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, their preparation methods and uses Technical Field

[0001] This application belongs to the field of biotechnology, particularly the field of immunotherapy, and specifically relates to an osteoclast, its preparation method, and its uses. Background Technology

[0002] Chimeric antigen receptor-T cell (CAR-T cell) therapy is a type of cellular immunotherapy that involves transferring genetic material with specific antigen recognition domains and T cell activation signals into T cells using gene cloning technology. This allows the T cells to directly activate by binding to specific antigens on the surface of tumor cells. CAR-T cells have been successfully used to treat malignant tumors of the circulating system, such as B-cell-derived malignancies. Following the success of CAR-T therapy, chimeric antigen receptor-natural killer cell (CAR-NK cell) therapy was developed. However, the efficacy of existing CAR-T and CAR-NK technologies for treating solid tumors remains limited.

[0003] On the other hand, tumors have different characteristics at different stages, especially when they metastasize to the bone in the later stages. Traditional CAR-T and CAR-NK technologies are difficult to be effective in these cases. At this time, patients urgently need a drug to suppress the disease. Therefore, it is also urgent to develop a drug that can target patients with bone metastasis in the late stage of tumors.

[0004] Summary of the Invention

[0005] Therefore, it is necessary to provide at least one osteoclast that targets tumor cells, its preparation method, and its uses.

[0006] In a first aspect of this application, an osteoclast that targets tumor cells is provided, the osteoclast comprising a targeting unit; the targeting unit having at least one or more of the following functions:

[0007] (i) Specifically recognizes tumor cells;

[0008] (ii) Specific killing of tumor cells.

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

[0010] (i) Specifically recognizes tumor cells;

[0011] (ii) Specific killing of tumor cells.

[0012] The aforementioned engineered osteoclast is a modified osteoclast.

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

[0014] In some implementations, the antigen-binding domain specifically recognizes tumor antigens.

[0015] In some embodiments, 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.

[0016] In some embodiments, the intracellular signal transduction domains are derived from the group consisting of FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, CD66d, CD32α, CD3EBRS, CD28CD, CD19, 2B4, DAP12, and DAP10.

[0017] In some embodiments, the antigen-binding domain is selected from the group consisting of antibodies, single-domain antibody fragments, and single-chain antibodies.

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

[0019] In some embodiments, the intracellular signal transduction domain further includes a co-stimulatory 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, CD8α, LFA-1, NKG2C, LAT, SLP-76, DAP10, PD-1, TRIM, and ZAP70 ligands.

[0020] In some embodiments, the chimeric antigen receptor comprises, in sequence, an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain: a single-chain antibody that specifically recognizes HER2, CD8α, and CD32α.

[0021] In some embodiments, the single-chain antibody comprises an amino acid sequence as shown in 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 this application, a method for preparing osteoclasts as described in the first aspect is provided, the method comprising:

[0026] The nucleic acid expressing the target unit is introduced into osteoclasts using a viral vector. In some embodiments, the osteoclasts are unengineered cells during the step of introducing the nucleic acid expressing the target unit into osteoclasts.

[0027] In some embodiments, the viral vector is selected from the group consisting of lentiviral vectors, retroviral vectors, adenovirus vectors, and adeno-associated virus vectors.

[0028] In a third aspect of this application, the use of osteoclasts as described in the first aspect in the preparation of medicaments for the prevention and treatment of diseases is provided.

[0029] In some implementations, the disease includes one or more of tumors, bone metastases, and bone hyperplasia.

[0030] In some implementations, the tumor includes a solid tumor.

[0031] In a fourth aspect of this application, a cell-based pharmaceutical composition is provided, comprising osteoclasts as described in the first aspect, and a pharmaceutically acceptable carrier.

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

[0033] In some embodiments, the subject is deemed to require administration of the osteoclasts or the cellular drug composition.

[0034] In some implementations, the disease includes one or more of tumors, bone metastases, and bone hyperplasia.

[0035] In some implementations, the tumor includes a solid tumor.

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

[0037] In some embodiments, the osteoclasts or the cell-pharmaceutical composition are administered via intravenous injection.

