimulatory domain of 4-1BB; or It consists of the extracellular and intramembrane domains of CD27, the intracellular activation domain of CD3ζ, and the intracellular costimulatory domain of 4-1BB. One of the NK cells described in descriptions 1-10.

[0179] Description 12. CAR is: Contains at least 80% identical amino acid sequence to Sequence ID No. 1; Essentially identical to the amino acid sequence of sequence number 1 by at least 80%; The amino acid sequence is at least 80% identical to sequence number 1; Contains at least 85% identical amino acid sequence to SEQ ID NO: 1; Essentially identical to the amino acid sequence of sequence number 1 by at least 85%; The amino acid sequence is at least 85% identical to sequence number 1; Contains at least 90% identical amino acid sequence to SEQ ID NO: 1; Essentially identical to the amino acid sequence of sequence number 1 by at least 90%; The amino acid sequence is at least 90% identical to sequence number 1; Contains at least 95% identical amino acid sequence to SEQ ID NO: 1; Essentially identical to the amino acid sequence of sequence number 1 by at least 95%; The amino acid sequence is at least 95% identical to sequence number 1; Contains an amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1; Essentially, it consists of an amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1; Consists of an amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1; Contains the amino acid sequence shown in Sequence ID No. 1; Essentially consisting of the amino acid sequence shown in Sequence ID No. 1; or The amino acid sequence shown in Sequence ID No. 1 is: One of the NK cells described in descriptions 1-11.

[0180] Description 13. NK cells isolated from the subject, from any of the descriptions in 1-12.

[0181] Description 14. NK cells isolated from the umbilical cord blood or peripheral blood of the subject, as described in any of Descriptions 1-13.

[0182] Description 15. NK cells as described in Description 13 or 14, which are cultured in vitro as desired.

[0183] Description 16. NK cells from any of descriptions 1-13, which have been differentiated in vitro from hematopoietic stem cells or induced pluripotent stem (iPS) cells.

[0184] Description 17. NK cells from any of the descriptions 1-13, where the NK cells are from a cloned NK cell line.

[0185] Description 18. Any NK cell described in Descriptions 1-13, where the NK cell is an NK-92 cell.

[0186] Description 19. Any NK cells described in Descriptions 1-18, which have been engineered to express one or more immunostimulatory cytokines.

[0187] Description 20. NK cells as described in Description 19, wherein one or more immunostimulatory cytokines are immunostimulatory interleukins (ILs).

[0188] Description 21. NK cells of Description 20, wherein the immunostimulatory interleukin is IL-15, IL-12 and / or IL-21, preferably human IL-15, IL-12 and / or IL-21, more preferably IL-15 and / or IL-21, even more preferably human IL-15 and / or IL-21, even more preferably IL-15, particularly preferably human IL-15.

[0189] Description 22. NK cells of any of Descriptions 1-21, wherein the expression of CAR and one or more optional immunostimulatory cytokines is independently constitutive or inducible.

[0190] Description 23. A method for producing NK cells according to any of Descriptions 1 to 22, comprising introducing a nucleic acid encoding any of Descriptions 1 to 22 in an expressible form, and optionally a nucleic acid encoding one or more immunostimulatory cytokines, into a starting population of NK cells.

[0191] Description 24. The method described in Description 23, wherein one or more nucleic acids are introduced into a starting population of NK cells by electroporation.

[0192] Description 25. The method of Description 23 or 24, wherein one or more nucleic acids are mRNA.

[0193] Description 26. Any method of Descriptions 23 to 25, further comprising selecting and / or expanding NK cells that contain one or more nucleic acids and are capable of expressing CARs and optionally one or more immunostimulatory cytokines.

[0194] Description 27. A pharmaceutical composition comprising any NK cells from Descriptions 1 to 22 and a pharmaceutically acceptable carrier.

[0195] Description 28. Any NK cells from Descriptions 1 to 22 or the pharmaceutical composition of Description 27 for use in treatment.

[0196] Description 29. Any NK cells from Descriptions 1 to 22 or a pharmaceutical composition from Description 27 for use in a method of treating neoplastic diseases.

