Osteoclast, and preparation method therefor and use thereof

Abstract

Provided are an osteoclast, and a preparation method therefor and a use thereof. The present invention specifically relates to an engineered osteoclast, a cell pharmaceutical composition containing same and a use thereof. The engineered osteoclast has a targeting unit; the targeting unit has at least one or more of the following functions: (i) specifically recognizing a tumor cell; and (ii) specifically killing the tumor cell.

Description

Osteoclasts, methods of making and uses thereof TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, in particular the field of immunotherapy, and specifically relates to an osteoclast and a method of making and uses thereof. BACKGROUND

[0002] Chimeric antigen receptor-T cell (CAR-T cell) therapy is a cellular immunotherapy, which refers to a method of transferring genetic material with specific antigen recognition domain and T cell activation signal to T cells through gene cloning technology, so as to directly activate T cells combined with specific antigens on the surface of tumor cells. CAR-T cells have been successfully used to treat circulatory system malignancies, such as B cell-derived malignancies. After the success of CAR-T therapy, chimeric antigen receptor-natural killer cell (CAR-NK cell) therapy has been developed, but the existing CAR-T and CAR-NK technologies have limitations in treating solid tumors.

[0003] On the other hand, tumors have different characteristics at different stages, especially when they develop to the late stage and metastasize to the bone. Traditional CAR-T and CAR-NK technologies are difficult to play a role at this time, and patients urgently need a drug to inhibit the disease at this time, so it is necessary to develop a drug for patients with bone metastasis at the late stage of tumor.

[0004] SUMMARY

[0005] Therefore, it is necessary to provide at least an osteoclast targeting tumor cells and a method of making and uses thereof.

[0006] In a first aspect of the present application, an osteoclast targeting tumor cells is provided, which contains a targeting unit; the targeting unit has at least one or more of the following functions:

[0007] (i) specifically recognizes tumor cells;

[0008] (ii) specifically kills tumor cells.

[0009] In the first aspect of the present application, an engineered osteoclast is also provided, which contains a targeting unit; the targeting unit has at least one or more of the following functions:

[0010] (i) specifically recognizes tumor cells;

[0011] (ii) specifically kills tumor cells.

[0012] The aforementioned engineered osteoclast is an engineered osteoclast.

[0013] In some embodiments, the targeting unit is a chimeric antigen receptor; the chimeric antigen receptor comprises an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain.

[0014] In some embodiments, the antigen binding domain specifically recognizes a tumor antigen.

[0015] In some embodiments, the transmembrane domain is selected from the group consisting of CD8a, CD28, CD3zeta, CD3gamma, CD3delta, CD3epsilon, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, TCRa, TCRp, TCRy, TCRd, TCRzeta, OX40, ICOS, LAG-3, 2B4, BTLA, CTLA-4, and PD-1.

[0016] In some embodiments, the intracellular signaling domain is derived from the group consisting of FcRy, FcRp, CD3y, CD3d, CD3e, CD3zeta, CD22, CD79a, CD79b, CD66d, CD32a, CD3EBRS, CD28CD, CD19, 2B4, DAP12, and DAP10.

[0017] In some embodiments, the antigen binding domain is selected from the group consisting of an antibody, a single domain antibody fragment, and a single chain antibody.

[0018] In some embodiments, the antigen recognized by the antigen binding domain is selected from the group consisting of HER2, CLDN6, FAP, GPC3, MLSN, uPAR, and PSMA.

[0019] In some embodiments, the intracellular signaling domain further comprises a costimulatory signaling domain derived from the group consisting of RANKL, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD134 (OX40), CD2, CD7, CD27, CD28, CD30, CD40, CD83, ICAM, 4-1BB (CD137), CD276 (B7-H3), CD278 (ICOS), GITR, LIGHT, HVEM (LIGHTR), BTLA, CD8a, LFA-1, NKG2C, LAT, SLP-76, DAP10, PD-1, TRIM, and ZAP70 ligand.

[0020] In some embodiments, the chimeric antigen receptor comprises, in the following order, an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain: a single-chain antibody that specifically recognizes HER2, CD8a, and CD32a.

[0021] In some embodiments, the single-chain antibody comprises an amino acid sequence of SEQ ID NO: 1.

[0022] In some embodiments, the chimeric antigen receptor further comprises a reporter gene.

[0023] In some embodiments, the reporter gene is a fluorescent reporter gene.

[0024] In some embodiments, the fluorescent reporter gene is selected from the group consisting of GFP, EGFP, RFP, mCherry, mStrawberry, Luciferase, mApple, mRuby, and EosFP.

[0025] In a second aspect of the present application, a method for preparing the osteoclast as described in the first aspect is provided, the method comprising:

[0026] introducing a nucleic acid expressing the targeting unit into the osteoclast using a viral vector. In some embodiments, the step of introducing a nucleic acid expressing the targeting unit into the osteoclast, the osteoclast is an unengineered cell.

[0027] In some embodiments, the viral vector is selected from the group consisting of a lentiviral vector, a retroviral vector, an adenoviral vector, and an adeno-associated viral vector.

[0028] In a third aspect of the present application, the osteoclast as described in the first aspect is provided for use in the preparation of a medicament for preventing or treating a disease.

[0029] In some embodiments, the disease comprises one or more of a tumor, bone metastasis, and osteopetrosis.

[0030] In some embodiments, the tumor comprises a solid tumor.

[0031] In a fourth aspect of the present application, a pharmaceutical composition of cells is provided, comprising the osteoclast as described in the first aspect, and a pharmaceutically acceptable carrier.