[0038] In some embodiments, the osteoclasts or the cell-pharmaceutical composition are administered via a multiple-dose or single-dose regimen.

[0039] In one embodiment of this application, a target gene fragment is introduced into osteoclasts via a virus (e.g., Ad5F35), thereby modifying the osteoclasts into cells capable of targeted disease treatment. The aforementioned virus is modified to carry a gene encoding a target cell recognition gene. Through this viral modification, the osteoclasts are transformed into osteoclasts capable of targeting and killing target cells. Furthermore, the target cells can be cells causing disease, tumor cells, or bone metastases.

[0040] The targeting unit in the aforementioned osteoclast-based CAR-OC targeting tumor cells can be a chimeric antigen receptor (CAR), which can then be used for immunotherapy. Taking CAR-modified osteoclasts as an example, they have a strong targeted killing ability. 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] Furthermore, osteoclasts can be derived from peripheral blood, for example, by inducing monocytes in peripheral blood to become osteoclasts. Attached Figure Description

[0042] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without creative effort. It should also be noted that the drawings are all drawn in a simplified form and are only used to conveniently and clearly assist in illustrating this application.

[0043] Figure 1 is a construction map of the plasmid used to infect macrophages and osteoclasts in one embodiment of this application.

[0044] Figure 2 shows the mCherry fluorescence detection results after 48 hours of infection of mouse BMDM and osteoclasts with Ad5F35-CAR-HER2-CD32a-mCherry in one embodiment of this application.

[0045] Figure 3 shows the TRAP detection results of mice BMDM and osteoclasts 48 h after infection with Ad5F35-CAR-HER2-CD32a-mCherry in one embodiment of this application.

[0046] Figure 4 shows the G4S flow cytometry results of mice BMDM and osteoclasts infected with Ad5F35-CAR-HER2-CD32a-mCherry 48 h after one embodiment of this application.

[0047] Figure 5 shows the G4S flow cytometry results of mice BMDM and osteoclasts infected with Ad5F35-CAR-HER2-CD32a-mCherry in one embodiment of this application for 48 hours.

[0048] Figure 6 shows the G4S flow cytometry results of mice BMDM and osteoclasts infected with Ad5F35-CAR-HER2-CD32a-mCherry 48 h after one embodiment of this application.

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

[0050] Figure 8 shows the killing results of CAR-BMDM and CAR-OC cells after 24 hours when the E / T ratio is 1:1 and 2:1 in one embodiment of this application.

[0051] Figure 9 shows the killing results of CAR-BMDM and CAR-OC cells 48 when the E / T ratio is 1:10 in one embodiment of this application.

[0052] Figure 10 shows the killing results of CAR-BMDM and CAR-OC cells 72 when E / T is 1:10 in one embodiment of this 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 “containing,” “comprising,” and “including” as used in this application are synonyms and are inclusive or open-ended, not excluding additional, uncited members or features. Members or features include, for example, materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions under which actions occur, timing, states, etc.

[0061] In this application, the technical features or solutions described in open-ended language include both closed-ended technical features or solutions consisting of the listed contents and open-ended technical features or solutions that include the listed contents.

[0062] In this application, the exemplary descriptions such as "in some implementations (or embodiments)" and "in one implementation (or embodiment)" may cover, but are 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 this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only a non-exhaustive enumeration purpose and should be understood not to constitute a closed limitation on quantity.

[0064] In this application, when numerical intervals (i.e., numerical ranges) are involved, unless otherwise specified, the distribution of selectable numerical values ​​within the numerical interval is considered continuous, and includes the two endpoints of the numerical interval (i.e., the minimum and maximum values), as well as every numerical value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. The term "numerical interval" can be broadly included to include numerical interval types such as percentage intervals, ratio intervals, and proportion intervals.

[0065] In this application, where the method flow involves multiple steps, unless otherwise explicitly stated herein, there is no strict order restriction on the execution of these steps; they can be executed in any order other than those described. Moreover, any step may include multiple sub-steps or multiple stages, which are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately or simultaneously with other steps or parts of the sub-steps or stages of other steps.