[0197] Description 30. Any NK cells from Descriptions 1-22 or a pharmaceutical composition from Description 27 for use in a method of treating cancer.

[0198] Description 31. A method for treating a subject in need of treatment, comprising administering to the subject a therapeutically effective amount of any NK cells from Descriptions 1 to 22 or the pharmaceutical composition from Description 27.

[0199] Description 32. A method for treating a subject with a neoplasm, comprising administering to the subject a therapeutically effective amount of any NK cells from Descriptions 1 to 22 or the pharmaceutical composition from Description 27.

[0200] Description 33. A method for treating a subject with cancer, comprising administering to the subject a therapeutically effective amount of any NK cells from Descriptions 1 to 22 or the pharmaceutical composition from Description 27.

[0201] Description 34. NK cells or pharmaceutical compositions or methods of Description 32 or 33 for use in neoplasms such as cancer, comprising CD70-positive cancer cells.

[0202] Description 35. NK cells or pharmaceutical composition or method of Description 34 for use in Description 34, wherein a neoplasm such as cancer contains 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or up to 100% CD70-positive cancer cells, and the percentage of CD70-positive cancer cells represents the ratio to total cancer cells in a representative sample of the neoplasm lesion.

[0203] Description 36. NK cells or pharmaceutical compositions or methods of Description 34 for use in which neoplasms such as cancer contain less than 10% CD70-positive cancer cells, e.g., 8% or less, 6% or less, 4% or less, 2% or less, or at least 0%, and the percentage of CD70-positive cancer cells represents the ratio of total cancer cells in a representative sample of the neoplasm lesion.

[0204] Description 37. NK cells or pharmaceutical compositions or methods of Description 32-36 for use in any of Descriptions 29, 30, or 34-36, wherein neoplasms such as cancer contain CD70-positive cancer-associated fibroblasts (CAFs).

[0205] Description 38. NK cells or pharmaceutical compositions or methods of Description 37 for use in a neoplasm such as cancer, which contains 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or up to 100% CD70-positive CAFs, where the percentage of CD70-positive CAFs represents the ratio to total CAFs in a representative sample of the neoplasm lesion.

[0206] Description 39. NK cells or pharmaceutical compositions or methods of Description 32 or 33 for use in Description 29 or 30, wherein neoplasms such as cancer include CD70-positive cancer cells and CD70-positive cancer-associated fibroblasts (CAFs).

[0207] Description 40. Neoplasmic diseases: CD70-positive cancer cells less than 10%, e.g., 8% or less, 6% or less, 4% or less, 2% or less, or at least 0% (the percentage of CD70-positive cancer cells represents the ratio of all cancer cells in a representative sample of neoplastic disease lesions); and CD70-positive CAFs 10% or more, e.g., 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or at most 100% (the percentage of CD70-positive CAFs represents the ratio of all CAFs in a representative sample of CD70-positive CAFs in neoplastic disease lesions). NK cells or pharmaceutical compositions or methods of description 39 for use in description 39.

[0208] Description 41. NK cells or pharmaceutical composition or any method of Description 32-40 for use in any of Descriptions 29, 30 or 34-40, wherein the neoplasm is colorectal cancer.

[0209] Description 42. NK cells or pharmaceutical composition or method of Description 41 for use in colorectal cancer comprising CD70-positive CAF.

[0210] Description 43. NK cells or pharmaceutical composition or method of Description 42 for use in Description 42, wherein colorectal cancer contains 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or up to 100% CD70-positive CAFs, and the percentage of CD70-positive CAFs represents the ratio of total CAFs in a representative sample of colorectal cancer lesions.

