Application of chimeric antigen receptors targeting non-classical HLA molecules for cancer therapy

Abstract

The present invention relates to the field of biotechnology, and specifically, to a chimeric antigen receptor targeting non-classical HLA molecules for cancer treatment, its modified immune cells, preparation methods and applications. The chimeric antigen receptor specifically targeting HLA-G provided by the present invention has an extracellular domain that is the antigen binding domain of the ILT4 receptor. Compared with CAR-modified immune cells with the antigen binding region of an antibody as the extracellular region, the HLA-G targeted immune response cells provided by the present invention can, on the one hand, broadly identify numerous tumor cells, greatly improving the killing efficiency and accuracy of various types of tumor cells; on the other hand, the extracellular domain is the antigen binding domain of the ILT4 receptor, which is the binding domain of a natural receptor. Compared with artificially synthesized antibodies, it is safe and reliable and can truly reflect the target binding ability in the natural state.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a chimeric antigen receptor targeting non-classical HLA molecules for cancer treatment, immune cells modified therefrom, a preparation method and applications thereof. Background Art

[0002] With the rapid development of biotechnology, immunotherapy has become one of the main therapies in the field of cancer treatment. Cancer immunotherapy mainly includes adoptive cell therapy, immunomodulators, tumor vaccines, antibody therapy, and immune checkpoint blockade therapy. Among them, chimeric antigen receptor-modified immune cell therapy, especially chimeric antigen receptor-modified T cell (CAR-T) therapy, is currently very popular and is a star treatment in this field.

[0003] The principle of CAR-T cell immunotherapy is to modify the patient's own T cells with chimeric antigen receptors through genetic engineering to form CAR-T cells. Through the modified chimeric antigen receptors, the CAR-T cells can specifically recognize tumor surface-associated antigens (tumor cell markers), thereby targeting and killing the tumor. Compared with ordinary immune cells, CAR-T cells have higher targeting, killing activity, and persistence. Currently, modified immune cell therapy represented by CAR-T cells targeting CD19 and BCMA has a significant effect in the treatment of hematological tumors such as lymphoma and multiple myeloma, and is considered one of the most promising tumor treatment methods.

[0004] However, since over 90% of cancer patients suffer from solid tumors, further identification of more types of solid tumors and more tumor-specific surface target antigens is needed. One of the greatest challenges in applying CAR-T immunotherapy to the treatment of solid tumors is the extremely high specificity required for CAR-T cells to express antigens on tumor cells. Failure to do so can easily lead to persistent activation of T cells, killing normal cells, or releasing large amounts of cytokines, causing serious side effects. Although CAR-T cell immunotherapy requires very high specificity for tumor cell antigen expression, there are only a few tumor-specific target antigens to choose from. Furthermore, most antigens expressed by tumors are not tumor-specific. CAR-T cell immunotherapy targeting tumor-associated antigens is subject to issues such as "off-target" effects and drug resistance.

[0005] Therefore, finding a more broad-spectrum yet specific, more efficient and safer molecule as a target for CAR therapy is an urgent problem to be solved in chimeric antigen receptor-modified immune cell therapy. The key to finding such a target is that the target antigen is highly expressed on the surface of tumor cells, but not expressed or expressed at a low level on the surface of normal cells. Summary of the Invention

[0006] Human leukocyte antigen G (HLA-G) is a non-classical HLA class I molecule. HLA-G is widely expressed on the surface of numerous tumor cells (particularly solid tumors). By interacting with the ILT4 receptor on the surface of immune cells, HLA-G activates downstream pathways in immune cells, transmitting inhibitory signals to them, leading to immunosuppression and thus escaping the immune system's attack. Therefore, it is considered a key immune checkpoint molecule. Furthermore, HLA-G is rarely expressed or only transiently expressed in healthy tissues. This is a tumor cell-specific alteration during tumor development, making it a precise target for immunotherapy of tumors, especially solid tumors, and providing insights into the development of related new therapies and drugs.

[0007] CARs typically consist of an extracellular targeting domain, a transmembrane spacer, and an intracellular signaling domain. The targeting domain of CARs is typically derived from the antigen-binding region of an antibody, namely the immunoglobulin heavy and light chains that form the antibody binding site. Common examples include the single-chain variable fragment (scFv) of a monoclonal antibody responsible for recognizing and binding to antigens. CAR-modified immune cells with the antibody's antigen-binding region as the extracellular region have advantages such as strong specificity and high sensitivity. However, there are also the following problems: Due to the high specificity of antibodies, an antibody can usually only bind to one target, making it prone to "off-target" effects when targeting tumor cells. For most tumors, the target molecules available for CAR immune cell therapy are scarce, and no target molecule is suitable for broad-spectrum tumor treatment.

[0008] The present invention aims to solve one of the technical problems in the above-mentioned technology at least to a certain extent.

[0009] To this end, the first aspect of the present invention provides a chimeric antigen receptor that specifically targets and binds to HLA-G, the chimeric antigen receptor comprising:

[0010] An extracellular domain comprising at least one binding domain for ILT4 targeting and binding to HLA-G;

[0011] a transmembrane domain, the transmembrane domain being connected to the extracellular domain;

[0012] An intracellular domain is connected to the transmembrane domain.