[0066] Existing cell therapies cannot address the complex microenvironment of tumors. The acidic microenvironment is not conducive to the function of cell therapies such as CAR-M, and macrophages cannot be well digested in the body of the subjects.

[0067] Therefore, there is an urgent need in this field for a new type of CAR-immune cell that not only has a good killing effect, can adapt to the acidic tumor microenvironment, but can also be well digested by the human body.

[0068] In a first aspect of this application, an osteoclast that targets tumor cells is provided, the osteoclast comprising a targeting unit; the targeting unit having at least one or more of the following functions:

[0069] (i) Specifically recognizes tumor cells;

[0070] (ii) Specific killing of tumor cells.

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

[0072] (i) Specifically recognizes tumor cells;

[0073] (ii) Specific killing of tumor cells.

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

[0075] Unless otherwise specified, the term chimeric antigen receptor (CAR) in this application refers to a molecule that binds an antibody-based specificity against a desired antigen (e.g., a tumor antigen) to a T-cell receptor-activated intracellular domain to produce a chimeric protein exhibiting specific anti-tumor cellular immune activity. CARs have now evolved to the fourth generation. First-generation CARs contain an extracellular single-chain variable fragment scFv, a transmembrane region, and a single intracellular activation signal CD3ζ or FcεRIγ. First-generation CAR-T cells can only induce transient T cell proliferation and limited cytokine secretion, greatly limiting their in vivo anti-tumor activity. Reduced T cell proliferation eventually leads to T cell apoptosis. Second-generation CARs introduce a co-stimulatory molecule to improve tumor-killing efficacy. Third-generation CARs carry multiple co-stimulatory factors, such as CD28, CD134 (OX40), and CD137 (4-1BB). These co-stimulatory molecules can activate signaling pathways such as JNK, ERK, and NF-κB in T cells, significantly improving T cell anti-tumor activity, proliferation, lifespan, and cytokine secretion (such as IL-2, TNF-α, and IFN-γ). Fourth-generation CARs add selective labeling and promoters encoding CAR amplification and suicide based on the third generation.

[0076] The term "co-stimulatory molecule" refers to a homologous binding partner on a T cell that specifically binds to a co-stimulatory ligand, thereby mediating a co-stimulatory response in the T cell, such as, but not limited to, proliferation.

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

[0078] The CAR (including its functional moiety and functional variants) in this application can be obtained by methods known in the art. CARs can be prepared by any suitable method for preparing peptides or proteins. Suitable methods for de novo synthesis of peptides and proteins 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*, edited by Reid, R., Marcel Dekker Inc., 2000; *Epitope Mapping*, edited by Westwood et al., Oxford University Press, Oxford, United Kingdom, 2001; and U.S. Patent 5,449,752. Additionally, peptides and proteins can be generated using standard recombinant methods with nucleic acid recombination as described in this application. See, for example, Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition, 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 their functional moieties and functional variants) described in this application can be isolated from and / or purified from sources such as plants, bacteria, insects, mammals such as rats, humans, etc. Isolation and purification methods are well known in the art. Optionally, the CARs (including their functional moieties and functional variants) described in this application can be commercially synthesized by companies such as Synpep (Dublin, CA), Peptide Technologies Corp. (Gaithersburg, MD), and Multiple Peptide Systems (San Diego, CA).

[0079] Methods for testing the ability of an antigen to bind to any functional part of a CAR are known in the art and include any antibody-antigen binding assay, such as radioimmunoassay (RIA), ELISA, Western blotting, immunoprecipitation, and competitive inhibition assay (see, for example, Janeway et al., hereinafter and U.S. Patent Application No. 2002 / 0197266A1).

[0080] This application also includes, within the scope of this application, functional variants of the CAR described herein. As used herein, the term "functional variant" refers to a CAR, polypeptide, or protein having a large or significant sequence identity or similarity to the parent CAR, said functional variant retaining the biological activity of the CAR variant. Functional variants encompass, for example, those variants of the CAR (parent CAR) described herein, which retain the ability to recognize target cells to a similar degree, the same degree, or a higher degree than the parent CAR. Regarding the parent CAR, the amino acid sequence of the functional variant may, for example, have at least about 30%, about 50%, about 75%, about 80%, about 90%, about 98%, about 99%, or higher identity with the amino acid sequence of the parent CAR.

