Anti-CD5 nanobodies and their application in in vivo CAR cell therapy

Abstract

The present invention relates to anti-CD5 nanobodies and their use in in vivo CAR cell therapy. Specifically, the present invention provides 10 nanobodies targeting mouse CD5, which have the function of binding to CD5 with high affinity and high specificity, and can be recognized by CD5-positive cells (such as T cells) and internalized into cells, and can be used to specifically target T cells in mice. The nanobodies of the present invention can be coupled with delivery vectors to prepare delivery vectors with immune cell targeting, which are used for research and application of in vivo CAR therapy, etc.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to anti-CD5 nanoantibodies and their application in in vivo CAR cell therapy. Background Art

[0002] Chimeric antigen receptor-modified T cell and NK cell therapy (CAR-T and CAR-NK) has a wide range of therapeutic uses in tumors and autoimmune diseases. The existing technology is to engineer autologous or donor-derived immune cells in vitro and then infuse them back into the patient to exert therapeutic effects. This method faces clinical bottlenecks such as high treatment costs and low accessibility. Generating CAR-modified immune cells directly in the patient's body, that is, in vivo or in situ CAR therapy (such as invivo CAR-T), is a new strategy that is expected to break through the bottleneck of existing technologies.

[0003] In vivo CAR therapy delivers nucleic acids encoding CAR molecules into target immune cells through delivery vectors or delivery systems. When screening in vivo CAR therapy, it is necessary to evaluate in vivo whether the delivery system can accurately deliver CAR molecules (in the form of DNA or mRNA) to target immune cells (such as T cells) in animals. Humanized mice are often used as a model for studying or evaluating the in vivo delivery of CAR molecules, but T cells in humanized mice are usually humanized and reconstructed by intravenous injection of human PBMCs, and the distribution of T cells in their bodies cannot reflect the distribution of their own T cells in normal mice. Therefore, there is an urgent need in the field to develop a delivery system for T cell targeting in normal mice for screening and studying the in vivo efficacy, metabolism, and distribution characteristics of in vivo CAR therapy.

[0004] CD5 is a specific marker on the surface of immune cells such as T cells and NK cells. Anti-CD5 antibodies can be used as targeting elements to couple with delivery vectors to achieve targeted delivery to T cells, etc. Therefore, there is an urgent need in the art to develop antibodies that can specifically target CD5 and be endocytosed by cells as targeting elements of delivery vectors. Summary of the invention

[0005] The purpose of the present invention is to provide a nano antibody and a targeted delivery vector that specifically targets CD5.

[0006] In the first aspect of the present invention, an anti-CD5 nanobody is provided, wherein the anti-CD5 nanobody has one or more sets of complementary determining regions (CDRs) selected from the following groups: (1) CDR1 shown in SEQ ID NO: 14; CDR2 shown in SEQ ID NO: 15; CDR3 shown in SEQ ID NO: 16 or SEQ ID NO: 29; (2) CDR1 shown in SEQ ID NO: 17; CDR2 shown in SEQ ID NO: 18; CDR3 shown in SEQ ID NO: 19; (3) CDR1 shown in SEQ ID NO: 20; CDR2 shown in SEQ ID NO: 21; CDR3 shown in SEQ ID NO: 22; (4) CDR1 shown in SEQ ID NO: 23; CDR2 shown in SEQ ID NO: 24; CDR3 shown in SEQ ID NO: 25; (5) CDR1 shown in SEQ ID NO: 26; CDR2 shown in SEQ ID NO: 27; CDR3 shown in SEQ ID NO: 28; (6) CDR1 shown in SEQ ID NO: 30; CDR2 shown in SEQ ID NO: 31; CDR3 shown in SEQ ID NO: 32 or SEQ ID NO: 36; (7) CDR1 shown in SEQ ID NO: 33; CDR2 shown in SEQ ID NO: 34; CDR3 shown in SEQ ID NO: 35; (8) CDR1 shown in SEQ ID NO: 37; CDR2 shown in SEQ ID NO: 38; CDR3 shown in SEQ ID NO: 39.

[0007] In another preferred embodiment, the CDR1, CDR2 and CDR3 are separated by the framework regions FR1, FR2, FR3 and FR4 of the VHH chain.

[0008] In another preferred example, the amino acid sequence of the VHH chain of the anti-CD5 nanobody is as shown in any one of SEQ ID NOs: 1-10.

[0009] In another preferred example, the VHH chain of the anti-CD5 nanobody comprises an amino acid sequence having a sequence similarity of at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% to any one of the amino acid sequences of SEQ ID NOs: 1-10.

[0010] In another preferred example, the amino acid sequence of the VHH chain of the anti-CD5 nanobody comprises one or more amino acid substitutions, preferably conservative amino acid substitutions, compared with any one of the amino acid sequences in SEQ ID NO: 1-10.

[0011] In another preferred embodiment, any one of the above amino acid sequences also includes a derivative sequence that is optionally subjected to addition, deletion, modification and / or substitution of at least one (such as 1-3, preferably 1-2, more preferably 1) amino acid and can retain the ability to specifically bind to CD5.

[0012] In another preferred embodiment, the anti-CD5 nanobody includes animal-derived antibodies (such as mouse-derived antibodies), humanized antibodies, and chimeric antibodies.

[0013] In another preferred embodiment, the anti-CD5 nanobody has a function selected from the following group: (1) Specific binding to CD5 protein; (2) Specific targeting of CD5-positive cells; (3) It is taken up by CD5-positive cells and enters the cell interior through endocytosis.

[0014] In another preferred embodiment, the CD5-positive cells are T cells, NK cells, or dendritic cells (DC).

[0015] In the second aspect of the present invention, an anti-CD5 multivalent antibody or an antibody against multiple epitopes is provided, wherein the multivalent antibody or the antibody against multiple epitopes comprises at least one antibody element against a CD5 epitope, and the antibody element is the anti-CD5 nanobody described in the first aspect of the present invention.

[0016] In another preferred embodiment, the anti-CD5 multivalent antibody or antibody targeting multiple epitopes includes one or more anti-CD5 nanobodies.

[0017] In another preferred embodiment, the anti-CD5 multivalent antibody includes a monomer, a bivalent body (bivalent antibody), a tetravalent body (tetravalent antibody), and / or a multivalent body (multivalent antibody).