[0013] The chimeric antigen receptor specifically targeting HLA-G provided by the present invention has an extracellular domain comprising the antigen-binding domain of the ILT4 receptor. Compared to the prior art CAR-modified immune cells using the antigen-binding region of an antibody as the extracellular region, the engineered HLA-G-targeted immune response cells of the present invention can, on the one hand, broadly recognize a wide range of tumor cells, especially solid tumor cells, greatly improving the efficiency and accuracy of killing various tumor cells. On the other hand, the extracellular domain comprises the antigen-binding domain of the ILT4 receptor, which is the binding domain of the natural receptor. Compared with synthetic antibodies, it is safe and reliable and can truly reflect the target binding ability in its natural state. Therefore, the present invention provides a new means of tumor treatment with application prospects.

[0014] According to a specific embodiment of the present invention, the ILT4-HLA-G targeting binding domain has an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2, or an amino acid sequence having at least 80% identity thereto.

[0015] According to a specific embodiment of the present invention, the extracellular domain further includes a signal peptide, and the signal peptide is connected to the binding domain of the ILT4 targeting HLA-G.

[0016] According to a specific embodiment of the present invention, the signal peptide comprises at least one of a CD8α signal peptide, an IgG signal peptide, and a CD28 signal peptide.

[0017] According to a specific embodiment of the present invention, the signal peptide is derived from CD8α signal peptide.

[0018] According to a specific embodiment of the present invention, the signal peptide has the amino acid sequence shown in SEQ ID NO:5.

[0019] According to a specific embodiment of the present invention, the extracellular domain is connected to the transmembrane domain via a hinge domain.

[0020] According to a specific embodiment of the present invention, the hinge domain includes a hinge domain derived from the CD8α hinge region, the IgG hinge region or at least a portion of an immunoglobulin, or the hinge domain is a variant of the CD8α hinge region, the IgG hinge region or at least a portion of an immunoglobulin hinge domain having one or more amino acid modifications.

[0021] According to a specific embodiment of the present invention, the hinge domain is derived from the CD8α hinge region.

[0022] According to a specific embodiment of the present invention, the hinge domain has an amino acid sequence as shown in SEQ ID NO: 6, or an amino acid sequence having at least 80% identity thereto.

[0023] According to a specific embodiment of the present invention, the transmembrane domain includes any one of the transmembrane structural region derived from CD8α, CD28, CD3ζ, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, BTLA, and synthetic peptides, wherein the synthetic peptide does not contain proteins associated with immune response.

[0024] According to a specific embodiment of the present invention, the transmembrane domain is derived from the CD8α transmembrane region.

[0025] According to a specific embodiment of the present invention, the transmembrane domain has an amino acid sequence as shown in SEQ ID NO: 7.

[0026] According to a specific embodiment of the present invention, the intracellular domain includes an immunoreceptor tyrosine-based activation motif and a co-stimulatory signaling domain, and the co-stimulatory signaling domain and the immunoreceptor tyrosine-based activation motif are connected via a connecting peptide.

[0027] According to a specific embodiment of the present invention, since HLA-G usually transmits inhibitory signals to immune cells after binding to immune cells and inhibits the function of immune cells, the extracellular segment of the chimeric antigen receptor designed in the present invention is the extracellular region of the inhibitory receptor ILT4 that recognizes HLA-G. The intracellular segment of the inhibitory receptor is replaced with a co-stimulatory signal domain, which can convert the signal recognizing HLA-G into an activation signal to activate immune cells. The CAR-modified immune cells prepared by the present invention can resist the inhibition from the tumor immune microenvironment and reverse the exhaustion of immune cells.

[0028] According to a specific embodiment of the present invention, the immunoreceptor tyrosine-based activation motif is derived from the intracellular signaling domain of the CD3ζ chain or the FcεRIγ intracellular signaling domain.

[0029] According to a specific embodiment of the present invention, the immunoreceptor tyrosine-based activation motif is derived from the intracellular signaling domain of the CD3ζ chain.

[0030] According to a specific embodiment of the present invention, the immunoreceptor tyrosine-based activation motif has an amino acid sequence as shown in SEQ ID NO:9.

[0031] According to a specific embodiment of the present invention, the co-stimulatory signaling domain includes at least one of a CD28 intracellular signaling domain, a 4-1BB intracellular signaling domain, an OX40 intracellular signaling domain, a CD40 or CD40L intracellular signaling domain, and an ICOS intracellular signaling domain.

[0032] According to a specific embodiment of the present invention, the costimulatory signaling domain is derived from the 4-1BB intracellular signaling domain.

[0033] According to a specific embodiment of the present invention, the costimulatory signal domain has an amino acid sequence as shown in SEQ ID NO:8.

[0034] According to a specific embodiment of the present invention, the chimeric antigen receptor has an amino acid sequence as shown in SEQ ID NO: 17 or SEQ ID NO: 18.

[0035] The second aspect of the present invention provides an isolated polynucleotide encoding the chimeric antigen receptor according to the first aspect.

[0036] The third aspect of the present invention provides an expression vector, which carries the polynucleotide described in the second aspect.

[0037] The fourth aspect of the present invention provides a recombinant virus, which carries the polynucleotide described in the second aspect or the expression vector described in the third aspect, and is capable of expressing the chimeric antigen receptor described in the first aspect.