[0081] The functional variant may, for example, comprise the amino acid sequence of the parent CAR having at least one conserved amino acid substitution. Alternatively or additionally, the functional variant may comprise the amino acid sequence of the parent CAR having at least one non-conserved amino acid substitution. In this case, non-conserved amino acid substitutions that do not interfere with or inhibit the biological activity of the functional variant are preferred. Non-conserved amino acid substitutions can enhance the biological activity of the functional variant, resulting in an increase in the biological activity of the functional variant compared to the parent CAR.

[0082] The amino acid substitutions in the CAR of this application are preferably conservative amino acid substitutions. Conservative amino acid substitutions are those 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, conservative amino acid substitutions can include replacing an acidic / negatively charged polar amino acid with another acidic / negatively charged polar amino acid (e.g., Asp or Glu), replacing an amino acid with a nonpolar side chain with another amino acid with a nonpolar side chain (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Tip, Cys, Val, etc.), replacing a basic / positively charged polar amino acid with another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), replacing an uncharged amino acid with a polar side chain with another uncharged amino acid with a polar side chain (e.g., Asn, Gln, Ser, Thr, Tyr, etc.), replacing an amino acid with a β-branched side chain with another amino acid with a β-branched side chain (e.g., Ile, Thr, and Val), and replacing an amino acid with an aromatic side chain with another amino acid with an aromatic side chain (e.g., His, Phe, Trp, and Tyr, etc.).

[0083] The CAR (including the functional portion and functional variants of this application) of the embodiments of this application may contain 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, aminocyclohexanecarboxylic acid, leucine, α-aminodecanoic acid, homoserine, S-acetaminomethylcysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2- Carboxylic acids, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norborneane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[0084] In this application, osteoclasts expressing CAR may also be referred to as CAR-OC cells, or CAR-modified OC cells.

[0085] In some implementations, the antigen-binding domain specifically recognizes tumor antigens.

[0086] In some embodiments, 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.

[0087] In some embodiments, the intracellular signal transduction domains are derived from the group consisting of FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, CD66d, CD32α, CD3EBRS, CD28CD, CD19, 2B4, DAP12, and DAP10.

[0088] In some embodiments, the antigen-binding domain is selected from the group consisting of antibodies, single-domain antibody fragments, and single-chain antibodies.

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

[0090] In some embodiments, the intracellular signal transduction domain further includes a co-stimulatory 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, CD8α, LFA-1, NKG2C, LAT, SLP-76, DAP10, PD-1, TRIM, and ZAP70 ligands.

[0091] In some embodiments, the chimeric antigen receptor comprises, in sequence, an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain: a single-chain antibody that specifically recognizes HER2, CD8α, and CD32α.

[0092] In some embodiments, the heavy chain variable region (VH) and the light chain variable region (VL) are operatively linked. For example, they are linked via 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, from the N-terminus to the C-terminus, VH-Linker-VL.

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

[0095] In some embodiments, the single-chain antibody comprises an amino acid sequence as shown 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 gene is selected from the group consisting of GFP, EGFP, RFP, mCherry, mStrawberry, Luciferase, mApple, mRuby, and EosFP.

[0099] In a second aspect of this application, a method for preparing osteoclasts as described in the first aspect is provided, the method comprising:

[0100] The nucleic acid expressing the target unit is introduced into osteoclasts using a vector.

[0101] In some embodiments, in the step of using a vector to introduce nucleic acid expressing the target unit into osteoclasts, the osteoclasts are unengineered cells.