[0032] In a fifth aspect of the present application, a method for preventing or treating a disease is provided, the method comprising administering to a subject an effective dose of the osteoclast as described in the first aspect or the pharmaceutical composition of cells as described in the fourth aspect.

[0033] In some embodiments, the subject is considered to be in need of administration of the osteoclasts or the cell pharmaceutical composition.

[0034] In some embodiments, the disease comprises one or more of a tumor, bone metastasis, and osteopetrosis.

[0035] In some embodiments, the tumor comprises a solid tumor.

[0036] In some embodiments, the dosage form of the cell pharmaceutical composition is selected from the group consisting of a solution type, an emulsion type, and a colloidal solution type.

[0037] In some embodiments, in the method, the administration method of the osteoclasts or the cell pharmaceutical composition is intravenous injection.

[0038] In some embodiments, in the method, the administration regimen of the osteoclasts or the cell pharmaceutical composition is multiple administration or single administration.

[0039] In one embodiment of the present application, a gene fragment of interest is introduced into an osteoclast by a virus (e.g., Ad5F35) to modify the osteoclast into a cell capable of targeted treatment of a disease. The aforementioned virus is a virus modified to carry a gene encoding a target cell-recognizing gene. After modification by the virus, the osteoclast is modified into an osteoclast capable of targeted recognition and killing of target cells. Further, the target cells can be cells that cause a disease, and further, the target cells can be tumor cells or bone metastasis cells.

[0040] The targeting unit in the aforementioned osteoclasts targeting tumor cells (CAR-OC) can be a chimeric antigen receptor (CAR), which is further used for immunotherapy. The CAR-modified osteoclasts have strong targeting and killing abilities. Compared with CAR-modified macrophages (CAR-M), the same amount of CAR-OC can kill more target cells and has a better killing effect.

[0041] Further, the osteoclasts can be derived from peripheral blood, such as peripheral blood mononuclear cells induced into osteoclasts. BRIEF DESCRIPTION OF DRAWINGS

[0042] In order to more clearly illustrate the technical solutions in the embodiments of the present application and the examples, and more completely understand the present application and its beneficial effects, the attached drawings needed to be used in the description of the embodiments or examples will be briefly introduced. Obviously, the attached drawings in the following description are only some embodiments of the present application, and other attached drawings can be obtained by those skilled in the art without any creative effort on the basis of these attached drawings. It should be noted that the attached drawings are drawn in a simplified form and are only used to conveniently and clearly assist in the description of the present application.

[0043] Figure 1 is a construction map of a plasmid for infecting macrophages and osteoclasts in an embodiment of the present application.

[0044] Figure 2 is a result of mCherry fluorescence detection after Ad5F35-CAR-HER2-CD32a-mCherry infects mouse BMDM and osteoclasts for 48 hours in an embodiment of the present application.

[0045] Figure 3 is a result of TRAP detection after Ad5F35-CAR-HER2-CD32a-mCherry infects mouse BMDM and osteoclasts for 48 hours in an embodiment of the present application.

[0046] Figure 4 is a result of G4S flow in an embodiment of the present application after Ad5F35-CAR-HER2-CD32a-mCherry infects mouse BMDM and osteoclasts for 48 hours.

[0047] Figure 5 is a result of G4S flow in an embodiment of the present application after Ad5F35-CAR-HER2-CD32a-mCherry infects mouse BMDM and osteoclasts for 48 hours.

[0048] Figure 6 is a result of G4S flow in an embodiment of the present application after Ad5F35-CAR-HER2-CD32a-mCherry infects mouse BMDM and osteoclasts for 48 hours.

[0049] Figure 7 is a well plate distribution diagram of different cell groups in a cell killing detection experiment in an embodiment of the present application.

[0050] Figure 8 is a result of killing of CAR-BMDM and CAR-OC cells for 24 hours when E / T is 1:1 and 2:1 in an embodiment of the present application.

[0051] Figure 9 is a result of killing of CAR-BMDM and CAR-OC cells for 48 hours when E / T is 1:10 in an embodiment of the present application.

[0052] Figure 10 is a result of killing of CAR-BMDM and CAR-OC cells for 72 hours when E / T is 1:10 in an embodiment of the present application. Detailed Implementation

[0053] To facilitate understanding of this application, a more complete description will be provided below with reference to the accompanying drawings. Preferred embodiments of this application are shown in the drawings. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of this application.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0055] In this application, unless otherwise specified, "one or more" means any one of the listed items or any combination of the listed items. Similarly, "one or more" and other instances that otherwise indicate "one or more" shall be understood in the same way unless otherwise specified.

[0056] The terms “combinations thereof,” “any combination thereof,” and “any combination thereof” as used in this application include all suitable combinations of any two or more of the listed items.

[0057] In this application, the word "suitable" in "suitable combination", "suitable method", "any suitable method" etc., shall be defined as being able to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0058] In this application, terms such as "further," "even more," "particularly," "for example," "like," "example," and "exemplary" are used for descriptive purposes to indicate that different technical solutions preceding and following each other are related in terms of their coverage, but should not be construed as limiting the preceding technical solution or restricting the scope of protection of this application. In this application, unless otherwise specified, A (e.g., B) indicates that B is a non-limiting example of A, and it can be understood that A is not limited to B.

[0059] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it refers to either "with" or "without" a parallel solution. If multiple "options" appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, each "option" is independent. Unless otherwise specified, the descriptions such as "optionally include" and "optionally contain" in this application, taking "optionally include" as an example, mean "may include or not include."