[0018] In another preferred embodiment, the anti-CD5 multivalent antibody comprises one or more VHH chains having an amino acid sequence as shown in any one of SEQ ID NOs: 1-10.

[0019] In another preferred embodiment, the anti-CD5 multivalent antibody comprises two VHH chains having the amino acid sequences shown in SEQ ID NOs: 1-10.

[0020] In another preferred embodiment, the anti-CD5 multivalent antibody includes humanized antibodies, animal-derived antibodies, and chimeric antibodies.

[0021] In the third aspect of the present invention, a recombinant protein is provided, wherein the recombinant protein has: (i) the anti-CD5 nanobody described in the first aspect of the invention, or the anti-CD5 multivalent antibody or antibody against multiple epitopes described in the second aspect of the invention; and (ii) optionally a tag sequence to facilitate expression and / or purification.

[0022] In another preferred embodiment, the tag sequence includes a Flag tag, an Fc tag, an HA tag and a His tag.

[0023] In another preferred embodiment, the tag sequence is a His tag.

[0024] In another preferred embodiment, the recombinant protein specifically binds to CD5 protein.

[0025] In a fourth aspect of the present invention, a targeted delivery vector for CD5-positive cells is provided, the targeted delivery vector comprising: (1) a delivery vector; and (2) an antibody coupled to the surface of the delivery vector; Wherein, the antibody is the anti-CD5 nanobody as described in the first aspect of the present invention, or the anti-CD5 multivalent antibody or antibody against multiple epitopes as described in the second aspect of the present invention.

[0026] In another preferred embodiment, the delivery carrier includes: lipid nanoparticles (LNP), polymer nanoparticles, nanomicelles, gold nanoparticles, and liposomes.

[0027] In another preferred embodiment, the targeted delivery vector is an antibody-LNP coupled vector.

[0028] In another preferred embodiment, the targeted delivery vector targets CD5 positive cells.

[0029] In another preferred embodiment, the CD5-positive cells are T cells, NK cells, or dendritic cells (DC).

[0030] In a fifth aspect of the present invention, a pharmaceutical composition is provided, comprising: (i) the targeted delivery vector according to the fourth aspect of the present invention; (ii) a biologically active substance encapsulated in the targeted delivery vehicle; and (iii) a pharmaceutically acceptable carrier, excipient or diluent.

[0031] In another preferred embodiment, the bioactive substance is selected from the following group: nucleic acid, small molecule compound, protein, or a combination thereof.

[0032] In another preferred embodiment, the bioactive substance is a bioactive substance used for in vivo generation of CAR-T cells and / or CAR-NK cell therapy.

[0033] In another preferred embodiment, the bioactive substance is nucleic acid.

[0034] In another preferred embodiment, the nucleic acid is RNA, DNA, antisense nucleic acid, aptamer, ribozyme, immunostimulatory nucleic acid or PNA.

[0035] In another preferred embodiment, the RNA is linear RNA or circular RNA.

[0036] In another preferred embodiment, the RNA includes: mRNA, siRNA, saRNA, tRNA, snRNA, microRNA inhibitor, microRNA activator or shRNA.

[0037] In another preferred embodiment, the mRNA is mRNA encoding CAR.

[0038] In another preferred embodiment, the CAR targets disease-related targets, including but not limited to: CD19, BCMA, CD20, CD22, CD33, CD38, CD70, CD123, HER2, MSLN, EGFR, CLDN18.2, GPC3, and GD2.

[0039] In another preferred embodiment, the pharmaceutical composition is used for targeted delivery of the biologically active substance into cells.

[0040] In another preferred embodiment, the cells are CD5 positive cells.

[0041] In another preferred embodiment, the cell is an immune cell, preferably a T cell.

[0042] In the sixth aspect of the present invention, an antibody-drug conjugate is provided, wherein the antibody-drug conjugate comprises: (a) the Nanobody as described in the first aspect of the invention, the multivalent antibody or antibody against multiple epitopes as described in the second aspect of the invention, or the recombinant protein as described in the third aspect of the invention; and (b) a conjugated moiety conjugated to the antibody portion, wherein the conjugated moiety is selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

[0043] In another preferred embodiment, the antibody portion and the coupling portion are coupled via a chemical bond or a linker.

[0044] In the seventh aspect of the present invention, a chimeric antigen receptor (CAR) is provided, wherein the CAR contains an extracellular domain, and the extracellular domain comprises the anti-CD5 nanobody as described in the first aspect of the present invention, or the anti-CD5 multivalent antibody as described in the second aspect of the present invention.

[0045] In another preferred embodiment, the extracellular domain also includes a signal peptide.

[0046] In another preferred embodiment, the extracellular domain also includes other exogenous proteins.

[0047] In another preferred embodiment, the CAR has a structure shown in formula (I): L-Nb-H-TM-C-CD3ζ (I) In the formula, The "-" is a connecting peptide or peptide bond; L is none or a signal peptide sequence; Nb is the antigen binding domain that specifically binds to CD5; H is none or hinge region; TM is the transmembrane domain; C is the co-stimulatory signaling domain; CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ (including wild type, or mutant / modified form thereof).

[0048] In another preferred embodiment, the L is selected from the signal peptides of the following proteins: CD28, 4-1BB, GM-CSF, CD3, CD8a or a combination thereof.

[0049] In another preferred embodiment, the sequence of Nb is shown in any one of SEQ ID NOs: 1-10.

[0050] In another preferred embodiment, the H is selected from the hinge region of the following histones: CD8, CD28, CD137, IgG, or a combination thereof.

[0051] In another preferred embodiment, the C is selected from the co-stimulatory domains of the following proteins: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD-1, Dap10, LIGHT, NKG2C, B7-H3, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, OX40L, 2B4, TLR, or their mutants / modified forms, or their combinations.

[0052] In the eighth aspect of the present invention, a polynucleotide is provided, which encodes a protein selected from the following group: the nanobody described in the first aspect of the present invention, or the multivalent antibody or antibody against multiple epitopes described in the second aspect of the present invention, the recombinant protein described in the third aspect of the present invention, or the CAR described in the seventh aspect of the present invention.

[0053] In the ninth aspect of the present invention, an expression vector is provided, wherein the expression vector contains the polynucleotide described in the eighth aspect of the present invention.

[0054] In another preferred embodiment, the expression vector is selected from the following group: DNA, RNA, viral vector, plasmid, transposon, other gene transfer systems, or a combination thereof.