[0102] Unless otherwise specified, the term "vector" in this application refers to a delivery vehicle that can operatively insert a genetic element (such as the aforementioned nucleic acid molecule) therein and enable the expression of that genetic element. Vectors can be, for example, plasmids, granules, viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses), RNA vectors, or linear or circular DNA or RNA molecules, which may include chromosomal, non-chromosomal, semi-synthetic, or synthetic nucleic acid molecules. This term includes vectors that serve as self-replicating nucleic acid structures as well as vectors incorporated into the host cell genome. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to as "expression vectors" in this application.

[0103] As used in this application, the term "expression" refers to the process by which a polypeptide is generated based on the coding sequence of a nucleic acid molecule (such as a gene). This process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post-translational modification, or any combination thereof.

[0104] In some implementations, a viral vector is used to introduce nucleic acids expressing the target unit into osteoclasts.

[0105] Unless otherwise specified, "viral vector" in this application refers to a vector obtained through viral modification that carries foreign genes and related gene elements, can be packaged into viral particles, and can mediate the transfer and expression of foreign genes. The viral vector can be a recombinant viral vector or a viral vector without viral genes. Non-limiting examples of viral vectors include adenovirus vectors, adeno-associated virus vectors, lentiviral vectors, etc.

[0106] In some embodiments, the viral vector is selected from the group consisting of lentiviral vectors, retroviral vectors, adenoviral vectors, and adeno-associated virus vectors. As used herein, the term "adenovirus" refers to a single-stranded DNA virus with a small (approximately 20 nm) protein shell, belonging to the Parvoviridae family, and specifically to viruses of the genus Adenoviridae. The term Adenoviridae generally refers to animal adenoviruses of the genus Adenovirus of mammals, including but not limited to the subgenus Adenovirus of humans, cattle, sheep, horses, dogs, pigs, mice, and simians. As used herein, the term "lentivirus" refers to a genus of the family Retroviridae. Lentivirals are unique among retroviruses in that they can infect non-dividing cells; they can deliver significant amounts of genetic information into the DNA of host cells, and are one of the most efficient methods of gene delivery vectors. Human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), and feline immunodeficiency virus (FIV) are examples of lentiviruses.

[0107] In a third aspect of this application, the use of osteoclasts as described in the first aspect in the preparation of medicaments for the prevention and treatment of diseases is provided.

[0108] In some implementations, the disease includes one or more of tumors, bone metastases, and bone hyperplasia.

[0109] In some implementations, the tumor includes a solid tumor.

[0110] In a fourth aspect of this application, a cell-based pharmaceutical composition is provided, comprising osteoclasts as described in the first aspect, and a pharmaceutically acceptable carrier.

[0111] "Pharmaceutical acceptable" means those ligands, materials, compositions, and / or dosage forms that are appropriate for administration to patients within the bounds of reasonable medical judgment and that are commensurate with a reasonable benefit / risk ratio.

[0112] "Pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or medium, such as liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials. As used herein, the term "pharmaceutically acceptable carrier" includes buffers compatible with drug administration, sterile water for injection, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delay agents, and the like. Each carrier must be "pharmaceutically acceptable" in the sense of compatibility with other components in the formulation and harmlessness to the patient.

[0113] In a fifth aspect of this application, a method for preventing and treating a disease is provided, the method comprising administering to a subject an effective dose of osteoclasts as described in the first aspect or a cell-pharmaceutical composition as described in the fourth aspect.

[0114] In some embodiments, the subject is deemed to require administration of the osteoclasts or the cellular drug composition.

[0115] Unless otherwise specified, the term "prevention and control" includes, but is not limited to, interventions such as prevention and treatment of diseases.

[0116] As used in this application, the term "treatment" refers to a therapeutic intervention aimed at reversing, alleviating, improving, suppressing, slowing, or stopping the progression or severity of a condition associated with a disease or symptom. The term "treatment" includes reducing or alleviating at least one side effect or symptom of a disease or symptom. Treatment is generally "effective" if it reduces one or more symptoms or clinical markers. Alternatively, treatment is "effective" if the progression of the disease is reduced or stopped; that is, "treatment" includes not only improvement of symptoms but also the cessation, or at least slowing, of the expected progression or worsening of symptoms in the absence of treatment. Beneficial or desired clinical outcomes include, but are not limited to, reduction of one or more symptoms, reduction of disease severity, stabilization (i.e., non-worsening) of the disease state, delay or slowing of disease progression, improvement or relief of the disease state, and relief (whether partial or complete), whether detectable or undetectable.