[0060] The terms "comprise", "contain", and "include", as used herein, are synonymous with each other, are inclusive or open-ended and do not exclude additional, unrecited members, or features. Members or features, for example, materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features also like actions, conditions, timing, states, etc.

[0061] In the present application, the technical features or technical solutions described in open language include closed technical features or technical solutions composed of listed contents, and also include open technical features or technical solutions containing listed contents.

[0062] In the present application, the exemplary description involving "in some embodiments (or examples)", "in an embodiment (or example)", etc. can cover but is not limited to the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0063] In the present application, in "first aspect", "second aspect", "third aspect", "fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are only for descriptive purposes, and cannot be understood as indicating or implying relative importance or quantity, nor can it be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration description, and should be understood as not constituting a closed limitation on the quantity.

[0064] In the present application, with respect to the numerical interval (i.e. numerical range), if no special instructions are given, the distribution of the optional values in the numerical interval is considered to be continuous, and includes both numerical endpoints (i.e. minimum value and maximum value) of the numerical interval, and every value between the two numerical endpoints. If no special instructions are given, when the numerical interval only points to the integers in the numerical interval, including the two endpoint integers of the numerical range and every integer between the two endpoints, it is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe a feature or property, these numerical ranges can be combined. In other words, unless otherwise indicated, the numerical ranges disclosed herein should be understood to include any and all sub-ranges therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. The "numerical interval" is allowed to be broadly included in the types of numerical intervals such as percentage interval, ratio interval, ratio interval, etc.

[0065] In the present application, unless there is an explicit different description in the present application, the execution of the steps involved in the method flow has no strict order limitation, and can be executed in other order than described. Moreover, any step can include multiple sub-steps or multiple stages, which are not necessarily executed at the same time, but can be executed at different times, and the execution order is not necessarily sequential, but can be executed alternately or simultaneously with other steps or sub-steps or stages of other steps.

[0066] The existing cell therapy cannot cope with the complex microenvironment of the tumor, the acidic microenvironment is not conducive to the function of the cell therapy such as CAR-M, and the macrophages cannot be well digested in the body of the subject.

[0067] Therefore, there is an urgent need in the art for a new CAR-immune cell which has good killing effect, can adapt to the acidic tumor microenvironment, and can be well digested by the human body.

[0068] In the first aspect of the present application, a tumor cell-targeted osteoclast is provided, the osteoclast contains a targeting unit; the targeting unit has at least one or more of the following functions:

[0069] (i) specifically recognizes tumor cells;

[0070] (ii) specifically kills tumor cells.

[0071] In the first aspect of the present application, an engineered osteoclast is also provided, the osteoclast contains a targeting unit; the targeting unit has at least one or more of the following functions:

[0072] (i) specifically recognizes tumor cells;

[0073] (ii) specifically kills tumor cells.

[0074] In some embodiments, the targeting unit is a chimeric antigen receptor; the chimeric antigen receptor comprises an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain.

[0075] Unless otherwise indicated, the term chimeric antigen receptor (CAR) in the present application is a molecule that combines the antibody-based specificity for a desired antigen (e.g., a tumor antigen) with a T cell receptor-activating intracellular domain to create a chimeric protein that displays specific anti-tumor cell immune activity. CARs have evolved to the fourth generation so far. The first generation of CARs contains an extracellular single-chain variable fragment scFv, a transmembrane region and a single intracellular activation signal CD3 zeta or Fc epsilon RI gamma, the first generation of CAR-T cells can only cause transient T cell proliferation and less cytokine secretion, and its in vivo anti-tumor activity is greatly limited, and the decrease of T cell proliferation eventually leads to apoptosis of T cells; the second generation of CARs introduces a costimulatory molecule on the basis of the first generation, which improves the tumor killing efficiency; the third generation of CARs carries multiple costimulatory factors such as CD28, CD134 (OX40) and CD137 (4-1BB), etc., and the costimulatory molecules can activate the JNK, ERK, NF-κB, etc. signaling pathways in T cells, which significantly improves the T cell anti-tumor activity, proliferation activity, survival cycle and secretion of cytokines (such as IL-2, TNF-α and IFN-γ), etc.; the fourth generation of CARs increases the selectable markers and promoters encoding CAR amplification and suicide on the basis of the third generation.

[0076] The term "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds with a costimulatory ligand to mediate a costimulatory response by the T cell, such as but not limited to proliferation.

[0077] In one embodiment, the CAR of the present application comprises an extracellular region having an antigen recognition domain, a transmembrane region and an intracellular region.

[0078] The CARs (including functional portions and functional variants thereof) in the present application can be obtained by methods known in the art. The CARs can be prepared by any suitable method of making polypeptides or proteins. Suitable methods of synthesizing polypeptides and proteins de novo are described in references such as Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2000; Peptide and Protein Drug Analysis, Reid, R., Ed., Marcel Dekker Inc., 2000; Epitope Mapping, Westwood et al., Eds., Oxford University Press, Oxford, United Kingdom, 2001; and U.S. Patent 5,449,752. In addition, polypeptides and proteins can be recombinantly produced using standard recombinant methods using the nucleic acids described in the present application. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rded., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Furthermore, some of the CARs (including functional portions and functional variants thereof) of the present application can be isolated and / or purified from sources such as plants, bacteria, insects, mammals such as rats, humans, and the like. Methods of isolation and purification are well known in the art. Alternatively, the CARs (including functional portions and functional variants thereof) described in the present application can be synthesized commercially by companies such as Synpep (Dublin, CA), Peptide Technologies Corp. (Gaithersburg, MD), and Multiple Peptide Systems (San Diego, CA).