[0211] Although the present invention is described in relation to its specific embodiments, it will be obvious to those skilled in the art that many modifications, alterations, and variations are apparent in light of the foregoing. Accordingly, all such modifications, alterations, and variations are intended to be encompassed in accordance with the spirit and scope of the appended claims, as follows:

[0212] The aspects and embodiments of the present invention disclosed herein are further supported by the following non-limiting embodiments. [Examples]

[0213] Cell line: NK-92 cell lines (purchased from German Collection of Microorganisms and Cell Cultures GmbH (DSMZ, Leibniz Institute, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, #ACC 488)) were cultured in α-Minimal Essential Medium (α-MEM, Life Technologies, #32561037) supplemented with 12.5% ​​fetal bovine serum (FBS, Life Technologies, #10270106), 12.5% ​​equine serum (Life Technologies; #16050122), 1% penicillin / streptomycin (P / S, Life Technologies; #15140122), 2 mM L-glutamine (Life Technologies; #25030024), and 150 U / mL recombinant interleukin-2 (IL-2, ImmunoTools, #11340028). Cells were grown in suspension and maintained exponential growth in a humidified incubator at 37°C in 5% CO2 + 95% air.

[0214] Various CD70 + Target cell lines used: Burkitt lymphoma cell line (Raji), colorectal cancer cell line (LIM2099), pancreatic ductal carcinoma cell line (PANC-1), and pancreatic cancer-associated fibroblast (CAF) cell line (RLT-PSC).

[0215] Raji cell lines (purchased from DSMZ, #ACC319) and LIM2099 cell lines (purchased from Sigma-Aldrich, #CBA-0164) were cultured in Roswell Park Memorial Laboratory (RPMI, Life Technologies, #52400025) medium supplemented with 10% FBS, 1% P / S, and 2 mM L-glutamine. PANC-1 cell lines were purchased from ATCC (#CRL-1469) and cultured in Dulbecco's modified Eagle medium (DMEM, Life Technologies, #10938025) supplemented with 10% FBS, 1% P / S, and 2 mM L-glutamine. RLT-PSC cell lines (donated by Prof. M. Loehr and R. Jesenofsky, University of Heidelberg, Mannheim, Germany) were cultured in DMEM-F12 (Life Technologies, #11320074) supplemented with 10% FBS, 1% P / S, and 2 mM L-glutamine. The cells were grown as a monolayer and maintained at exponential growth in a humidified incubator at 37°C in 5% CO2 + 95% air.

[0216] CD70-directed CAR mRNA production : The CD27-CAR construct (shown in SEQ ID NO: 21 and encoded by SEQ ID NO: 31) comprising (1) the extracellular and transmembrane portions of the CD27 receptor, (2) the 41BB costimulatory domain, (3) the CD3ζ intracellular T cell activation domain, and (4) the IL-15 cytokine cassette was developed by CellRapeutics at Creative Biolabs (Shirley, NY, USA). TM The development was carried out using the Chimeric Antigen Receptor Technology platform. To produce constructs with and without IL-15, the IL-15 cytokine cassette was cleaved with EcoRI restriction enzyme (Life Technologies, #ER0271).

[0217] CD27-CAR construct-containing plasmids (with or without IL-15 cytokine cassette) were amplified in Escherichia coli bacteria, purified using the NucleoBond Xtra Midi Plus EF kit (Macherey Nagel; #740422.50), and linearized using compatible PmeI restriction enzyme (Life Technologies; #ER1342). CD27-CAR messenger RNA (mRNA) was extracted using mMESSAGE mMACHINE TM Using the T7 Transcription Kit (Life Technologies, #AM1344), the product was produced starting from 1 μg of linearized CD27-CAR DNA and stored at -80°C until use.

[0218] Development of CD70-oriented CAR-NK-92 cells: To optimize electroporation efficiency, NK-92 cells were stimulated with 150 U / mL IL-2 for 24 hours, followed by electroporation, and OptiMEM was used immediately before electroporation. TM Washed and resuspended with culture medium (Life Technologies, #11058021). 5–20 × 10⁶ per condition. 6 NK-92 cells were electroporated in 200 μL OptiMEM medium with 20 μg of CAR mRNA using a GenePulser (Bio-Rad, Hercules, CA, USA) with the following timed protocol: 300 V, 12 ms, and cuvette 4. NK-92 cells electroporated in the absence of mRNA (MOCK) were used as a negative control. After electroporation, the cells were resuspended in IL-2-free α-MEM medium as a recovery medium.