[0117] In some implementations, the disease includes one or more of tumors, bone metastases, and bone hyperplasia.

[0118] In some implementations, the tumor includes a solid tumor.

[0119] There are no particular restrictions on the dosage form and administration method of the cell drug composition.

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

[0121] Representative administration methods include, but are not limited to, oral, intratumoral, rectal, parenteral (intravenous, intramuscular, or subcutaneous) injection, and local administration. In some embodiments, the osteoclasts or the cell-pharmaceutical composition are administered via intravenous injection.

[0122] In some embodiments, the osteoclasts or the cell-pharmaceutical composition are administered via multiple doses or a single dose.

[0123] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where conditions are not specified, reference should be made to the guidelines given in this application, or to experimental manuals or conventional conditions in the art, or to the conditions recommended by the manufacturer, or to experimental methods known in the art.

[0124] The reagents and consumables used in the examples were sourced from the following sources:

[0125] Table 1. Reagent and Consumable Information

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

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

[0128] Table 2

[0129] Specifically as follows:

[0130] 1. Isolation of primary mouse cells

[0131] a. Five C57 mice were euthanized by dislocation and immersed in a beaker containing 75% alcohol for 3-5 minutes;

[0132] b. Lay a plastic bag flat inside the biosafety cabinet and place the mice on the plastic bag for the experiment;

[0133] c. Carefully pinch the abdominal skin between the mouse's two hip joints with ophthalmic forceps, carefully cut the skin with ophthalmic scissors, and separate the skin of the two lower limbs. Cut downwards at the ankles and upwards at the hip joints to free the mouse's two lower limbs.

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

[0135] e. Remove the mouse carcass, clean the biosafety cabinet, and put on new gloves;

[0136] f. Take one 5mL sterile syringe, draw up the prepared DMEM, gently insert it into the bone marrow cavity, align it with a sterile 15mL centrifuge tube, and flush the cells out; [It is necessary to cut open both ends of the femur to facilitate flushing]

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

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

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

[0140] Centrifuge at 700g for 5 minutes, discard the supernatant, and obtain BM cell pellet;

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

[0142] l. Take 6.35×10 7 4.35 × 10⁻⁶ cells 7 One cell was placed in two new 50 mL centrifuge tubes, centrifuged at 700 g for 5 min, and the supernatant was discarded;

[0143] m. The cell pellet was resuspended in macrophage culture medium and osteoclast culture medium respectively (see below) for induction culture.

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

[0145] 2.1 Culture medium preparation

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

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

[0148] 2.2 Cell Culture

[0149] Mouse BMDM cells were cultured in 10cm culture dishes, with 10 million cells per dish; 6 dishes were induced to induce osteoclasts and 4 dishes to induce macrophages. (Both cell types were simultaneously seeded in two-well six-well plates, with 1.75 million cells per well, for counting during viral infection; the bottom area of ​​the 10cm dish is approximately 5.7 times that of the six-well plate).

[0150] The culture medium was changed every two days according to the experimental groups.

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

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

[0153] On day 4 of induction culture, macrophages and osteoclasts were infected with Ad5F35-CAR-HER2-CD32a-mCherry. The specific steps are as follows:

[0154] a. The macrophages and osteoclasts in the pre-coated 6-well plates were digested, resuspended, and counted;

[0155] b. The number of cells per well × 5.7 equals the number of cells in a 10cm dish;

[0156] c. Replace the culture medium in the 10cm dish and add Ad5F35-CAR-HER2-CD32a-mCherry at an MOI of 2000. Mix thoroughly and then return to the incubator.

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

[0158] 4. Flow cytometry and cell counting

[0159] When the cells were seeded into 48 wells according to the experimental groups, the cells in each dish were digested and resuspended, and counted separately. The remaining cells were then analyzed by flow cytometry. The detection groups are shown in Table 3 below.