[0079] Methods of testing the ability of an antigen to bind to any functional portion of a CAR are known in the art and include any antibody-antigen binding assay, e.g., radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, and competitive inhibition assay (see, e.g., Janeway et al., infra, and U.S. Patent Application No. 2002 / 0197266 Al).

[0080] Also included within the scope of the application are functional variants of the CARs described herein. The term "functional variant" as used herein refers to a CAR, polypeptide, or protein having substantial or significant sequence identity or similarity to a parent CAR that retains the biological activity of the CAR variant. Functional variants encompass, for example, those variants of the CARs (parent CARs) described herein that retain the ability to recognize a target cell to a similar degree, to the same degree, or to a higher degree than the parent CAR. The amino acid sequence of the functional variant can, for example, have at least about 30%, about 50%, about 75%, about 80%, about 90%, about 98%, about 99%, or more identity to the amino acid sequence of the parent CAR.

[0081] The functional variant may, for example, comprise the amino acid sequence of the parent CAR with at least one conservative amino acid substitution. Alternatively or additionally, the functional variant can comprise the amino acid sequence of the parent CAR with at least one non-conservative amino acid substitution. In this case, it is preferred that the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution can enhance the biological activity of the functional variant such that the biological activity of the functional variant is increased compared to the parent CAR.

[0082] The amino acid substitution of the CARs of the application is preferably a conservative amino acid substitution. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid having certain physical and / or chemical properties is exchanged for another amino acid having the same or similar chemical or physical properties. For example, a conservative amino acid substitution can be the substitution of an acidic / negatively charged polar amino acid for another acidic / negatively charged polar amino acid (e.g., Asp or Glu), an amino acid having a nonpolar side chain for another amino acid having a nonpolar side chain (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Tip, Cys, Val, etc.), a basic / positively charged polar amino acid for another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), an uncharged amino acid having a polar side chain for another uncharged amino acid having a polar side chain (e.g., Asn, Gin, Ser, Thr, Tyr, etc.), an amino acid having a beta-branched side chain for another amino acid having a beta-branched side chain (e.g., He, Thr, and Val), an amino acid having an aromatic side chain for another amino acid having an aromatic side chain (e.g., His, Phe, Trp, and Tyr), etc.

[0083] CARs of embodiments of the application, including functional portions and functional variants of the application, can comprise synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexane carboxylic acid, norleucine, alpha-amino normal decanoic acid, homoserine, S-acetylamino-methyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, beta-phenylserine, beta-hydroxyphenylalanine, phenylglycine, alpha-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, Ν',Ν'-dibenzyl-lysine, 6-hydroxylysine, ornithine, alpha-aminocyclopentane carboxylic acid, alpha-aminocyclohexane carboxylic acid, alpha-aminocycloheptane carboxylic acid, alpha-(2-amino-2- norbornane)-carboxylic acid, alpha, gamma-diaminobutyric acid, alpha, beta-diaminopropionic acid, homophenylalanine, and alpha-tert-butylglycine.

[0084] Osteoclasts expressing CARs in the present application can also be referred to as CAR-OC cells, or CAR-modified OC cells.

[0085] In some embodiments, the antigen binding domain specifically recognizes a tumor antigen.

[0086] In some embodiments, the transmembrane domain is selected from the group consisting of CD8a, CD28, CD3y, CD35, CD3s, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, TCRa, TCRp, TCRy, TCR5, TCRz, OX40, ICOS, LAG-3, 2B4, BTLA, CTLA-4, and PD-1.

[0087] In some embodiments, the intracellular signaling domain is derived from the group consisting of FcRy, FcRP, CD3y, CD35, CD3s, CD3z, CD22, CD79a, CD79b, CD66d, CD32a, CD3EBRS, CD28CD, CD19, 2B4, DAP12, and DAP10.

[0088] In some embodiments, the antigen binding domain is selected from the group consisting of an antibody, a single domain antibody fragment, and a single chain antibody.

[0089] In some embodiments, the antigen recognized by the antigen binding domain is selected from the group consisting of HER2, CLDN6, FAP, GPC3, MLSN, uPAR, and PSMA.

[0090] In some embodiments, the intracellular signaling domain further comprises a costimulatory signaling domain derived from the group consisting of RANKL, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD134 (OX40), CD2, CD7, CD27, CD28, CD30, CD40, CD83, ICAM, 4-1BB (CD137), CD276 (B7-H3), CD278 (ICOS), GITR, LIGHT, HVEM (LIGHTR), BTLA, CD8a, LFA-1, NKG2C, LAT, SLP-76, DAP10, PD-1, TRIM, and ZAP70 ligands.

[0091] In some embodiments, the chimeric antigen receptor comprises an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain connected in sequence, which are: a single chain antibody specifically recognizing HER2, CD8a, and CD32a.

[0092] In some embodiments, the heavy chain variable region (VH) and the light chain variable region (VL) are operably linked. For example, by a linker. The linker can be a linker commonly used in the art, such as a flexible linker peptide.

[0093] In some embodiments, the single chain antibody comprises, in sequence from N-terminus to C-terminus: VH-Linker-VL.

[0094] In some embodiments, the single chain antibody comprises, in sequence from N-terminus to C-terminus: VL-Linker-VH.

[0095] In some embodiments, the single chain antibody comprises an amino acid sequence as set forth in SEQ ID NO: 1.

[0096] In some embodiments, the chimeric antigen receptor further comprises a reporter gene.

[0097] In some embodiments, the reporter gene is a fluorescent reporter gene.