[0219] Verification of CD70-oriented CAR-NK-92 cells : CD27-CAR expression (extracellular portion of the CAR construct, CD27 expression) was determined using a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA) 24 hours after electroporation, using a monoclonal PE conjugate anti-human CD27 antibody (Cell Signaling Technology, Danvers, CA, USA; clone 0323; #55584S) and the corresponding IgG1 isotype control (Cell Signaling Technology; clone MOPC-21; #63630). 7-AAD (BioLegend, #420403) fluorescent insertion viability staining was used to analyze CD27 expression on viable cells. MOCK electroporated NK-92 cells were used as a negative control sample. The results are shown in Figures 3 and 4.

[0220] The production and secretion of IL-15 cytokines into the culture supernatant were verified using multiplex electrochemiluminescence (Meso Scale Discovery Inc., Rockville, USA, #K151URK-1).

[0221] In vitro cytotoxicity of CD70-oriented CAR-NK-92 cells : The in vitro killing ability of CD70-targeted CAR-NK-92 cells was investigated under various effector conditions (MOCK, CD27-CAR, and CD27-CAR with IL-15 cytokine cassette, 24 hours after electroporation), and various CD70 +Target cell lines—Burkitt lymphoma cell line (Raji), colorectal cancer and pancreatic cancer cell lines (LIM2099 and PANC-1, respectively), and pancreatic CAF cell line (RLT-PSC)—were evaluated by co-culturing them in sterile FACS tubes for 4 hours in a 5:1 effector:target ratio. After 4 hours of co-culturing, cell death in the target cell lines was measured using a CytoFLEX flow cytometer, stained with the viability insertion dye 7-AAD and the apoptotic cell death marker Annexin V (BD Bioscience, #51-65875X). To distinguish effector cells from target cells, the latter were transiently stained with the green fluorescent dye PKH67 (Sigma-Aldrich, #MIDI67-KT). The results are shown in Figure 5.

[0222] To analyze whether the observed death by CD70-CAR-NK-92 cells is CAR-specific, we used the extracellular antigen recognition domain, CD27, and CD70. + Blocked during co-culture with the Raji cell line. Various effector conditions (MOCK, CD27-CAR, and CD27-CAR with IL-15 cytokine cassette) were incubated overnight with three different concentrations (10 μg / mL, 50 μg / mL, and 100 μg / mL) of monoclonal neutralizing anti-CD27 antibody (R&D Systems; #MAB382) and corresponding IgG1 isotype controls (R&D Systems; #MAB002) 6 hours after electroporation. These effector cells were co-cultured with the Raji cell line in sterile FACS tubes at a 5:1 effector:target ratio for 4 hours 24 hours after electroporation. Raji cells incubated with the neutralizing antibody and isotype were used as control conditions. The amount of target cell death was detected as previously described. The results are shown in Figure 6.

[0223] In vitro cytotoxicity of CD70-oriented CAR-NK-92 cells and further cytokines: The ability of IL-12, IL-15, and IL-21 cytokines to improve the cytotoxic performance of CD70-directed CAR-NK-92 cells was determined. CD27-CAR cells without an IL-15 cytokine cassette were incubated overnight, 6 hours after electroporation, with effector doses of recombinant IL-12 (R&D Systems, #219-IL-005, 0.05 ng / mL), recombinant IL-15 (R&D Systems, #247-ILB-005, 2.60 ng / mL), and recombinant IL-21 (R&D Systems, #8879-ILB-010, 8 ng / mL) cytokines (ED50; provided by R&D Systems) to mimic or emulate the effects of each cytokine when co-expressed with CAR cells by NK-92 cells. Three effector conditions (MOCK, CD27-CAR, and CD27-CAR with IL-15 cytokine cassette) and stimulated CD27-CAR-92 cells were electroporated 24 hours later, and various CD70 cells were subjected to different treatments. + Target cell lines (Raji, LIM2099, PANC-1, and RLT-PSC) were co-cultured with the target cells in a 5:1 effector:target ratio in sterile FACS tubes for 4 hours. The amount of target cell death was detected as described earlier. The results are shown in Figure 7.