[0038] The fifth aspect of the present invention provides a recombinant cell carrying the polynucleotide described in the second aspect or the expression vector described in the third aspect or infected by the recombinant virus described in the fourth aspect, and capable of expressing the chimeric antigen receptor described in the first aspect.

[0039] According to a specific embodiment of the present invention, the recombinant cell is derived from an immune cell.

[0040] According to a specific embodiment of the present invention, the immune cells are obtained by separation from peripheral blood, umbilical cord blood, tissues, or organs, or the immune cells are obtained by differentiation of stem cells or immune cell progenitor cells.

[0041] According to a specific embodiment of the present invention, the stem cells include induced pluripotent stem cells, hematopoietic stem cells, or embryonic stem cells within 14 days of fertilization that have not undergone in vivo development.

[0042] According to a specific embodiment of the present invention, the immune cells include at least one selected from T cells, B cells, monocytes, NK cells, dendritic cells, macrophages, regulatory T cells, helper T cells, cytotoxic T cells, NKT cells and γδT cells.

[0043] A sixth aspect of the present invention provides a composition comprising at least one of the following:

[0044] The chimeric antigen receptor of the first aspect;

[0045] The polynucleotide of the second aspect;

[0046] The expression vector according to the third aspect;

[0047] The recombinant virus according to the fourth aspect;

[0048] The recombinant cell described in the fifth aspect.

[0049] The seventh aspect of the present invention provides use of the composition described in the sixth aspect in the preparation of anti-tumor drugs.

[0050] According to a specific embodiment of the present invention, HLA-G is expressed on the cell surface of the tumor.

[0051] To overcome the problem of scarcity of effective targets for most solid tumors in existing cell therapy clinical applications, the present invention provides a chimeric antigen receptor targeting human HLA-G, its gene and recombinant expression vector, an engineered HLA-G-targeting chimeric antigen receptor-modified immune response cell, and its application.

[0052] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0054] Figure 1 The results of the validation of ILT4 receptor expression on CAR-NK cells in Example 3 of the present invention are shown;

[0055] Figure 2 Shows the results of screening of HLA-G positive tumor cell lines in Example 4 of the present invention;

[0056] Figure 3 The results of CAR-NK cells killing K562 cells in Example 5 of the present invention are shown;

[0057] Figure 4 The results of killing NCI-H716 cells by CAR-NK cells in Example 5 of the present invention are shown;

[0058] Figure 5 The results of CAR-NK cells killing SKOV3 cells in Example 5 of the present invention are shown;

[0059] Figure 6 The results of CAR-NK cells killing Huh7 cells in Example 5 of the present invention are shown;

[0060] Figure 7The results of the treatment of subcutaneous colon cancer transplanted tumors in mice with CAR-NK cells in Example 6 of the present invention are shown, wherein: Figure 7 A in the figure shows the images of tumors in each experimental group after the experiment was terminated. Figure 7 B in the figure shows the statistical results of the tumor weights of each experimental group after the experiment was terminated. DETAILED DESCRIPTION

[0061] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0062] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0063] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0064] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which the present invention belongs.

[0065] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0066] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0067] In the present invention, the term "effector-target ratio" refers to the ratio of the number of effector cells (specific CAR-NK cells targeting HLA-G) to target cells (tumor cells).

[0068] In this invention, the term "chimeric antigen receptor" is abbreviated as CAR. It is an artificial receptor molecule manufactured by genetic engineering technology, including an antigen-binding domain (usually derived from a single-chain antibody variable region gene fragment, scFV; or from a single-domain antibody), a hinge domain, a transmembrane domain, and an intracellular signaling domain.

[0069] In the present invention, the term "reduced expression" refers to the elimination or reduction of expression of one or more gene products encoded by a gene in cells, compared to the normal expression level of the gene's mRNA or protein. In some cases, this effect on gene expression is temporary or reversible, such as by inhibiting gene expression through small molecule compounds, nucleic acids such as RNAi, siRNA, or shRNA, or specific proteins. In some cases, this effect on gene expression is permanent, such as by gene editing to introduce base insertions or deletions into the coding region of a target gene, resulting in a frameshift mutation, or by inserting a DNA sequence into the coding or noncoding region of a target gene through homologous recombination, resulting in a frameshift mutation, premature termination of transcription and / or translation, or translation of an entirely different polypeptide, thereby permanently inactivating the gene. The proportion of gene expression reduction achieved by different methods can range from 100%, greater than 90%, greater than 80%, greater than 70%, greater than 60%, greater than 50%, greater than 40%, greater than 30%, or greater than 20%.

[0070] In the present invention, the term "hinge region" refers to the region between the CH1 and CH2 domains of an antibody heavy chain. This region includes inter-H chain disulfide bonds, is rich in proline, does not form an α-helix, and is prone to stretching and some degree of twisting, which facilitates complementary binding between the antibody's antigen-binding site and the antigen epitope. In the heavy chain single-domain antibodies of the present invention, the heavy chain variable region is connected to the Fc region via the hinge region.

[0071] In the present invention, the term "cell" refers to a eukaryotic cell, in particular a mammalian cell, such as a human cell.