[0098] In some embodiments, the fluorescent reporter is selected from the group consisting of GFP, EGFP, RFP, mCherry, mStrawberry, Luciferase, mApple, mRuby, and EosFP.

[0099] In a second aspect of the application, there is provided a method of making the osteoclast cell of the first aspect, the method comprising:

[0100] introducing the nucleic acid expressing the targeting unit into the osteoclast cell using a vector.

[0101] In some embodiments, the step of introducing the nucleic acid expressing the targeting unit into the osteoclast cell using a vector, the osteoclast cell is an unengineered cell.

[0102] The term "vector", as used herein, refers to a vehicle into which a genetic element (e.g., the aforementioned nucleic acid molecule) can be operatively inserted and expressed, unless otherwise indicated. Vectors can be, for example, plasmids, cosmids, viruses (e.g., lentivirus, retrovirus, adenovirus, and adeno-associated virus), RNA vectors, or linear or circular DNA or RNA molecules, which can include chromosomal, non-chromosomal, semi-synthetic, or synthetic nucleic acid molecules. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors".

[0103] As used herein, the term "expression" refers to the process by which a polypeptide is produced based on the coding sequence of a nucleic acid molecule (e.g., a gene). The process can include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post-translational modification, or any combination thereof.

[0104] In some embodiments, the step of introducing the nucleic acid expressing the targeting unit into the osteoclast cell using a vector, the vector is a viral vector.

[0105] As used herein, "viral vector" refers to a vector based on a virus that has been engineered to carry foreign and related genetic elements, which can be packaged into a viral particle and can mediate the transfer and expression of the foreign gene, unless otherwise indicated. The viral vector can be a recombinant viral vector, or a viral vector without viral genes. Non-limiting examples of viral vectors are adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, and the like.

[0106] In some embodiments, the viral vector is selected from the group consisting of a lentiviral vector, a retroviral vector, an adenoviral vector, and an adeno-associated viral vector. As used herein, the term "adenovirus" refers to a single-stranded DNA virus with a small (about 20 nm) protein coat, belonging to the family Parvoviridae, and specifically refers to viruses of the genus Adenoviridae. The term Adenoviridae refers collectively to animal adenoviruses of the genus Mastadenovirus, including but not limited to human, bovine, ovine, equine, canine, porcine, murine, and simian adenovirus subgenera. As used herein, the term "lentivirus" refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in their ability to infect non-dividing cells; they can deliver significant amounts of genetic information into the DNA of host cells, making them one of the effective methods in gene delivery vectors. Human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), and feline immunodeficiency virus (FIV) are all examples of lentiviruses.

[0107] In a third aspect of the present application, there is provided use of the osteoclast as described in the first aspect in the preparation of a medicament for preventing or treating a disease.

[0108] In some embodiments, the disease comprises one or more of a tumor, bone metastasis, and osteopetrosis.

[0109] In some embodiments, the tumor comprises a solid tumor.

[0110] In a fourth aspect of the present application, there is provided a cell pharmaceutical composition comprising the osteoclast as described in the first aspect, and a pharmaceutically acceptable carrier.

[0111] "Pharmaceutically acceptable" refers to those ligands, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for administration to patients, and which are compatible with the reasonable benefit / risk ratio.

[0112] "Pharmaceutically acceptable carrier" refers to pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. As used herein, the language "pharmaceutically acceptable carrier" includes buffers, sterile water for injection, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Each carrier must be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.

[0113] In a fifth aspect of the present application, there is provided a method for preventing or treating a disease, the method comprising administering to a subject an effective amount of the osteoclast as described in the first aspect or the cell pharmaceutical composition as described in the fourth aspect.

[0114] In some embodiments, the subject is considered in need of the administration of the osteoclast or the cell pharmaceutical composition.

[0115] The term "prevention" includes, but is not limited to, prophylactic, therapeutic, and the like interventions to a disease, unless otherwise specified.

[0116] As used herein, the term "treatment" refers to therapeutic treatment, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder. The term "treatment" embraces both reduction or lessening of at least one side effect or symptom of a disease or disorder. A treatment is generally "effective" if there is an improvement in one or more symptoms or clinical markers. Alternatively, a treatment is "effective" if the progression of disease is reduced or halted, that is, "treatment" includes not only the improvement of symptoms but also the cessation or at least slowing of the progression or worsening of symptoms that would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or remission of the disease state, and remission (whether partial or total), whether detectable or undetectable.

[0117] In some embodiments, the disease comprises one or more of a tumor, bone metastasis, and osteopetrosis.

[0118] In some embodiments, the tumor comprises a solid tumor.

[0119] The dosage form and administration method of the cell pharmaceutical composition are not particularly limited.

[0120] In some embodiments, the dosage form of the cell pharmaceutical composition is selected from the group consisting of a solution type, an emulsion type, and a colloidal solution type.

[0121] Representative administration methods include, but are not limited to, oral, intratumoral, rectal, parenteral (intravenous, intramuscular, or subcutaneous) injection, and topical administration. In some embodiments, the administration method of the osteoclast or the cell pharmaceutical composition is intravenous injection.

[0122] In some embodiments, the administration regimen of the osteoclast or the cell pharmaceutical composition is multiple administration or single administration.

[0123] Embodiments of the present application will be described in detail below with reference to Examples. It should be understood that these Examples are intended to illustrate but not limit the scope of the present application. The experimental methods in the following Examples, for which no conditions are specified, are preferably referred to the guidelines given in the present application, but can also be performed according to the protocols or conditions known in the art, or according to the protocols or conditions suggested by the manufacturers, or according to the protocols or conditions known in the art.