[0224] Statistical analysis: Prism 9.1.2 software (GraphPad) was used for data comparison, data graphing, and statistical calculations. The Kruskal-Wallis test was used for mean comparisons between more than two groups. The Mann-Whitney U test was used for mean comparisons between two groups. All statistical analyses were performed on a minimum of three independent experiments. A p-value ≤ 0.05 was considered statistically significant. The present invention provides, for example, the following: [Section 1] Natural killer (NK) cells engineered to express a chimeric antigen receptor (CAR), wherein the CAR contains the extracellular domain of CD27 or its CD70 binding portion. [Section 2] The NK cells of item 1, wherein the CAR further comprises the intracellular domain of CD27 and / or lacks all or part of the intracellular domain of CD27, preferably lacks all of the intracellular domain of CD27. [Item 3] The NK cells of item 1 or 2, wherein the intracellular portion of the CAR comprises an intracellular activation domain such as at least one CD3ζ or FcRγ intracellular activation domain and optionally and preferably an intracellular co-stimulation domain such as at least one CD28, 4-1BB, DAP10, OX40 and / or ICOS intracellular co-stimulation domain. [Item 4] The NK cells of item 3, wherein the CAR comprises a CD3ζ intracellular activation domain and / or a 4-1BB intracellular co-stimulation domain. [Item 5] The NK cells of any one of items 1 to 4, wherein the CAR comprises, consists essentially of or consists of the extracellular and intracellular domains of CD27, a CD3ζ intracellular activation domain and a 4-1BB intracellular co-stimulation domain. [Item 6] The NK cells of any one of items 1 to 5, wherein the CAR comprises an amino acid sequence that is at least 80% identical, preferably at least 90% identical, more preferably at least 95% identical, particularly preferably 100% identical to SEQ ID NO: 1, consists essentially of or consists of it. [Item 7] The NK cells of any one of items 1 to 6, wherein the NK cells are isolated from a subject such as from umbilical cord blood or peripheral blood of the subject and optionally cultured in vitro; or the NK cells are differentiated from hematopoietic stem cells or induced pluripotent stem (iPS) cells in vitro; or preferably the NK cells are from a cloned NK cell line, for example, particularly preferably NK-92 cells. [Item 8] The NK cells of any one of items 1 to 7, wherein the NK cells are engineered to further express one or more immune-stimulatory cytokines, such as one or more immune-stimulatory interleukins (ILs), preferably IL-15, IL-12 and / or IL-21, more preferably IL-15 and / or IL-21, particularly preferably human IL-15. [Item 9] Any NK cell according to any of claims 1 to 8, wherein the expression of the CAR and any one or more immunostimulatory cytokines is each independently constitutive or inducible. [Claim 10] A nucleic acid encoding any of the CARs according to any of claims 1 to 9 in an expressible form and, optionally, a nucleic acid encoding one or more immunostimulatory cytokines in an expressible form are introduced into a starting population of NK cells, optionally by electroporation or the like; and optionally including the selection and / or expansion of NK cells containing the one or more nucleic acids and capable of expressing the CAR and optionally one or more immunostimulatory cytokines, a method for producing an NK cell according to any of claims 1 to 9. [Claim 11] A pharmaceutical composition comprising an NK cell according to any of claims 1 to 9 and a pharmaceutically acceptable carrier. [Claim 12] An NK cell according to any of claims 1 to 9 or the pharmaceutical composition of claim 11 for use in therapy. [Claim 13] An NK cell according to any of claims 1 to 9 or the pharmaceutical composition of claim 11 for use in a method of treating a neoplastic disease, particularly cancer. [Claim 14] An NK cell or pharmaceutical composition for use according to claim 13, wherein the neoplastic disease comprises CD70-positive cancerous cells, for example, comprising 10% or more of CD70-positive cancerous cells and / or CD70-positive cancer-associated fibroblasts (CAFs), for example, comprising 10% or more of CD70-positive CAFs, for example, the neoplastic disease comprises less than 10% of CD70-positive cancerous cells and 10% or more of CD70-positive CAFs. [Claim 15] An NK cell or pharmaceutical composition for use according to claim 12 or 13, wherein the neoplastic disease is colorectal cancer comprising CD70-positive CAFs.