[0160] Table 3

[0161] Note: The abbreviation "WT" stands for wild type (the WT corresponding to "CAR-Osteoclast" above refers to osteoclasts induced by RANKL differentiation, and the WT corresponding to "CAR-BMDM" refers to BMDMs induced by RANKL differentiation); BMDM stands for bone marrow-derived macrophages.

[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 both infected groups did not adhere firmly to the plate, but they were not dead cells. After changing the culture medium and coating 48-well plates before the killing experiment, the cells in all 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, with the isotype control group serving as the negative control group, the G4S positivity rate in the CAR-OC group was 65.68%, which was higher than that in the CAR-BMDM group (63.04%). In this experiment, the G4S detection nonspecificity in the BMDM group was relatively high (35.75%), indicating that the G4S positivity rate in the mouse CAR-BMDM group was lower than the detected value.

[0164] 5. TRAP detection

[0165] After 48 hours (day 6 of induction culture) of mouse BMDM and osteoclasts infected with Ad5F35-CAR-HER2-CD32a-mCherry, the cell induction status was detected by TRAP.

[0166] The specific steps are as follows:

[0167] a. Remove the culture medium from the 12-well plate, add 500 μl of PBS to each well and wash the plate 3 times;

[0168] b. Remove any residual PBS from the well plate, add 500 μl of TRAP fixative (pre-cooled at 4°C), and fix at room temperature for 1 min;

[0169] c. Remove the fixative from each well, add 500 μl ddH2O to wash each well, 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 ddH2O to each well to wash it, then drain the water.

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

[0173] TRAP assays showed that osteoclasts were successfully induced in the mice in this experiment, and adenovirus infection did not affect their differentiation (Figure 3).

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

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

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

[0177] NCI-N87 cells were purchased from Shanghai Binsui Biotechnology Co., Ltd., and 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 table below, and the distribution of the well plates is shown in Figure 7.

[0179] Table 4

[0180] Note: The abbreviation "WT" stands for wild type (the WT corresponding to "CAR-boneclast" above refers to osteoclasts induced by RANKL in Example 1, and the WT corresponding to "CAR-BMDM" refers to BMDMs induced by RANKL in Example 1); BMDM stands for bone marrow-derived macrophages.

[0181] c. After culturing for 6 h and 24 h according to the groups, add one volume of D-fluorescein potassium (0.6 mg / ml) directly to the well plate to be tested, mix well and incubate at 37℃ for 5 min;

[0182] d. Imaging is performed using a small animal live imaging system.

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

[0184] This demonstrates that CAR-OC has a lethal effect on N87-Fluc.

[0185] Furthermore, at E / T ratios of 1:1 and 2:1, the killing efficiency of CAR-OC and CAR-BMDM in killing N87-Fluc cells was close to the upper limit of detection, making it impossible to compare the killing effects of CAR-OC and CAR-BMDM. Therefore, the experiment was further adjusted, and the killing effects of CAR-OC and CAR-BMDM were further compared through Experiment 7 below.

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

[0187] This experiment is similar to Experiment 7 above, with the main difference being the E / T ratio. Specifically:

[0188] a. After digesting the four groups of cells (see Table 5 for grouping details), the cells were plated in 48-well plates according to the experimental grouping, with 100,000 cells per well, and cultured in a carbon dioxide incubator for 24 hours.

[0189] b. Add NCI-N87-Fluc-GFP cells (hereinafter referred to as N87-Fluc) to each well according to the experimental group; change the culture medium daily;

[0190] The ratio of target cells to effector cells is shown in the table below.

[0191] Table 5

[0192] Note: The abbreviation "WT" stands for wild type (the WT corresponding to "CAR-OC" above refers to osteoclasts induced by RANKL differentiation, and the WT corresponding to "CAR-BMDM" above refers to BMDMs induced by RANKL differentiation); BMDM stands for bone marrow-derived macrophages.

[0193] c. After culturing for 48h and 72h according to the groups, add one volume of D-fluorescein potassium (0.6mg / ml) directly to the well plate to be tested, mix well and incubate at 37℃ for 5min;

[0194] d. Imaging is performed using a small animal live imaging system.