[0124] The sources of reagents and materials used in the Examples are as follows:

[0125] Table 1 Reagent Consumable Information

[0126] Example 1 Preparation of CAR-containing osteoclasts and macrophages

[0127] The experimental procedures are summarized in Table 2.

[0128] Table 2

[0129] The details are as follows:

[0130] 1. Mouse primary cell isolation

[0131] a. Dislocate 5 C57 mice and immerse them in a beaker containing 75% alcohol for 3-5 minutes;

[0132] b. Lay a plastic bag flat in the safety cabinet and place the mouse on the plastic bag for experimental operation;

[0133] c. Carefully pinch the abdominal skin between the two hip joints of the mouse with an ophthalmic forceps, carefully cut the skin with an ophthalmic scissors, and separate the skin of the two lower limbs, cutting off at the ankle and cutting off at the hip joint, so that the two lower limbs of the mouse are isolated;

[0134] d. Carefully peel off the muscles of the lower limbs, remove the tibia and femur, and place them in a culture dish containing 75% alcohol;

[0135] e. Take out the mouse carcass, clean the safety cabinet, and put on new gloves;

[0136] f. Take 5 mL of sterile syringes, use the syringes to suck the prepared DMEM, gently insert the bone marrow cavity, and align a sterile 15 mL centrifuge tube to flush out the cells; [It is beneficial to cut the ends of the femur to facilitate washing]

[0137] g. After flushing out the bone marrow cells (BM cells), centrifuge at 700 x g for 5 min, and discard the supernatant;

[0138] h. Add 3-4 mL of red blood cell lysis solution to blow off ice for 10 min to lyse red blood cells, add 30-40 mL of sterile PBS, and wash twice (add PBS at a ratio of 1:9 between the addition of red blood cell lysis solution and PBS);

[0139] i. Filter the mixture containing BM cells through a 100 μm cell sieve into a new 50 mL centrifuge tube;

[0140] j. Centrifuge at 700 g for 5 min, discard the supernatant, and obtain the BM cell precipitate;

[0141] k. Resuspend the BM cell pellet in 40 mL PBS and count the cells;

[0142] l. Pipette 6.35 x 106 cells and 4.35 x 106 cells into two new 50 mL centrifuge tubes, respectively, and centrifuge at 700 g for 5 min, discard the supernatant; 7 7

[0143] m. Resuspend the cell pellets with macrophage medium and osteoclast medium (see below), respectively, for induction culture.

[0144] 2. RANKL-induced differentiation of BMDM and osteoclasts

[0145] 2.1 Medium preparation

[0146] Macrophage medium: 79% DMEM medium + 20% FBS + 1% P / S + 25 ng / mL M-CSF.

[0147] Osteoclast medium: Osteoclast basal medium + Mouse RANKL recombinant protein (30 ng / ml) (purchased from Protech, item number 315-11).

[0148] 2.2 Cell culture

[0149] Culture mouse BMDM cells in 10 cm dishes, 100 million cells per dish; among them, 6 dishes are induced to be osteoclasts, and 4 dishes are induced to be macrophages. (Two kinds of cells are plated in two wells of a six-well plate, 17.5 million cells per well, for counting when infected with viruses, the bottom area of a 10 cm dish is about 5.7 times that of a six-well plate).

[0150] Change the medium every 2 days according to the experimental grouping.

[0151] 3. Ad5F35-CAR-HER2-CD32a-mCherry infection of macrophages and osteoclasts

[0152] Construct Ad5F35-CAR-HER2-CD32a-mCherry according to the plasmid map in Figure 1.

[0153] On the 4th day of induction culture, use Ad5F35-CAR-HER2-CD32a-mCherry to infect macrophages and osteoclasts. The specific steps are as follows:

[0154] a. Digest and resuspend the macrophages and osteoclasts in the 6-well plate plated in advance, and count the cells;

[0155] b. The number of cells per well x 5.7 is the number of cells in a 10 cm dish; ​​

[0156] c.10 cm dish, medium was changed and Ad5F35-CAR-HER2-CD32a-mCherry was added at MOI = 2000, mixed well and put back into the incubator.

[0157] The macrophages and osteoclasts obtained after infection are hereinafter referred to as CAR-BMDM and CAR-OC (Osteoclast, OC), respectively.

[0158] 4. Flow cytometry and cell counting

[0159] According to the experimental grouping, the cells in each dish were resuspended after digestion and counted, and the remaining cells were subjected to flow cytometry detection. The detection groups are shown in Table 3 below.

[0160] Table 3

[0161] Note: The full name of the abbreviation "WT" is wild type, i.e., wild type (the WT corresponding to "CAR-osteoclast" above refers to the osteoclasts obtained by RANKL induction differentiation above, and the WT corresponding to CAR-BMDM refers to the BMDM obtained by RANKL induction differentiation above); the full name of BMDM is bone marrow-derived macrophage.

[0162] After 48 hours of infection of mouse BMDM and osteoclasts with Ad5F35-CAR-HER2-CD32a-mCherry, the mCherry fluorescence detection results showed that the fluorescence of the CAR-OC group was stronger than that of the CAR-BMDM group (Figure 2); the cells in the two infection groups were not firmly adherent, but were not dead cells. After replacing the medium and plating in a 48-well plate for the killing experiment, the cells in the four groups adhered well on the second day.