[0055] In another preferred embodiment, the expression vector is a lentiviral vector.

[0056] In the tenth aspect of the present invention, a host cell is provided, which contains the expression vector described in the ninth aspect of the present invention, or the polynucleotide described in the eighth aspect of the present invention is integrated into its genome, or expresses the nanobody described in the first aspect of the present invention, the multivalent antibody or antibody against multiple epitopes described in the second aspect of the present invention, the recombinant protein described in the third aspect of the present invention, the pharmaceutical composition described in the fifth aspect of the present invention, or the CAR described in the seventh aspect of the present invention.

[0057] In another preferred embodiment, the cell is an isolated cell, and / or the cell is a genetically engineered cell.

[0058] In another preferred embodiment, the cell is a mammalian cell.

[0059] In another preferred embodiment, the cell is a T cell.

[0060] In another preferred embodiment, the host cell is an engineered immune cell.

[0061] In another preferred embodiment, the engineered immune cells include autologous or allogeneic αβT cells, γδT cells, NKT cells, NK cells, or a combination thereof.

[0062] In another preferred embodiment, the engineered immune cells are CAR-T cells.

[0063] In the eleventh aspect of the present invention, a kit is provided, wherein the kit contains the polynucleotide described in the eighth aspect of the present invention, the CAR described in the seventh aspect of the present invention, or the vector described in the ninth aspect of the present invention.

[0064] In another preferred embodiment, the kit is used to prepare immune cells expressing the CAR described in the seventh aspect of the present invention.

[0065] In the twelfth aspect of the present invention, there is provided the use of the nanobody described in the first aspect of the present invention, the multivalent antibody or the antibody against multiple epitopes described in the second aspect of the present invention, the recombinant protein described in the third aspect of the present invention, the antibody-drug conjugate described in the sixth aspect of the present invention, or the immune cell expressing the CAR described in the seventh aspect of the present invention for the preparation of a medicament for preventing and / or treating CD5-related diseases or conditions.

[0066] In another preferred embodiment, the drug contains a pharmaceutically acceptable carrier.

[0067] In another preferred embodiment, the disease or condition associated with CD5 is selected from the following group: immune-related diseases, tumors, or a combination thereof.

[0068] In the thirteenth aspect of the present invention, there is provided use of the targeting vector described in the fourth aspect of the present invention or the pharmaceutical composition described in the fifth aspect of the present invention for preparing a drug for preventing and / or treating a disease.

[0069] In another preferred embodiment, the disease is a disease suitable for CAR-T therapy, including but not limited to: tumor, autoimmune disease, or a combination thereof.

[0070] In another preferred embodiment, the tumor includes: a hematological tumor, a solid tumor, or a combination thereof.

[0071] In the fourteenth aspect of the present invention, a method for treating a disease is provided, the method comprising administering the pharmaceutical composition according to the fifth aspect of the present invention to a subject in need of treatment.

[0072] In another preferred embodiment, the subject includes a mammal, such as a human or a mouse.

[0073] In another preferred embodiment, the disease includes a disease suitable for CAR-T therapy.

[0074] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

[0075] The beneficial effects of the present invention include: (1) The anti-CD5 nanobody of the present invention has high affinity binding to CD5.

[0076] (2) The anti-CD5 nanobody of the present invention can specifically bind to positive cells expressing CD5, but not to cells that do not express CD5, and has high specificity.

[0077] (3) The anti-CD5 nanobody of the present invention can be efficiently internalized by T cells and can be used as a targeting element in a delivery vector.

[0078] (4) The anti-CD5 nanoantibody of the present invention has a simple structure and is easy to prepare, and has broad application prospects in tumor treatment, especially in the field of in vivo CAR therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1 The results of SDS-PAGE (A) and SEC-HPLC (B) of the antigen Mouse CD5-8XHis recombinant protein are shown.

[0080] Figure 2 The construction of yeast display library is shown. A is an agarose gel electrophoresis of RNA; B is a 500 bp fragment recovered by agarose electrophoresis of RT-PCR one-step PCR product; C is the linearization of yeast display vector pYDisplay.

[0081] Figure 3 The first (A) and second (B) rounds of sorting of the yeast display library are shown.

[0082] Figure 4 The ELISA test results of the binding activity of the first group of antibodies to Mouse-CD5 are shown.

[0083] Figure 5 The results of FACS detection of the binding activity of the first group of antibodies to EL4 are shown.

[0084] Figure 6 The results of FACS detection of the binding activity of the second group of antibodies to EL4 are shown.

[0085] Figure 7 The results of the candidate antibody endocytosis assay are shown.

[0086] Figure 8 The results of the efficiency test of candidate antibody-coupled LNP transfection into mouse T cells are shown. DETAILED DESCRIPTION

[0087] After extensive and in-depth research, the inventors screened and identified a series of nanobodies targeting mouse CD5, and obtained 10 nanobodies with high affinity and high specificity, whose sequences are shown in SEQ ID NO: 1-10. The nanobodies of the present invention can specifically bind to CD5, and can be recognized by CD5-positive cells (such as T cells) and internalized into the cell body, so they can be used to specifically target T cells in mice. The nanobodies of the present invention can be coupled with delivery vectors to prepare delivery vectors with immune cell targeting, which are used for research and application of in vivo CAR therapy, etc.

[0088] The present invention has been completed on this basis.

[0089] the term In order to more easily understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms should have the meaning given below. Other definitions are set forth throughout the application.

[0090] The term "about" can refer to a value or composition that is within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0091] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0092] Sequence identity is determined by comparing two aligned sequences along a predetermined comparison window (which can be 50%, 60%, 70%, 80%, 90%, 95% or 100% of the length of the reference nucleotide sequence or protein) and determining the number of positions at which identical residues occur. Typically, this is expressed as a percentage. The measurement of sequence identity of nucleotide sequences is a method well known to those skilled in the art.

[0093] Antibody As used herein, the term "antibody" refers to immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds.

[0094] As used herein, the terms "single domain antibody (VHH)" and "nanobody" have the same meaning, referring to cloning the variable region of the antibody heavy chain to construct a single domain antibody (VHH) consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete functions. Usually, an antibody naturally lacking the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single domain antibody (VHH) consisting of only one heavy chain variable region.