[0072] In the present invention, the term "NK cells" refers to natural killer cells, which are generally CD56+CD45+ double positive and express multiple activating receptors, such as NKG2D, NKp44, NKp46, etc. NK cells can be differentiated from pluripotent stem cells (such as iPSCs), isolated from peripheral blood, differentiated from CD34+ cells (such as umbilical cord blood or placental blood), or obtained from tumor cell lines (such as NK92).

[0073] As used herein, the term "signal peptide," also called a signal sequence, is a localization tag for membrane, secretory, and lysosomal proteins. It is typically located at the amino terminus of a protein and consists of 13-26 residues. Signal peptides do not have a conserved amino acid sequence, but rather possess conserved structural features.

[0074] As used herein, the term "immune cell" in the present invention refers to a cell from a human. More specifically, the immune cells in the present invention can be derived from pluripotent stem cells through directed differentiation. Depending on the different differentiation conditions provided, these immune cells can be T cells, NK cells and macrophages. The immune cells in the present invention can also be derived from human hematopoietic stem cells. These hematopoietic stem cells can be isolated from peripheral blood, umbilical cord blood, placental blood, or differentiated from pluripotent stem cells. Depending on the different differentiation conditions provided, these immune cells can be T cells, NK cells and macrophages. The immune cells in the present invention can also be isolated from human blood, bone marrow, lymph or lymphoid organs, specific tissues (including but not limited to tumor tissue, abdominal cavity, liver, lungs and other organs, muscles, etc.). For example, cells of innate or adaptive immunity, for example, bone marrow or lymphocytes, are typically T cells, NK cells, macrophages, etc. Preferably, according to the present invention, immune cells include T cells, NK cells and macrophages. The immune cells of the present invention can also be differentiated from immune cell progenitor cells, such as lymphocyte progenitor cells.

[0075] In the present invention, the term "pluripotent stem cells" generally refers to human embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC). Human ESCs are isolated from blastocysts during the development of human fertilized eggs, and can also be isolated from embryos obtained by nuclear transplantation (embryonic stem cells within 14 days of fertilization that have not undergone in vivo development); human iPSCs can be reprogrammed from human cells by overexpressing specific transcription factors (such as OCT4, SOX2, KLF4, and MYC). Both human ESCs and iPSCs express OCT4, NANOG, Tra-1-60, and SSEA-4, but do not express SSEA-1. A notable feature of pluripotent stem cells is that they can differentiate into three germ layer tissues or cells in vitro or in vivo.

[0076] In the present invention, the term "hematopoietic stem cells" refers to CD34+ cells. These cells can be isolated from peripheral blood, umbilical cord blood, placental blood, bone marrow, spleen, lymph nodes, or other tissues, or can be differentiated from pluripotent stem cells.

[0077] In the present invention, the term "progenitor cells" refers to cells that can differentiate into various types of immune cells, such as myeloid stem cells, lymphoid stem cells, and further, also refers to erythroid stem cells, granulocyte and monocytic stem cells, megakaryocyte stem cells, precursor B cells, precursor T cells, NK progenitor cells, and macrophage progenitor cells.

[0078] In the present invention, the term "vector" refers to a vehicle into which a genetic element (e.g., the aforementioned nucleic acid molecule) can be operatively inserted and the genetic element can be expressed, for example, to produce a protein, RNA, or DNA encoded by the genetic element, or to replicate the genetic element. A vector can be used to transform, transduce, or transfect a host cell so that the genetic element it carries is expressed in the host cell. For example, vectors include: plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses. A vector can contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector can also contain a replication initiation site. A vector can also include components that assist its entry into cells, including, but not limited to, viral particles, liposomes, or protein coats. A vector can be an expression vector or a cloning vector. In some embodiments, the vector provided by the present invention (e.g., an expression vector) contains the nucleic acid sequence encoding the fusion protein described in the present invention, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selection marker.

[0079] In the present invention, when the term "identity" is used to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 70:173-187 (1997); and BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs that utilize these algorithms are also available and include, but are not limited to, ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0080] Chimeric antigen receptor

[0081] The present invention provides a chimeric antigen receptor that specifically targets and binds to HLA-G, wherein the chimeric antigen receptor comprises:

[0082] An extracellular domain, comprising a binding domain for ILT4 targeting and binding to HLA-G;

[0083] a transmembrane domain, the transmembrane domain being connected to the extracellular domain;

[0084] An intracellular domain is connected to the transmembrane domain.

[0085] The ILT4 receptor is a receptor on the surface of immune cells that is responsible for binding to the HLA-G molecule. ILT4 is only expressed on immune cells of myeloid origin. The ILT4 receptor is divided into an extracellular region, a transmembrane region, and an intracellular region, of which the extracellular region is the functional region responsible for recognizing and binding to HLA-G. The extracellular region of the ILT4 receptor is divided into four domains, named Domain 1 (D1), Domain 2 (D2), Domain 3 (D3), and Domain 4 (D4). According to reports, D1 and D2 of the receptor are responsible for binding to HLA-G, while D3 and D4 are only responsible for supporting the external structure formed after D1 and D2 bind to HLA-G.

[0086] In a specific embodiment of the present invention, the present application provides a synthetic polypeptide that targets and binds to HLA-G, wherein the synthetic polypeptide sequence is derived from the ILT4 protein. Generally speaking, the synthetic polypeptide includes, but is not limited to, the following compositions: ILT4-D1D2, ILT4-ECD, ILT4-D1, ILT4-D2, ILT4-D1D1, ILT4-D2D1, ILT4-D2D2, preferably: ILT4-D1D2, ILT4-ECD.