[0163] After 48 hours of infection of mouse BMDM and osteoclasts with Ad5F35-CAR-HER2-CD32a-mCherry, the G4S flow cytometry results are shown in Figures 4, 5 and 6. Figures 4 to 6 show that the isotype control group is the negative group, the G4S positive rate of the CAR-OC group is 65.68%, which is higher than that of the CAR-BMDM group (63.04%). The non-specificity of the BMDM group in this experiment is 35.75%, and the G4S positive rate of the mouse CAR-BMDM group is lower than the detection value.

[0164] 5. TRAP detection

[0165] After 48 hours of infection of mouse BMDM and osteoclasts with Ad5F35-CAR-HER2-CD32a-mCherry (sixth day of induction culture), TRAP was used to detect the induction of cells.

[0166] The specific steps are:

[0167] a. Suck the culture medium in the 12-hole plate, add PBS to wash the hole plate according to 500 μl / hole, and wash 3 times;

[0168] b. Suck the residual PBS in the hole plate, add 500 μl of TRAP fixing solution (4°C pre-cooling), and fix at room temperature for 1 min;

[0169] c. Suck the fixing solution in each hole, add 500 μl of ddH2O for washing, and then drain the water;

[0170] d. Add 500 μl of TRAP incubation solution to cover the cells, and incubate at 37°C for 60 min;

[0171] e. After incubation, add 500 μl of ddH2O to each hole for washing, and then drain the water;

[0172] f. Add 500 μl of PBS to transfer to the microscope for observation and counting.

[0173] After TRAP detection, the mouse osteoclasts in this experiment were successfully induced, and the adenovirus infection did not affect their differentiation (Figure 3).

[0174] 6. Test of killing N87-Fluc cells by mouse osteoclasts after CAR introduction (E:T = 2:1 or 1:1) in vitro

[0175] a. After 48 hours of adenovirus infection, the four groups of cells (see Table 4 for grouping) were digested and plated in a 48-hole plate according to the experimental grouping, with 200,000 cells per hole, and cultured in a carbon dioxide incubator for 16 h;

[0176] b. Add NCI-N87-Fluc-GFP cells (hereinafter referred to as N87-Fluc) to each hole according to the experimental grouping

[0177] NCI-N87 cells were purchased from Shanghai Binsui Biotechnology Co., Ltd., and N87-Fluc cells were constructed by transfecting the firefly luciferase (Fluc) and green fluorescent protein (GFP) genes with lentivirus.

[0178] The ratio of target cells to effector cells and cell grouping are shown in the following table, and the hole plate distribution is shown in Figure 7.

[0179] Table 4

[0180] Note: the full name of the abbreviation "WT" is wild type, i.e. wild type (the WT corresponding to "CAR-OC" above refers to the osteoclasts differentiated by RANKL in Example 1, and the WT corresponding to "CAR-BMDM" refers to the BMDMs differentiated by RANKL in Example 1); the full name of BMDM is bone marrow-derived macrophage.

[0181] c. After 6h and 24h of incubation according to the grouping, one volume of D-luciferin potassium (0.6mg / ml) was directly added to the well plate to be tested, mixed and incubated at 37°C for 5min;

[0182] d. The small animal live imaging system was used for imaging.

[0183] The 24h killing results showed that the CAR-BMDM cells had stronger killing effect on N87-Fluc cells than the CAR-OC cells when the E / T ratio was 1:1; when the E / T ratio was 2:1, there was no significant difference between the two (Figure 8).

[0184] It was proved that CAR-OC had killing effect on N87-Fluc.

[0185] Further, when the E / T ratio was 1:1 and 2:1, the killing efficiency of CAR-OC and CAR-BMDM on N87-Fluc cells was close to the upper limit of detection, and it was impossible to compare the killing effect of CAR-OC and CAR-BMDM, so further adjustment was made to further compare the killing effect of CAR-OC and CAR-BMDM through the following experiment 7.

[0186] 7. Killing test of N87-Fluc cells in vitro by mouse osteoclasts after introduction of CAR (E / T ratio was 1:10 or 1:100)

[0187] This experiment was similar to the above experiment 7, and the main difference was the E / T ratio. Specifically:

[0188] a. Four groups of cells (grouping is shown in Table 5) were digested and plated in 48-well plates according to the experimental grouping, 100,000 cells per well, and incubated in a carbon dioxide incubator for 24h;

[0189] b. NCI-N87-Fluc-GFP cells (hereinafter referred to as N87-Fluc) were added to each well according to the experimental grouping; the culture medium was replaced daily;

[0190] The E / T ratio of the target cells and the effector cells is shown in the following table.

[0191] Table 5

[0192] Note: the full name of the abbreviation "WT" is wild type, i.e. wild type (the WT corresponding to the above "CAR-OC" is the osteoclast obtained by the above RANKL-induced differentiation, and the WT corresponding to the above CAR-BMDM is the BMDM obtained by the above RANKL-induced differentiation); the full name of BMDM is bone marrow-derived macrophage.

[0193] c. After 48h and 72h of culture according to the grouping, one volume of D-luciferin potassium (0.6mg / ml) was directly added to the well plate to be detected, mixed and incubated at 37°C for 5min;

[0194] d. The small animal live imaging system was used for imaging.

[0195] According to the cell test of the above experiment 6 and experiment 7:

[0196] CAR-OC has a significant killing effect on N87-Fluc (Figure 8). As shown in Figures 9 and 10, when the E / T is increased to 1:10, it can be seen that the killing effect of CAR-OC on N87-Fluc is significantly better than that of CAR-BMDM cells after 24h and 48h of killing.

[0197] It is proved that a small number of CAR-OC cells can kill N87-Fluc cells.