[0095] The term "antigen-binding fragment of an antibody" (or simply "antibody fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., CD5). It has been shown that fragments of a full-length antibody can be used to perform the antigen-binding function of an antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment of an antibody" include;

[0096] (i) Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) F(ab') 2 Fragment, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge on the hinge region; (iii) Fd fragment consisting of VH and CH1 domains; (iv) The Fv fragment consists of the VH and VL domains of a single arm of an antibody.

[0097] Fv antibody contains the variable region of antibody heavy chain and variable region of light chain, but no constant region, and has the smallest antibody fragment of all antigen binding sites. Generally, Fv antibody also includes a polypeptide linker between VH and VL domains, and can form the structure required for antigen binding.

[0098] The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody that are primarily responsible for antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat EA et al. (1991) Sequences of proteins of immunological interest. NIH Publication, No. 91-3242).

[0099] The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds (e.g., a specific site on a CD5 molecule). An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-consecutive amino acids in a unique spatial conformation.

[0100] The terms "specific binding", "selective binding", "selectively binds" and "specifically binds" refer to the binding of an antibody to a predetermined epitope on an antigen. -7 M, for example, less than about 10 -8 M, 10 -9 M or 10 -10 Binds with an affinity (KD) of M or less.

[0101] As used herein, the term "antigenic determinant" refers to a discrete three-dimensional site on an antigen that is recognized by the antibodies or antigen-binding fragments of the present invention.

[0102] The present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.

[0103] In the present invention, the antibody of the present invention also includes its conservative variants, which refers to a polypeptide formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids of similar or similar properties compared to the amino acid sequence of the antibody of the present invention. These conservative variant polypeptides are preferably produced by amino acid substitution according to the following Table 1.

[0104] Table 1

[0105] Anti-CD5 Nanobody As used herein, the term "CD5" generally refers to natural or recombinant CD5 proteins, including human CD5 proteins as well as non-human homologs of CD5, including mouse CD5.

[0106] As used herein, the terms "Nanobody of the present invention", "single domain antibody of the present invention", and "anti-CD5 Nanobody of the present invention" are used interchangeably and all refer to the Nanobody of the present invention with high specificity and high affinity for CD5.

[0107] Compared with traditional monoclonal antibodies, the nanoantibodies of the present invention have small molecular weight, strong penetration, high sensitivity, strong specificity and good stability; when they enter the body, they can efficiently penetrate cells to quickly capture antigens to achieve targeting purposes.

[0108] In the present invention, the anti-CD5 nanobody includes a monomer, a bivalent body (bivalent antibody), a tetravalent body (tetravalent antibody), and / or a multivalent body (multivalent antibody).

[0109] In a preferred example of the present invention, the amino acid sequence of the VHH chain of the anti-CD5 nanobody is selected from one or more of SEQ ID NOs: 1-10; preferably selected from one or more of SEQ ID NOs: 1-7.

[0110] The preferred Nanobody names, VHH sequence numbers and corresponding CDR region sequence numbers of the present invention are shown in Table 2: Table 2

[0111] Targeted delivery vehicle The present invention also provides a targeted delivery vector using anti-CD5 nanoantibodies as targeting elements, which can achieve targeted delivery to CD5-positive cells and be used for applications such as in vivo CAR-T therapy.

[0112] Specifically, the targeted delivery vector of the present invention comprises: (1) a delivery vector; and (2) an antibody coupled to the surface of the delivery vector; wherein the antibody is the anti-CD5 nanobody, or the anti-CD5 multivalent antibody or an antibody against multiple epitopes described in the present invention.

[0113] As used herein, the term "delivery vector" refers to a tool or system for effectively delivering drugs, genes or other bioactive substances to specific target cells. In the present invention, the type of delivery vector is not limited, including: lipid-based carriers (e.g., lipid nanoparticles LNP), polymer-based carriers (e.g., cationic polymer nanoparticles), inorganic carriers (e.g., gold nanoparticles), etc.

[0114] The preferred carrier is a lipid nanoparticle. As used herein, the terms "lipid nanoparticle", "lipid nanoparticle" or "LNP" are used interchangeably and refer to particles with nanoscale size formed by self-assembly of biocompatible lipid molecules, with a diameter of about 5 to 500 nm. LNP is usually composed of a variety of functional lipid molecules, including but not limited to: ionizable lipids, electrostatically binding to the carried substance; auxiliary lipids, regulating the stability and release rate of LNP, and improving biocompatibility; PEGylated lipids (PEG-lipids), prolonging the circulation time of LNP in the blood, reducing the recognition and clearance of the reticuloendothelial system.

[0115] In the present invention, the components and preparation methods of LNP are not limited, and common components include: ionizable lipids, DSPC (distearoylphosphatidylcholine), DSPE-PEG (distearoylphosphatidylethanolamine-polyethylene glycol), DMG-PEG (dimyristoylglycerol-polyethylene glycol), and cholesterol. Ionizable lipids are positively charged under acidic conditions and can bind to negatively charged nucleic acid molecules to assist phospholipids DSPC, DOPC, POPC, DOPE, etc., to form stable lipid nanoparticles with cholesterol and PEG lipids.

[0116] The delivery vector of the present invention is used to load or encapsulate "cargo", which is usually a biologically active substance with a therapeutic effect. As used herein, the term "biologically active substance" includes, but is not limited to: RNA (mRNA, rRNA, circRNA, siRNA, saRNA, tRNA, snRNA, antagomir, microRNA inhibitors, microRNA activators or shRNA, etc.), DNA (including plasmids), natural and synthetic oligonucleotides (including antisense oligonucleotides, interfering RNA and small interfering RNA), nucleoproteins, peptides, nucleic acids, ribozymes, aptamers, immunostimulatory nucleic acids or PNA, DNA-containing nucleoproteins, etc. In some embodiments, the biologically active substance can be mixed with an adjuvant.

[0117] In the present invention, the bioactive substance may be mRNA for producing a target polypeptide or protein in vivo. In one embodiment of the present invention, the bioactive substance is mRNA for producing CAR in vivo.

[0118] As used herein, the term "CAR" refers to a chimeric antigen receptor, the basic structure of which includes an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain. The extracellular antigen recognition domain is usually composed of a derivative of a monoclonal antibody fragment or an antibody-like molecule, which can specifically recognize and bind to specific antigens on the surface of target cells, such as cancer-associated antigens on the surface of tumor cells. This feature enables CAR-T cells to accurately find target cells. The transmembrane domain serves to connect the extracellular antigen recognition domain and the intracellular signaling domain, ensuring that the signal can be transmitted from the extracellular to the intracellular, triggering subsequent cellular responses. The intracellular signaling domain contains one or more immunoreceptor tyrosine activation motifs (ITAMs). When the antigen binds to the extracellular domain, it triggers the phosphorylation of ITAMs, thereby initiating a series of signal transduction pathways, activating the function of T cells, causing T cells to proliferate, release cytotoxic factors, etc., and ultimately leading to the death of target cells.