[0087] According to a specific embodiment of the present invention, the ILT4-HLA-G targeting binding domain has an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2, or an amino acid sequence having at least 80% identity thereto.

[0088] According to a specific embodiment of the present invention, the extracellular domain further includes a signal peptide, and the signal peptide is connected to the binding domain of the ILT4 targeting HLA-G.

[0089] According to a specific embodiment of the present invention, the signal peptide comprises at least one of a CD8α signal peptide, an IgG signal peptide, and a CD28 signal peptide.

[0090] According to a specific embodiment of the present invention, the signal peptide has the amino acid sequence shown in SEQ ID NO:5.

[0091] According to a specific embodiment of the present invention, the extracellular domain is connected to the transmembrane domain via a hinge domain.

[0092] According to a specific embodiment of the present invention, the hinge domain includes a hinge domain derived from the CD8α hinge region, the IgG hinge region or at least a portion of an immunoglobulin, or the hinge domain is a variant of the CD8α hinge region, the IgG hinge region or at least a portion of an immunoglobulin hinge domain having one or more amino acid modifications.

[0093] According to a specific embodiment of the present invention, the hinge domain has an amino acid sequence as shown in SEQ ID NO: 6, or an amino acid sequence having at least 80% identity thereto.

[0094] According to a specific embodiment of the present invention, the transmembrane domain includes any one of the transmembrane structural region derived from CD8α, CD28, CD3ζ, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, BTLA, and synthetic peptides, wherein the synthetic peptide does not contain proteins associated with immune response.

[0095] According to a specific embodiment of the present invention, the transmembrane domain has an amino acid sequence as shown in SEQ ID NO: 7.

[0096] According to a specific embodiment of the present invention, the intracellular domain includes an immunoreceptor tyrosine-based activation motif and a co-stimulatory signaling domain, and the co-stimulatory signaling domain and the immunoreceptor tyrosine-based activation motif are connected via a connecting peptide.

[0097] According to a specific embodiment of the present invention, the immunoreceptor tyrosine-based activation motif is derived from the intracellular signaling domain of the CD3ζ chain or the FcεRIγ intracellular signaling domain.

[0098] According to a specific embodiment of the present invention, the immunoreceptor tyrosine-based activation motif is derived from the intracellular signaling domain of the CD3ζ chain.

[0099] According to a specific embodiment of the present invention, the immunoreceptor tyrosine-based activation motif has an amino acid sequence as shown in SEQ ID NO:9.

[0100] According to a specific embodiment of the present invention, the co-stimulatory signaling domain includes at least one of a CD28 intracellular signaling domain, a 4-1BB intracellular signaling domain, an OX40 intracellular signaling domain, a CD40 or CD40L intracellular signaling domain, and an ICOS intracellular signaling domain.

[0101] According to a specific embodiment of the present invention, the costimulatory signaling domain is derived from the 4-1BB intracellular signaling domain.

[0102] According to a specific embodiment of the present invention, the costimulatory signal domain has an amino acid sequence as shown in SEQ ID NO:8.

[0103] According to a specific embodiment of the present invention, the chimeric antigen receptor has an amino acid sequence as shown in SEQ ID NO: 17 or SEQ ID NO: 18.

[0104] According to an embodiment of the present invention, from N-terminus to C-terminus, the CAR may sequentially comprise a synthetic polypeptide derived from ILT4 that binds to HLA-G, the hinge region, the transmembrane domain, the co-stimulatory signaling domain, and the intracellular signaling domain.

[0105] According to a specific embodiment of the present invention, from N-terminus to C-terminus, the CAR may sequentially comprise a synthetic polypeptide binding to HLA-G derived from ILT4, a hinge region derived from CD8α, a transmembrane domain derived from CD8α, a costimulatory signaling domain derived from 4-1BB, and an intracellular signaling domain derived from CD3ζ.

[0106] According to a specific embodiment of the present invention, from N-terminus to C-terminus, the CAR may sequentially comprise a signal peptide, a synthetic polypeptide derived from ILT4 that binds to HLA-G, the hinge region, the transmembrane domain, the co-stimulatory signal domain, and the intracellular signaling domain.

[0107] According to a more specific embodiment of the present invention, the inventors designed two synthetic polypeptides that can target and bind to human HLA-G, as shown in Table 1. Both synthetic polypeptides are composed of polypeptide sequences derived from ILT4. The corresponding amino acid sequences are shown in SEQ ID NOs: 1-2, and the corresponding nucleotide sequences are shown in SEQ ID NOs: 3-4. Any one of the two synthetic polypeptides is used as the extracellular region of the chimeric antigen receptor, with a signal peptide sequence derived from CD8α connected to its N-terminus, and a hinge region sequence derived from CD8α, a transmembrane domain sequence derived from CD8α, a 4-1BB sequence, and a CD3ζ sequence connected to its C-terminus in sequence. The amino acid sequences are shown in SEQ ID NOs: 5-9, and the nucleotide sequences are shown in SEQ ID NOs: 10-14. The two chimeric receptors finally formed contain ILT4-D1D2 and ILT4-ECD, respectively, and are named CAR1 and CAR2, respectively.