[0198] Further, based on the above-mentioned few CAR-OC cells that can kill N87-Fluc cells, it can be seen that CAR-OC has advantages in treating diseases such as tumors, bone metastases, and hyperosteogeny. On the one hand, the application of CAR-OC technology in treating diseases can reduce the amount of cells isolated from patients, and on the other hand, fewer CAR-OC can achieve better killing effect, and also reduce the difficulty of preparation process, and at the same time, reduce the amount of cells required for reinfusion to achieve therapeutic effect.

[0199] In summary, the CAR-OC cells prepared in the present application can effectively kill N87-Fluc cells. Due to the characteristics of osteoclasts that can exist in an acidic environment, CAR-OC can effectively exert its targeted killing ability in the acidic microenvironment of tumors, and is a preferred cell therapy.

[0200] The technical features of the above-described embodiments can be combined in any manner. In order to make the description concise, all possible combinations of the technical features in the above-described embodiments are not described, however, as long as the combinations of the technical features do not exist contradictory, they should be considered as the scope of the present disclosure.

[0201] The above embodiments only express several implementation manners of the present application, and the description is relatively specific and detailed, but it should not be understood as a limitation on the patent scope of the application. It should be pointed out that for ordinary skilled persons in the art, several modifications and improvements can be made without departing from the concept of the present application, which are all within the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. An engineered osteoclast comprising a targeting unit; the targeting unit has one or more of the following functions: (i) specifically recognizes tumor cells; (ii) specifically kills tumor cells.

2. The osteoclast of claim 1, wherein, The osteoclast targets tumor cells.

3. The osteoclast of claim 1 or 2, wherein, The targeting unit is a chimeric antigen receptor; the chimeric antigen receptor comprises an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain.

4. The osteoclast of claim 3, wherein, The chimeric antigen receptor meets one or more of the following conditions: The antigen binding domain specifically recognizes a tumor antigen; The transmembrane domain is selected from the group consisting of CD8α, CD28, CD3ζ, CD3γ, CD3δ, CD3ε, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, TCRα, TCRβ, TCRγ, TCRδ, TCRζ, OX40, ICOS, LAG-3, 2B4, BTLA, CTLA-4 and PD-1; The intracellular signaling domain is derived from the group consisting of FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, CD66d, CD32α, CD3EBRS, CD28CD, CD19, 2B4, DAP12 and DAP10.

5. The osteoclast of claim 3 or 4, wherein, The antigen binding domain meets one or more of the following conditions: The antigen binding domain is selected from the group consisting of antibodies, single domain antibody fragments and single chain antibodies; The antigen recognized by the antigen binding domain is selected from the group consisting of HER2, CLDN6, FAP, GPC3, MLSN, uPAR and PSMA.

6. The osteoclast of any one of claims 3-5, wherein, The intracellular signaling domain further comprises a costimulatory signaling domain, which is derived from the group consisting of RANKL, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD134 (OX40), CD2, CD7, CD27, CD28, CD30, CD40, CD83, ICAM, 4-1BB (CD137), CD276 (B7-H3), CD278 (ICOS), GITR, LIGHT, HVEM (LIGHTR), BTLA, CD8α, LFA-1, NKG2C, LAT, SLP-76, DAP10, PD-1, TRIM and ZAP70 ligand.

7. The osteoclast of any one of claims 3-6, wherein, The chimeric antigen receptor comprises an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain connected in sequence: A single chain antibody that specifically recognizes HER2, CD8α and CD32α.

8. The osteoclast of claim 7, wherein, The single chain antibody comprises an amino acid sequence as shown in SEQ ID NO:

1.

9. The osteoclast of any one of claims 1-8, wherein, The chimeric antigen receptor further comprises a reporter gene.

10. The osteoclast of claim 9, wherein, The reporter gene is a fluorescent reporter gene.

11. The osteoclast of claim 10, wherein, The fluorescent reporter gene is selected from the group consisting of GFP, EGFP, RFP, mCherry, mStrawberry, Luciferase, mApple, mRuby and EosFP.

12. A method for preparing the osteoclast according to any one of claims 1-11, comprising: introducing a nucleic acid expressing the targeting unit into the osteoclast using a viral vector.

13. The method of claim 12, wherein, The viral vector is selected from the group consisting of a lentivirus vector, a retrovirus vector, an adenovirus vector and an adeno-associated virus vector.

14. Use of the osteoclast according to any one of claims 1-11 in the preparation of a medicament for preventing and treating a disease.

15. The use of claim 14, wherein, The disease comprises one or more of a tumor, bone metastasis and hyperostosis; optionally, the tumor comprises a solid tumor.

16. A cell pharmaceutical composition comprising the osteoclast according to any one of claims 1-11 and a pharmaceutically acceptable carrier.

17. A method of controlling a disease, wherein, The method comprises administering an effective dose of the osteoclast according to any one of claims 1-11 or the cell pharmaceutical composition according to claim 16 to a subject.

18. The method of claim 17, wherein, The disease comprises one or more of a tumor, bone metastasis and hyperostosis; optionally, the tumor comprises a solid tumor.

19. The method of claim 17 or 18, wherein, The subject is considered to be in need of administration of the osteoclast or the cell pharmaceutical composition.

20. The method of any one of claims 17-19, wherein, The dosage form of the cell pharmaceutical composition is selected from the group consisting of a solution type, an emulsion type and a colloidal solution type.

21. The method of any one of claims 17-20, wherein, The method satisfies one or more of the following conditions: The administration method of the osteoclast or the cell pharmaceutical composition is intravenous injection; The administration regimen of the osteoclast or the cell pharmaceutical composition is multiple administration or single administration.

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