[0195] Based on the cell tests in Experiments 6 and 7 above:

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

[0197] This demonstrates that a small number of CAR-OC cells can kill N87-Fluc cells.

[0198] Furthermore, based on the fact that a relatively small number of CAR-OC cells can kill N87-Fluc cells, it is evident that CAR-OC has advantages in treating diseases such as tumors, bone metastases, and bone hyperplasia. On the one hand, using CAR-OC technology to treat diseases can reduce the amount of cells separated from the patient; on the other hand, fewer CAR-OC cells can achieve better killing effects, and the preparation process can be simplified, thus reducing the amount of cells required to achieve the therapeutic effect.

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

[0200] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0201] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. An engineered osteoclast comprising a targeting unit; said targeting unit having at least one or more of the following functions: (i) Specifically recognizes tumor cells; (ii) Specific killing of tumor cells.

2. The osteoclasts as described in claim 1, wherein, The osteoclasts target tumor cells.

3. The osteoclasts as described in claim 1 or 2, wherein, The targeting unit is a chimeric antigen receptor; the chimeric antigen receptor includes an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain.

4. The osteoclast as described in claim 3, wherein, The chimeric antigen receptor satisfies one or more of the following conditions: The antigen-binding domain specifically recognizes tumor antigens; The transmembrane domains are 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 signal transduction domains are derived from a group consisting of FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, CD66d, CD32α, CD3EBRS, CD28CD, CD19, 2B4, DAP12, and DAP10.

5. The osteoclast as described in claim 3 or 4, wherein, The antigen-binding domain satisfies 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 antigens recognized by the antigen-binding domain are selected from the group consisting of HER2, CLDN6, FAP, GPC3, MLSN, uPAR, and PSMA.

6. The osteoclasts according to any one of claims 3-5, wherein, The intracellular signal transduction domain further includes a co-stimulatory signaling domain derived from a 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 ligands.

7. The osteoclasts according to any one of claims 3-6, wherein, The chimeric antigen receptor comprises, in sequence, an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain: Single-chain antibodies, CD8α, and CD32α that specifically recognize HER2.

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

1.

9. The osteoclasts according to any one of claims 1-8, wherein, The chimeric antigen receptor also includes a reporter gene.

10. The osteoclast as described in claim 9, wherein, The reporter gene is a fluorescent reporter gene.

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

12. A method for preparing osteoclasts as described in any one of claims 1-11, comprising: The nucleic acid expressing the target unit was introduced into osteoclasts using a viral vector.

13. The method of claim 12, wherein, The viral vectors are selected from the group consisting of lentiviral vectors, retroviral vectors, adenovirus vectors, and adeno-associated virus vectors.

14. The use of osteoclasts as described in any one of claims 1-11 in the preparation of medicaments for the prevention and treatment of diseases.

15. The application as described in claim 14, wherein, The disease includes one or more of tumors, bone metastases, and bone hyperplasia; optionally, the tumor includes solid tumors.

16. A cell-based drug composition comprising osteoclasts as described in any one of claims 1-11, and a pharmaceutically acceptable carrier.

17. A method for preventing and treating diseases, wherein, The method includes administering to a subject an effective dose of osteoclasts as described in any one of claims 1-11 or the cell drug composition as described in claim 16.

18. The method of claim 17, wherein, The disease includes one or more of tumors, bone metastases, and bone hyperplasia; optionally, the tumor includes solid tumors.

19. The method of claim 17 or 18, wherein, The subject was deemed to require administration of the osteoclasts or the cell-pharmaceutical composition.

20. The method according to any one of claims 17-19, wherein, The dosage form of the cell drug composition is selected from the group consisting of solution, emulsion and colloidal solution.

21. The method according to any one of claims 17-20, wherein, The method satisfies one or more of the following conditions: The osteoclasts or the cell-drug composition are administered via intravenous injection. The administration regimen for the osteoclasts or the cell-pharmaceutical composition is multiple administrations or a single administration.

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