[0119] The target targeted by the CAR of the present invention is not limited and can be any disease-related target known in the art. Common targets include: CD19, BCMA, CD20, CD22, CD33, CD38, CD70, CD123, HER2, MSLN, EGFR, CLDN18.2, GPC3, GD2, but are not limited thereto. The structure of the CAR of the present invention is not limited and can be a first generation, second generation, third generation or fourth generation CAR. The design or selection of CAR is routinely mastered by those skilled in the art.

[0120] The targeted delivery vector of the present invention specifically targets CD5 positive cells, including immune cells such as T cells and NK cells, and delivers the cargo into the cell through endocytosis. As used herein, the term "endocytosis" refers to the process in which the antibody binds to a specific antigen on the cell surface, and the cell is prompted to encapsulate and internalize the antigen and the antibody bound thereto into the cell through the cell's intrinsic mechanism. After the anti-CD5 antibody of the present invention binds to CD5 on the cell surface to form an antigen-antibody complex, the cell will initiate a series of signal transduction and cytoskeleton rearrangement processes, causing the cell membrane to partially invaginate, and eventually encapsulate the antigen and antibody in a vesicle, separate from the cell surface and enter the cytoplasm. When the anti-CD5 antibody is coupled to the delivery vector, the delivery vector and the cargo inside it can be delivered into the target cell through endocytosis. In a preferred embodiment, the target cells of the present invention are CD5 positive cells, especially T cells and NK cells.

[0121] Antibody-drug conjugates (ADCs) The present invention also provides an antibody-drug conjugate (ADC) based on the nanobody of the present invention.

[0122] Typically, the antibody-drug conjugate comprises the antibody and an effector molecule, wherein the antibody is coupled to the effector molecule, and preferably chemically coupled. The effector molecule is preferably a drug with therapeutic activity. In addition, the effector molecule may be one or more of a toxic protein, a chemotherapeutic drug, a small molecule drug or a radionuclide.

[0123] Certain residues on antibodies (such as Cys or Lys, etc.) are used to connect to a variety of functional groups, including imaging agents (such as chromophores and fluorescent groups), diagnostic agents (such as MRI contrast agents and radioisotopes), stabilizers (such as ethylene glycol polymers) and therapeutic agents. Antibodies can be coupled to functional agents to form antibody-functional agent conjugates. Functional agents (such as drugs, detection agents, stabilizers) are coupled (covalently linked) to antibodies. Functional agents can be directly or indirectly connected to antibodies through linkers.

[0124] The drug can be any cytotoxic, cytostatic or immunosuppressive drug. In an embodiment, a linker connects the antibody and the drug, and the drug has a functional group that can form a bond with the linker. For example, the drug can have an amino, carboxyl, sulfhydryl, hydroxyl, or keto group that can form a bond with the linker. In the case where the drug is directly connected to the linker, the drug has a reactive group before being connected to the antibody.

[0125] Useful drug classes include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, etc. In the present invention, drug-linkers can be used to form ADCs in a simple step. In other embodiments, bifunctional linker compounds can be used to form ADCs in a two-step or multi-step method. For example, a cysteine ​​residue reacts with a reactive portion of a linker in a first step, and in a subsequent step, a functional group on the linker reacts with a drug to form an ADC.

[0126] application The present invention provides an application of anti-CD5 nano antibodies for preparing a composition for treating a disease. In a preferred embodiment, the composition is a pharmaceutical composition, which contains the above-mentioned anti-CD5 nano antibodies or derivatives, including fusion proteins, ADCs or corresponding CAR-T cells, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value may vary with the properties of the formulated substance and the condition to be treated. The prepared pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or topical administration.

[0127] The anti-CD5 nanoantibodies and their derivatives of the present invention can be directly used to bind to CD5 protein molecules, and thus can be used to prevent and treat CD5-related diseases. CD5-related diseases include, but are not limited to, autoimmune diseases or tumors. In addition, other therapeutic agents can also be used simultaneously.

[0128] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned nano antibodies of the present invention (or their conjugates, derivatives, etc.) and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram / kg body weight to about 5 mg / kg body weight per day. In addition, the polypeptide of the present invention can also be used with other therapeutic agents.

[0129] When using a pharmaceutical composition, a safe and effective amount of the pharmaceutical composition is administered to a mammal, wherein the safe and effective amount is usually at least about 10 micrograms / kg body weight, and in most cases does not exceed about 50 milligrams / kg body weight, preferably the dosage is about 10 micrograms / kg body weight to about 20 milligrams / kg body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health status, which are all within the skill range of skilled physicians.

[0130] The antibody of the present invention can also be used as a targeting element to prepare a targeted delivery carrier for delivering biologically active substances such as drugs to CD5 positive cells for gene therapy or cell therapy. In a preferred embodiment, the delivered biologically active substance can be a nucleic acid molecule encoding CAR, such as mRNA encoding CAR, especially circular mRNA, for generating chimeric antigen receptor T cell immunotherapy (in vivo CAR-T) in vivo, etc.

[0131] Unlike traditional CAR-T therapy, in vivo CAR-T therapy does not require the extraction of T cells from the patient for in vitro transformation and amplification, but directly converts T cells into CAR-T cells with specific targeting in the patient. This process can be achieved by introducing the gene encoding CAR into CD5-positive cells using the targeted delivery vector of the present invention. The delivery vector containing anti-CD5 nano antibodies of the present invention is particularly suitable for studying T cell targeting systems in normal mice, and is used to screen and study the in vivo efficacy, metabolism and distribution characteristics of in vivo CAR therapy.

[0132] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually performed under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and fractions are weight percentages and weight fractions.

[0133] Example 1: Screening of anti-CD5 antibodies 1. Alpaca Immunization Mouse CD5-8XHis recombinant protein was used as an antigen to immunize two alpacas. The results of SDS-PAGE and SEC-HPLC detection of Mouse CD5-8XHis recombinant protein are shown in Figure 1 , showing that the protein purity is >95% and can be used for immunization and screening.