[0108] Under the premise of not substantially affecting the activity of the domain (retaining at least 95% of the activity), those skilled in the art can replace, add and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids in the sequence of the present invention to obtain variants of the sequence of the domain or its functional fragment. They are all considered to be included in the scope of protection of the present invention. The variant sequence of the present invention may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity (or homology) with the reference sequence. The sequence identity of the present invention can be measured using sequence analysis software. For example, the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. The amino acid sequences described in the present invention are all shown in an N-terminal to C-terminal manner.

[0109] Polynucleotide, expression vector, recombinant virus, cell, composition

[0110] The present invention also provides an isolated nucleic acid encoding CAR and an expression vector and a recombinant virus containing the nucleic acid. The nucleic acid molecule encodes the above-mentioned CAR, and the nucleic acid is preferably an expression cassette obtained by genetic engineering means.

[0111] The expression vector may refer to a cloning vector or a recombinant vector, which can be obtained by operably linking the nucleic acid to a commercially available vector (such as a plasmid or viral vector). Commonly used plasmids include pSeTag2, PEE14, pMH3, pUC57, etc.

[0112] According to an embodiment of the present invention, immune cells with enhanced function can be obtained by using any one of the following methods or a combination thereof:

[0113] 1) Randomly integrate one or more circular or linear exogenous DNA fragments into target cells;

[0114] 2) Integrate one or more circular or linear exogenous DNA fragments into target cells. Site-directed integration can enhance the efficiency of homologous recombination of exogenous DNA by introducing a nuclease at the target site. Other physical methods, such as electroporation, can also be used to enhance the efficiency of homologous recombination.

[0115] 3) One or more endonucleases that can recognize specific sites introduce one or more double-strand breaks into target cells at the specific sites, and the target cells generate base insertions or base deletions at the specific sites through non-homologous end joining;

[0116] 4) One or more endonucleases that can recognize specific sites introduce one or more double-strand breaks into target cells at specific sites, and exogenous circular or linear DNA fragments are provided at the same time. The cells generate base insertions or base deletions at the selected sites through homologous recombination.

[0117] In this case, the target cell can be the immune cell, stem cell, or progenitor cell mentioned above, or a donor cell for reprogramming into iPSCs, or any cell derived from a stem cell or progenitor cell during the process of differentiating into an immune cell. The exogenous DNA fragment may contain a gene expression cassette, i.e., at least components such as a promoter and an open reading frame, or may not contain a gene expression cassette.

[0118] In some preferred embodiments, the nucleic acid molecule is species-optimized and is more easily expressed in mammalian cells.

[0119] The present invention also provides an expression vector comprising the above-mentioned isolated nucleic acid molecule. When the above-mentioned isolated polynucleotide is connected to the vector, the polynucleotide can be directly or indirectly connected to the control elements on the vector, as long as these control elements can control the translation and expression of the polynucleotide. Of course, these control elements can come directly from the vector itself, or they can be exogenous, that is, not from the vector itself. Of course, the polynucleotide and the control element are operably connected. Herein, "operably connected" refers to connecting the exogenous gene to the vector so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can play their expected function of regulating the transcription and translation of the exogenous gene. Of course, the polynucleotides used to encode different domains of CAR can be independently inserted into different vectors, and it is common to insert them into the same vector. Commonly used vectors can be, for example, plasmids, phages, and the like.

[0120] The present application provides a recombinant virus, which can express a specific chimeric antigen receptor targeting HLA-G and can infect immune response cells.

[0121] The present invention also provides a recombinant cell comprising the expression vector. The expression vector can be introduced into mammalian cells to construct recombinant cells, and then these recombinant cells are used to express the chimeric antigen receptor provided by the present invention. The recombinant cells are derived from immune response cells, including at least one of T cells, B cells, monocytes, NK cells, dendritic cells, macrophages, regulatory T cells, helper T cells, cytotoxic T cells, NKT cells and γδT cells. The immune cells are obtained by separation from peripheral blood, umbilical cord blood or tissues and organs, or the immune cells are obtained by differentiation of stem cells or immune cell progenitor cells; the stem cells include induced pluripotent stem cells, hematopoietic stem cells or embryonic stem cells within 14 days of fertilization that have not undergone in vivo development.

[0122] The composition provided by the present invention contains at least one of the chimeric antigen receptor, polynucleotide, expression vector, recombinant virus or recombinant cell as described above, and further, the composition contains a pharmaceutically acceptable carrier. In certain embodiments, the composition includes a combination separated in time and / or space, as long as they can work together to achieve the purpose of the present invention. For example, the components contained in the composition can be administered to the subject as a whole or separately. When the components contained in the composition are administered to the subject separately, each component can be administered to the subject simultaneously or sequentially.

[0123] In some embodiments, the pharmaceutical carrier includes any solvent, solid excipient, diluent, binder, disintegrant, or other liquid excipient, dispersant, flavoring agent or suspending agent, surfactant, isotonic agent, thickener, emulsifier, preservative, solid binder, glidant or lubricant, etc., suitable for the specific target dosage form. Except for any conventional excipients that are incompatible with the compounds of the present invention, such as any adverse biological effects produced or any other components of the pharmaceutically acceptable composition that interact in a harmful manner, their use is also within the scope of the present invention.