Claims

1. A pharmaceutical composition comprising natural killer (NK) cells engineered to express a chimeric antigen receptor (CAR), wherein the CAR includes the extracellular domain of CD27 or its CD70 binding portion, for the treatment of a neoplasm, wherein the neoplasm is a solid tumor containing CD70-positive cancer-associated fibroblasts (CAFs).

2. The pharmaceutical composition of claim 1, wherein CAR further comprises an intramembrane domain of CD27 and / or lacks all or part of the intracellular domain of CD27.

3. The pharmaceutical composition of Claim 2, wherein CAR lacks all of the intracellular domains of CD27.

4. A pharmaceutical composition according to any one of claims 1 to 3, wherein the intracellular portion of CAR comprises at least one intracellular activation domain.

5. The pharmaceutical composition according to claim 4, wherein at least one intracellular activation domain is a CD3ζ or FcRγ intracellular activation domain.

6. The pharmaceutical composition of claim 4 or 5, wherein the intracellular portion of CAR comprises at least one intracellular costimulatory domain.

7. The pharmaceutical composition of claim 6, wherein at least one intracellular costimulatory domain is CD28, 4-1BB, DAP10, OX40 and / or ICOS intracellular costimulatory domain.

8. A pharmaceutical composition according to any one of claims 4 to 7, wherein CAR comprises a CD3ζ intracellular activation domain and / or a 4-1BB intracellular costimulatory domain.

9. A pharmaceutical composition according to any one of claims 1 to 8, wherein CAR comprises the extracellular and intramembrane domains of CD27, the CD3ζ intracellular activation domain, and the 4-1BB intracellular costimulatory domain.

10. A pharmaceutical composition according to any one of claims 1 to 9, wherein CAR contains an amino acid sequence that is at least 95% or 100% identical to that of SEQ ID NO:

1.

11. A pharmaceutical composition according to any one of claims 1 to 10, wherein the NK cells are isolated from a subject; or the NK cells are differentiated in vitro from hematopoietic stem cells or induced pluripotent stem (iPS) cells; or the NK cells are from a cloned NK cell line.

12. NK cells are isolated from the umbilical cord blood or peripheral blood of the subject; Isolated NK cells are cultured in vitro; or NK cells are NK-92 (trademark) cells. The pharmaceutical composition according to claim 11.

13. A pharmaceutical composition according to any one of claims 1 to 12, wherein NK cells are manipulated to further express one or more immunostimulatory cytokines.

14. The pharmaceutical composition according to claim 13, wherein one or more immunostimulatory cytokines are one or more immunostimulatory interleukins (ILs).

15. The pharmaceutical composition of claim 14, wherein one or more immunostimulant interleukins are IL-15, IL-12 and / or IL-21.

16. A pharmaceutical composition according to any one of claims 1 to 15, wherein the expression of CAR is constitutive or inducible, or the expression of CAR and one or more immunostimulatory cytokines is independently constitutive or inducible.

17. A pharmaceutical composition according to any one of claims 1 to 16, wherein the neoplasm is cancer.

18. A pharmaceutical composition according to any one of claims 1 to 17, wherein the solid tumor contains less than 10% CD70-positive cancer cells, and the percentage of CD70-positive cancer cells represents the ratio to total cancer cells in a representative sample of the neoplastic disease lesion.

19. A pharmaceutical composition according to any one of claims 1 to 18, wherein the solid tumor contains 10% or more CD70-positive CAF, and the percentage of CD70-positive CAF represents the ratio to total CAF in a representative sample of the neoplastic disease lesion.

20. A pharmaceutical composition according to any one of claims 1 to 19, wherein the neoplasm is colorectal cancer or pancreatic cancer.

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