[0134] The specific immunization scheme is shown in Table 3, where the adjuvant is Gerbu: Table 3 Alpaca immunization program

[0135] 2. Serum titer detection After the immunization, 5 mL of alpaca peripheral blood was collected, and the serum was separated for ELISA immunovalence detection. The method is as follows: 1ug / ml Mouse-CD5-His was used to coat the ELISA plate, overnight at 4°C; the gradient serum was diluted, the initial dilution was 1:1K, 5-fold gradient dilution was used for a total of 7 points, 100ul / well was added to the ELISA plate for incubation, and after washing the plate, 1:50000 diluted Goat antillama IgG (H+L) was added as the secondary antibody solution. After the incubation was completed, the stop solution was added, the plate was washed, the color was developed, and the detection was performed using an ELISA reader.

[0136] The results are shown in Tables 4 and 5. The results showed that the immune titers of the two alpacas were above 1:64K, meeting the requirements for library construction.

[0137] Table 4

[0138] Table 5

[0139] At the same time, FACS was used to detect the binding activity of serum to CD5-positive cells EL4, and MC38 cells that do not express CD5 were used as control cells, Rat-anti-mCD5 (SEQ ID NO: 40) was used as a positive control antibody, and its secondary antibody was Anti-rat-lgG-FITC.

[0140] The test results are shown in Table 6.

[0141] Table 6

[0142] The FACS results showed that the triple-immune serum could bind to EL4 cells, and blood sampling could be arranged for library construction.

[0143] 3. Construction of antibody display library PBMC cells were isolated from the peripheral blood of the alpaca after the above immunization, RNA was extracted, and after reverse transcription into cDNA, the VHH sequence was amplified from the sample and cloned into the yeast display vector, and the EYB100 competent cells were electroporated to construct a single domain antibody yeast display library, such as Figure 2 shown.

[0144] 4. Antibody display library selection Mouse CD5-8XHis recombinant protein was modified with Biotin. After the antibody display library was induced, Biotin-protein was first used for magnetic separation and enrichment. The results of the first and second rounds of separation are shown in Figure 2. Figure 3The enriched display library is further sorted using a sorting flow cytometer to obtain monoclonal cells that can recognize the target protein, and the candidate clones are sequenced, and the amino acid sequences of the candidate single-domain antibodies obtained by sequencing are compared.

[0145] A total of 13 different antibody sequences were obtained and divided into two batches for functional testing. The first group contained 9 antibodies: 3-C1, 4-E6, 4-F12, 5-F10, 3-F1, 4-C8, 5-D3, 4-D3, and 3-G8; the second group contained 4 antibodies: 6-C7, 6-E10, 6-G7, and 8-C6.

[0146] The candidate antibody sequences are as follows, where the CDR region sequences are underlined.

[0147] > 3-F1 (SEQ ID NO: 1) MGWSCIILFLVATATGVHSKAQPAMAQVQLVESGGGLVQPGGSLRLSCAASGFTLD YYAIG WFRQAPGKEREGVS CMSSSGGSTNYADSVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA VYSIGDPGAYFCSGHIPYDY WGQGTQVTVSSEPKTPKPGHEG >6-C7 (SEQ ID NO: 2) MGWSCIILFLVATATGVHSKAQPAMAQLQLVESGGGLVQPGGSLRLSCAGSGFSLD DYA WFRRAPGKEREGVS CISSNGRATNYADFVRG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA VYSLGDPGAYFCSGYVPYDY WGQGTQVTVSSEPKTPKPGHEG >4-E6 (SEQ ID NO: 3) MGWSCIILFLVATATGVHSKAQPAMAQLQLVESGGGLVQPGGSLRLSCTASESISN IYG WYRQAPGKEREWVA YITTKGGQYYTDSVKG RFTIARDDAKNTMYLQMANLKPEDTAVYYCST DQ WGQGTQVIVSSEPKTPKPGHEG >6-G7 (SEQ ID NO: 4) MGWSCIILFLVATATGVHSKAQPAMAQVQLVESGGGLVQPGGSLRLSCVASGPFFA ISIMR WYRQAPGKQRELVA AITNDGSTNYADSVED RFSISRDNAKKTLYLQMNDLKPEDTAVYYCNE ASPDWTNIKAYDF WGQGTQVRVSSEPKTPKPGHEG >3-C1 (SEQ ID NO: 5) MGWSCIILFLVATATGVHSKAQPAMAQVQLVESGGGLVQPGGSLRLSCAASGFTLD YYAIG WFRQAPGKEREGVS CMSSSGGSTNYADSVKG RFTISRDNAKNTVYLFMNCLKPEDTAVYYCAA VYSIGDLGAYFCSGYVPYDY WGQGTQVAVSSEPKTPKPGHEG >4-C8 (SEQ ID NO: 6) MGWSCIILFLVATATGVHSKAQPAMAQLQLVESGGGLAQPGGSLRISCAASLNIFA DTNLH WYRQAPGNQREWVA TMVPSGVAMYADPVRG RFSVSSDSAKKTAFLQMNSLRPEDTAVYYCAT QUR WGQGTQVTVSSEPKTPKPGHEG >5-F10 (SEQ ID NO: 7) MGWSCIILFLVATATGVHSKAQPAMAQVQLVESGGGSVQPGGSLKLSCAASGSIFS INAMN WYRQAPGKERELVA AISSGGSTYYADSVKG RFTISRDNAKNSLYLQMNSLKPEDTAMYYCAS PDGA WGQGTQVTVSSEPKTPKPGHEG >4-D3 (SEQ ID NO: 8) MGWSCIILFLVATATGVHSKAQPAMAQVQLVESGGGLVQPGGSLTLSCVASGFTFS SYAMS WYRQAPGKEREMVA FISNTGGNTYYPDSMKG RFTISRDNAKSTVYLQMNSLKPEDTAVYRCAC VNGV WGQGTQVTVPSEPKTPKPGHEG >5-D3 (SEQ ID NO: 9) MGWSCIILFLVATATGVHSKAQPAMAQVQLVESGGGLVQAGGSLRLSCAASGRTFS INAMN WYRQAPGKERELVA AISSGGSTYYADSVKG RFTISRDNAKNTLYLQMNSLKPEDTAMYYCAA QUR WGQGTQVTVSSEPKTPKPGHEG >4-F12 (SEQ ID NO: 10) MGWSCIILFLVATATGVHSKAQPAMAQVQLVESGGGLVQPGGSLTLSCVAPRGFIQ SNSIN WYHEAPGKEREWVA WIAPNGYTNYADAVLH HFTISRDNSKNTIYLEMDDLKPEDTGMYRCAS PSGI WGQGTQVTVSSEPKTPKPGHEG >8-C6 (SEQ ID NO: 11) MGWSCIILFLVATATGVHSKAQPAMAQLQLVESGGGLVQAGGSLKLSCAASKNIFN FATMA WYRQAPGKERELVA LITFRGGTNYADSVKG RFTISRDNARNMVYMQMNSLKPEDTAVYCNQ KLGTRDD WGQGTQVTVSSEPKTPKPGHEG >6-E10 (SEQ ID NO: 12) MGWSCIILFLVATATGVHSKAQPAMAQVQLVESGGGLVQPGGSLRLSCTASGMQFS IYDM WYRLAPGKEREWVG SVSFDGTTFYADSVKG RFTISRDSAKSTMYLQMSSLKADDTAVYLCST LqCy WGQGTQVTVSLEPKTPKPGHEG >3-G8 (SEQ ID NO: 13) MGWSCIILFLVATATGVHSKAQPAMAEVQLVESWGGLVQAGGSLRLSCAASGSIFN IYGIN WYRQAPGKQREFIA GITSDGSTTYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAAYRCSD PRGI WGQWTQVTVSSEPKTPKPGHEG Example 2: Antibody Function Detection The functional activity of the two groups of antibodies screened above was tested, and the positive control (PC) used was a mouse anti-CD5 antibody, the sequence of which is as follows: MGWSCIILFLVATATGVHSNIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLRWMGWINTHTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCTRRGYDW YFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFHHKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLDYEDMGIYYCQQYDE SPWTFGGGTKLEMKEPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 40) 1. The first group of antibody function tests (1) Supernatant detection For the first group of antibody construction vector expression antibody verification: construct pcDNA34-Fc (Human IgG1Fc tag) eukaryotic expression vector, transiently transfect 293F cells, collect the culture supernatant, and use ELISA to detect the binding of antibodies and MouseCD5 recombinant protein in the transfection supernatant. Coat the ELISA plate with 1ug / ml Mouse-CD5-His at 4°C overnight; after washing and blocking, add 100ul of transfection supernatant to each well as the primary antibody, wash after incubation; add Human IgG-Fc antibody diluted 1:1000 as the secondary antibody, 100ul / well, wash after incubation, develop color, and read the OD value.