[0124] In a specific embodiment of the present invention, the present invention screens out suitable target cell lines for testing the killing ability of newly produced CAR-NK cells, wherein the screened target cells can be used for both in vitro experiments and in vivo experiments in mice.

[0125] The scheme of the present disclosure will be explained below in conjunction with the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present disclosure and should not be considered to limit the scope of the present disclosure. Where specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in this area or according to the product instructions. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0126] Table 1 shows the nucleotide and amino acid sequences involved in the present invention. Based on the sequences provided by the present invention, those skilled in the art can easily obtain the CAR-NK cells provided by the present invention.

[0127] Table 1

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134] Example 1 Design of a chimeric antigen receptor specifically targeting human HLA-G

[0135] The inventors designed two synthetic polypeptides that can target and bind to human HLA-G, as shown in Table 1. Both synthetic polypeptides are composed of polypeptide sequences derived from ILT4. The corresponding amino acid sequences are shown in SEQ ID NO: 1-2, and the corresponding nucleotide sequences are shown in SEQ ID NO: 3-4. The two synthetic polypeptides are used as the extracellular region of the chimeric antigen receptor, and the signal peptide sequence derived from CD8α is connected at its N-terminus, and the hinge region sequence derived from CD8α, the transmembrane domain sequence derived from CD8α, the 4-1BB sequence and the CD3ζ sequence are sequentially connected at its C-terminus. The amino acid sequences are shown in SEQ ID NO: 5-9, and the nucleotide sequences are shown in SEQ ID NO: 10-14. The two chimeric receptors finally formed are named CAR1 and CAR2, respectively.

[0136] Example 2 Construction of an expression plasmid vector containing a chimeric antigen receptor specifically targeting human HLA-G

[0137] The construction of expression plasmid vectors (pCDH-CAR1 and pCDH-CAR2) containing specific chimeric antigen receptors targeting human HLA-G specifically includes the following steps:

[0138] (1) GENEWIZ synthesized DNA sequences of chimeric antigen receptors (CAR1 and CAR2) that target human HLA-G and inserted them into the pUC57 vector. The DNA sequences are shown in SEQ ID NOs: 15-16, and the corresponding encoded amino acid sequences are shown in SEQ ID NOs: 17-18.

[0139] (2) The pCDH-EV vector was double-digested with EocR1 and BamH1 endonucleases, and the double-digested vector was recovered by electrophoresis on a 1% agarose gel for 20 min.

[0140] (3) using primer pair 1 to obtain a nucleotide fragment of a chimeric antigen receptor that specifically targets and binds to human HLA-G by PCR from the pUC57 vector containing the DNA sequence of the chimeric antigen receptor that specifically targets and binds to human HLA-G synthesized in (1), wherein the nucleotide sequence of the primer pair (primer 1F, primer 1R) is as shown in SEQ ID NOs: 19-20;

[0141] (4) homologously recombining the double-enzyme-cleaved vector obtained in (2) and (3) with the nucleotide fragment of the chimeric antigen receptor that specifically targets and binds to human HLA-G;

[0142] (5) The constructed expression plasmid vector (pCDH-CAR1 / 2) containing a chimeric antigen receptor that specifically targets human HLA-G was sequenced and verified, and the sequencing results confirmed that the construction was successful.

[0143] Example 3 Construction and identification of a cell line containing a chimeric antigen receptor specifically targeting human HLA-G

[0144] The expression plasmids (pCDH-CAR1 and pCDH-CAR2) were co-transfected with pMD2G and psPAX2 into a 15 cm culture dish containing HEK293-T cells at a density of 90%. Three days later, the cell supernatant was collected, the virus solution was filtered using a 0.45 μm filter, and the virus was concentrated to obtain lentivirus expressing plasmids (pCDH-CAR1 and pCDH-CAR2).

[0145] NK92 cells were incubated with human recombinant IL-2 and 20 ng / mL human IL-21 at a final concentration of 1000 U / mL and cultured for 24 hours. The lentivirus and the infection-enhancing reagent polybrene were then added at an MOI of 1 and infected for 24 hours. The cells were then supplemented with appropriate medium, and the infection efficiency of the NK92 cells was determined by flow cytometry 48 hours later. The specific procedures are as follows.

[0146] (1) Blank control group: NK cells not infected with virus solution;

[0147] (2) Experimental group: 2 NK cells infected with different CAR1 or CAR2 viruses.

[0148] The experimental group and control group were washed twice with PBS and then resuspended in FACS solution (PBS containing 0.1% sodium azide and 0.4% BSA). According to the antibody instructions, APC-labeled anti-human ILT4 antibody was added to the cell suspension of the cell group to be tested and the control group, and incubated at 4°C for 30 minutes. The stained cells were obtained by flow cytometry and the results were analyzed using FlowJo software. The flow cytometry results are shown in Figure 2. Figure 1 As shown in the figure, the expression of CAR molecules was almost undetectable in the control group, while the expression rate of CAR molecules in each experimental group reached more than 90%. Figure 1 It can be seen from the flow cytometry results that the cells to be tested collected in this example expressed a specific chimeric antigen receptor targeting HLA-G.