[0148] The ELISA results are shown in Table 7.

[0149] Table 7

[0150] At the same time, FACS was used to detect the binding of antibodies to EL4 and MC38 in the transfection supernatant. The test results are shown in Table 8.

[0151] Table 8

[0152] The above results show that all the candidate antibodies tested can bind to EL4 cells expressing CD5, and 3-G8 and MC38 have non-specific binding. The remaining 8 antibodies except 3-G8 were subsequently purified and tested.

[0153] (2) Purified antibody detection The eukaryotic expression vector of the candidate antibody was transiently transfected into 293F cells, and the recombinant antibody was separated and purified by affinity chromatography. The purity of the recombinant antibody was analyzed by SDS-PAGE. The target recombinant protein was diluted to 1 μg / mL, coated on the ELISA plate, and the candidate was gradiently diluted. The ELISA experiment was performed, and a curve was drawn with the candidate antibody concentration as the horizontal axis and OD450 as the vertical axis. The results are shown in Figure 4 and as shown in Table 9.

[0154] Table 9

[0155] The EC50 of the binding activity of the purified antibody to EL4 cells was detected by FACS. Figure 5 As shown in Table 10.

[0156] Table 10

[0157] Among them, the binding activities of 3-C1, 4-E6, 3-F1, and 4-C8 antibodies were comparable to or better than the positive control antibody, showing excellent binding activity.

[0158] 2. Second set of antibody function tests For the second group of antibodies, ELISA or FACS detection was performed directly after purification, and the detection method was the same as above.

[0159] The binding activity of 6-C7, 6-E10, 6-G7 and 8-C6 to Mouse CD5 was detected by ELISA, and the results are shown in Table 11. Candidate antibodies 6-C7, 6-E10 and 6-G7 have strong binding activity.

[0160] Table 11

[0161] FACS detected the EC50 of 6-C7, 6-G7 and EL4 binding. The results are as follows Figure 6 As shown in Table 12. 6-C7 and 6-G7 have strong binding activity with EL4.

[0162] Table 12

[0163] Example 3: Internalization assay of candidate antibodies This example detects the efficiency of candidate antibodies being internalized by EL4 cells, and the detection steps are as follows: Add 3 × 10 5 EL4 cells, add 10ug / ml, 100ul / well candidate antibody, incubate at 4℃ for 30min; then centrifuge at 4℃, 800xg for 3min, remove the supernatant containing antibody, and wash the cells 3 times with pre-cooled 2% FBS. The cells were evenly divided into 2 groups, incubated at 4℃ and 37℃ for 120min and 240min, respectively. After the incubation, 2% FBS in ice bath was immediately added to terminate the endocytosis experiment, centrifuged at 4℃, 800xg for 3min, and washed the cells 3 times with pre-cooled 2% FBS; immediately add PE-Goat anti-Human IgG Fc secondary antibody (1:1000 dilution), incubate at 4℃ for 30min; centrifuge at 4℃, 800xg for 3min, remove the supernatant containing antibody, and wash the cells 3 times with pre-cooled 2% FBS. Use 200uL pre-cooled 2% FBS to resuspend the cells, prepare a corresponding number of 1.5ml EP tubes, add 300ul pre-cooled 2% FBS to each EP tube, transfer the corresponding samples to the EP tubes, place them on ice, and perform flow cytometry analysis.

[0164] The internalization level of cell surface-bound antibody at a certain time point was calculated according to the following formula: MFI% = MFI of sample incubated at 37°C / MFI of control sample incubated at 4°C; Internalization percentage = 100% - MFI%.

[0165] The experimental test results are as follows Figure 7 As shown in Table 13.

[0166] Table 13

[0167] The results showed that the candidate antibodies 6-C7, 3-F1 and 4-E6 all had high internalization percentages, indicating that they could be well internalized by cells and could be used as targeting elements for cell-specific delivery.