[0149] Example 4 Screening of target cell lines

[0150] In order to screen suitable target cell lines for testing the killing ability of newly produced CAR-NK cells, the inventors detected and analyzed the expression of HLA-G in tumor cell lines such as ovarian cancer cell SKOV3, colon cancer cell NCI-H716, and leukemia cell K562. The tumor cells for testing were collected, washed twice with PBS, and resuspended in FACS buffer (PBS containing 0.1% sodium azide and 0.4% BSA), counted, and the cell concentration was adjusted to 1×10 6 / ml; PE-HLA-G and isotype control antibodies were added and incubated at 4°C for 30 minutes; washed twice with PBS and resuspended in FACS solution, and cell fluorescence was analyzed by flow cytometry and FlowJo software was used to analyze the results. Figure 2 As shown, K562, NCI-H716, and SKOV3 cells in the tested tumor cell groups all showed high expression of HLA-G antigen, while the liver cancer cell line Huh7 did not express HLA-G.

[0151] Example 5 Verification of in vitro killing of target cells by chimeric antigen receptor cells specifically targeting human HLA-G

[0152] Next, the in vitro killing activity of CAR-NK cells was tested by PI and CFSE staining. Specifically, for the tumor cell lines K562, NCI-H716, and Huh7, the three target cell lines were stained with CSFE fluorescence at 2×10 4 The cells were plated on culture plates at an inoculum concentration of 100 cells / ml. Four experimental groups and one control group were set up for each target cell line. The experimental groups were supplemented with a cell suspension of the HLA-G-targeting CAR-NK cells obtained in Example 3; the blank control group was supplemented with NK cells infected with an empty vector virus. In the experimental groups described above, the HLA-G-targeting CAR-NK cells of Example 3 were mixed with target cells at a 1:1 effector-target ratio.

[0153] For the tumor cell line SKOV3, 5×10 3 After the target cells adhered to the E-plate, the experimental group cells and the control group cells were added, and then the growth of the target cells was detected by RTCA instrument.

[0154] Similarly, a blank control group was prepared by mixing NK cells with target cells at a 1:1 effector-target ratio. After 4 hours of incubation, the supernatant was removed by centrifugation, and the cell pellet was washed and stained with PI. The stained cells were acquired by flow cytometry and analyzed using FlowJo software. Figure 3 The results show the tumor cell killing rate test results using leukemia cells K562 as target cells. Figure 3It can be seen that the specific CAR-NK cells targeting HLA-G in Example 3 have a significant killing effect on leukemia cells K562 (significantly higher than the control group). Figure 4 The results show the tumor cell killing rate test results using colon cancer cell NCI-H716 as target cells. Figure 4 It can be seen that the specific CAR-NK cells targeting HLA-G in Example 3 have a significant killing effect on colon cancer cells NCI-H716 (significantly higher than the control group). Figure 5 The results show the tumor cell killing rate test results using ovarian cancer cell SKOV3 as target cells. Figure 5 It can be seen that the specific CAR-NK cells targeting HLA-G in Example 3 have a significant killing effect on ovarian cancer SKOV3 (significantly higher than the control group). Figure 6 It can be seen that the specific CAR-NK cells targeting HLA-G in Example 3 have no obvious killing enhancement effect on HLA-G negative Huh7 (the killing effect is similar to that of the control group).

[0155] The above results indicate that the CAR-NK cells provided by the present invention have the ability to specifically recognize and kill HLA-G positive tumor cells.

[0156] Example 6 In vivo experimental verification of the effective killing of target cells by chimeric antigen receptor NK cells targeting human HLA-G

[0157] Take 1×10 7 NCI-H716 cells were subcutaneously injected into 5-week-old NCG mice. After 10 days of tumor growth, the tumor size was measured with a vernier caliper. Mice with similar tumor formation were randomly divided into 4 groups, two of which were experimental groups and the remaining two groups were control groups (one group was untreated and the other group was treated with ordinary NK92). 7 CAR1 or CAR R2-NK92 cells were injected into the tail vein of a control group of mice with 1×10 7 Ordinary, non-genetically modified NK92 cells were injected into the tail vein of a control group of mice with an equal volume of PBS. The treatment was then continued once a week, and the size of the subcutaneous transplanted tumors was measured. IL2 was injected intraperitoneally every 3 days, with 5×10 per mouse injected each time. 4 U, a total of 3 treatments. Figure 7 The results show the killing rate test results of CAR1 and CAR2-NK cells after colon cancer cells NCI-H716 were used as target cells. Figure 7 It can be seen that specific CAR-NK cells targeting HLA-G have a significant tumor-suppressing effect on colon cancer cells NCI-H716 in vivo (significantly higher than the control group).

[0158] The above results indicate that the CAR-NK cells provided by the present invention have the ability to specifically recognize and kill HLA-G positive tumor cells both in vitro and in vivo.

[0159] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be construed as limiting the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. Persons skilled in the art will appreciate that improvements and modifications may be made without departing from the spirit and scope of the present invention, and such improvements and modifications should also be considered within the scope of protection of the present invention.

[0160] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", "some implementation plans" or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0161] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. Use of the composition in the preparation of an anti-tumor drug, wherein the tumor cell surface expresses HLA-G; The tumor is selected from at least one of leukemia, ovarian cancer and colon cancer; The composition contains NK cells expressing a chimeric antigen receptor having an amino acid sequence as shown in SEQ ID NO: 17 or SEQ ID NO: 18.

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