[0168] Example 4: Efficiency of transfection of mouse T cells by candidate antibody-coupled LNP Taking antibody 3-F1 as an example, the targeting of antibody-LNP conjugate to mouse T cells was verified. The experimental steps are as follows:

[0169] 1. Preparation of targeted lipid nanoparticle-circular mRNA complexes (tLNP-cmRNA) The preparation of T cell-targeted tLNP complexes loaded with circular mRNA (cmRNA) includes three main steps: LNP preparation, antibody-SH preparation, and antibody-LNP coupling. The specific process is as follows:

[0170] LNP preparation: ALC-0315: cholesterol: DSPC: ALC-0159: MAL-DSPE-PEG2000 (Aiweituo (Shanghai) Pharmaceutical Technology Co., Ltd.) was dissolved in an ethanol solution at a molar ratio of 46.3: 42.7: 9.4: 1.1: 0.5, and the circular RNA was dissolved in a 25 mM sodium citrate buffer at pH = 4.0. The two phases were quickly mixed using a microfluidic device with a total flow rate of 12 ml / min, and the flow rate ratio of the aqueous buffer (RNA) to the ethanol lipid mixture was 3:1. After preparation, the ethanol was removed by ultrafiltration using an ultrafiltration tube and replaced with a coupling buffer (PBS + 10 mM EDTA, pH 7.6).

[0171] Antibody-SH preparation: In the coupling buffer, 3-F1 antibody and SATA were mixed at a molar ratio of 1:10 for antibody:SATA, reacted at room temperature for 30 minutes, and then ultrafiltered or dialyzed into the coupling buffer. Deamidation solution (0.5M hydroxylamine) was added to the antibody-SATA to a final concentration of 0.05M, reacted at room temperature for 2 hours, and then ultrafiltered or dialyzed into the coupling buffer.

[0172] Antibody-LNP preparation: Antibody-SH and LNP were mixed at a molar ratio of antibody: maleimide (LNP) = 1:10 and reacted at room temperature for 1 h. After the reaction, L-cysteine ​​was added at a final concentration of 10 mM to block the unreacted maleimide groups for 30 min. Ab-LNP and unreacted antibody-SH were separated using a CL-4B gel column with PBS as the mobile phase. The antibody-LNP separation product was collected, concentrated by ultrafiltration, and dialyzed in the storage solution at 4°C overnight. After sterile filtration, the final product was quantified by ribogreen, and the particle size, PDI, and potential were measured simultaneously. The particle size, polydispersity index (PDI), and Zeta potential of the final product of the ring were detected by dynamic light scattering (DLS) using a laser particle size analyzer (Zetasizer Nano-ZSP, Malvern), and the content and encapsulation efficiency of circular RNA in the complex were detected using Quant-iT Ribogreen (ThermoFisher Scientific).

[0173] 2. Evaluation of the effect of tLNP-cmRNA transfection in T cells in vitro Circular mRNA encoding green fluorescent protein EGFP was used as the delivery cargo to prepare a tLNP-cmRNA complex containing the circular mRNA (referred to as tLNP-cmCAR). PBS was used as the negative control group, and the experimental groups were loaded with 0.4μg (tLNP-low) and 2μg (tLNP-high) of EGFP-cmRNA, respectively. tLNP-cmCAR was co-incubated with activated mouse T cells, and the proportion of EGFP-positive cells was detected by flow cytometry after 24 hours.

[0174] The experimental results are as follows Figure 8 As shown, no green fluorescence signal was detected in the control PBS group, while obvious green fluorescence signals were observed in the experimental groups of tLNP-encapsulated EGFP-cmRNA coupled with 3-F1, among which the proportion of EGFP-positive cells in the high-dose group was as high as 79%.

[0175] This experiment demonstrated that the antibody-LNP delivery system of the present invention can efficiently deliver circular mRNA into mouse T cells and express the target protein.

[0176] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. An anti-CD5 nanobody, characterized in that The anti-CD5 nanobody has the following complementary determining region CDR: CDR1 shown in SEQ ID NO: 14; CDR2 shown in SEQ ID NO: 15; CDR3 shown in SEQ ID NO: 16 or SEQ ID NO:

29.

2. The Nanobody according to claim 1, wherein The amino acid sequence of the VHH chain of the anti-CD5 nanobody is shown in SEQ ID NO: 1 or 5.

3. An anti-CD5 multivalent antibody or an antibody against multiple epitopes, characterized in that: The multivalent antibody or antibody against multiple epitopes comprises at least one antibody element against the CD5 epitope, and the antibody element is the anti-CD5 nanobody according to claim 1.

4. A targeted delivery vector for CD5-positive cells, characterized in that: The targeted delivery vector comprises: (1) a delivery vector; and (2) an antibody coupled to the surface of the delivery vector; Wherein, the antibody is the anti-CD5 nanobody as described in claim 1, or the anti-CD5 multivalent antibody or antibody against multiple epitopes as described in claim 3.

5. The targeted delivery vector according to claim 4, characterized in that The CD5 positive cells are T cells, NK cells, or dendritic cells (DC).

6. A pharmaceutical composition, characterized in that The pharmaceutical composition contains: (i) the targeted delivery vector according to claim 4; (ii) a biologically active substance encapsulated in the targeted delivery vehicle; and (iii) a pharmaceutically acceptable carrier, excipient or diluent.

7. The pharmaceutical composition according to claim 6, characterized in that The bioactive substance is a bioactive substance used to generate CAR cell therapy in vivo.

8. An antibody-drug conjugate, characterized in that: The antibody-drug conjugate contains: (a) the Nanobody of claim 1, the multivalent antibody of claim 3, or an antibody against multiple epitopes; and (b) a conjugated moiety conjugated to the antibody portion, wherein the conjugated moiety is selected from the group consisting of a detectable label, a drug, a toxin, or a combination thereof.

9. A polynucleotide, characterized in that The polynucleotide encodes a protein selected from the group consisting of the Nanobody of claim 1 or the multivalent antibody of claim 3 or an antibody against multiple epitopes.

10. An expression vector, characterized in that: The expression vector contains the polynucleotide according to claim 9.

11. A host cell, characterized in that The host cell contains the expression vector according to claim 10, or the polynucleotide according to claim 9 is integrated into its genome, or expresses the nanobody according to claim 1, the multivalent antibody according to claim 3, or an antibody against multiple epitopes.

Images