Anti-CD5 Nanobody and Its Application in In Vivo CAR Cell Therapy

Abstract

The present invention relates to anti-CD5 nanobodies and their applications in in vivo CAR cell therapy. Specifically, the present invention provides 10 nanobodies targeting murine CD5, which have the functions of high affinity and high specific binding to CD5, can be recognized by CD5-positive cells (such as T cells) and internalized into cells, and can be used for specific targeting of T cells in mice. The nanobodies of the present invention can be conjugated with delivery vectors to prepare delivery vectors with immunocyte targeting properties for research and application in in vivo CAR therapy and the like.

Description

Technical Field

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

[0002] Chimeric antigen receptor-modified T cell and NK cell therapies (CAR-T and CAR-NK) have extensive therapeutic uses in tumors and autoimmune diseases. The existing technology is to engineer autologous or donor-derived immune cells in vitro and then transfuse them into patients to exert therapeutic effects. This method faces clinical bottlenecks such as high treatment costs and low accessibility. Generating CAR-modified immune cells directly in patients, 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 bottlenecks of the existing technology.

[0003] In in vivo CAR therapy, nucleic acids encoding CAR molecules are delivered into target immune cells through a delivery vector or delivery system. When screening in vivo CAR therapy, it is necessary to evaluate in animals whether the delivery system can accurately deliver CAR molecules (in the form of DNA or mRNA) into target immune cells (such as T cells). Humanized mice are often used as models to study or evaluate the in vivo delivery of CAR molecules. However, T cells in humanized mice are usually humanized and reconstructed by intravenous injection of human PBMC, 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 targeting T cells in normal mice to screen and study the in vivo efficacy, metabolism, and distribution of in vivo CAR therapy.

[0004] CD5 is a specific marker on the surface of immune cells such as T cells and NK cells. Coupling an anti-CD5 antibody as a targeting element with a delivery vector can achieve targeted delivery to T cells and the like. Therefore, there is an urgent need in this field to develop an antibody that can specifically target CD5 and can be endocytosed by cells to serve as a targeting element for delivery vectors. Summary of the Invention

[0005] The object of the present invention is to provide nanobodies specifically targeting CD5 and targeted delivery vectors.

[0006] In a first aspect of the present invention, there is provided an anti-CD5 nanobody having one or more sets of complementarity-determining regions CDR selected from the following groups:

[0007] (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;

[0008] (2) CDR1 shown in SEQ ID NO: 17; CDR2 shown in SEQ ID NO: 18; CDR3 shown in SEQ ID NO: 19;

[0009] (3) CDR1 shown in SEQ ID NO: 20; CDR2 shown in SEQ ID NO: 21; CDR3 shown in SEQ ID NO: 22;

[0010] (4) CDR1 shown in SEQ ID NO: 23; CDR2 shown in SEQ ID NO: 24; CDR3 shown in SEQ ID NO: 25;

[0011] (5) CDR1 shown in SEQ ID NO: 26; CDR2 shown in SEQ ID NO: 27; CDR3 shown in SEQ ID NO: 28;

[0012] (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;

[0013] (7) CDR1 shown in SEQ ID NO: 33; CDR2 shown in SEQ ID NO: 34; CDR3 shown in SEQ ID NO: 35;

[0014] (8) CDR1 shown in SEQ ID NO: 37; CDR2 shown in SEQ ID NO: 38; CDR3 shown in SEQ ID NO: 39.

[0015] In another preferred example, the CDR1, CDR2 and CDR3 are separated by the framework regions FR1, FR2, FR3 and FR4 of the VHH chain.

[0016] 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.

[0017] In another preferred example, the VHH chain of the anti-CD5 nanobody comprises an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence similarity to any one of the amino acid sequences of SEQ ID NOs: 1-10.

[0018] In another preferred embodiment, the amino acid sequence of the VHH chain of the anti-CD5 nanobody contains one or more amino acid substitutions, preferably conservative amino acid substitutions, compared to any of the amino acid sequences of SEQ ID NO: 1-10.

[0019] In another preferred embodiment, any of the amino acid sequences further includes a derivative sequence that optionally has at least one (such as 1-3, preferably 1-2, more preferably 1) amino acid added, deleted, modified, and / or substituted and still retains the ability to specifically bind to CD5.

[0020] In another preferred embodiment, the anti-CD5 nanobody includes animal-derived antibodies (such as murine antibodies), humanized antibodies, and chimeric antibodies.

[0021] In another preferred embodiment, the anti-CD5 nanobody has a function selected from the following group:

[0022] (1) Specifically bind to the CD5 protein;

[0023] (2) Specifically target CD5-positive cells;

[0024] (3) Be internalized by CD5-positive cells and enter the cell interior through endocytosis.

[0025] In another preferred embodiment, the CD5-positive cells are T cells, NK cells, or dendritic cells (DC).

[0026] In the second aspect of the present invention, a multivalent anti-CD5 antibody or an antibody against multiple epitopes is provided. The multivalent antibody or the antibody against multiple epitopes includes at least one antibody element directed against a CD5 epitope, and the antibody element is the anti-CD5 nanobody described in the first aspect of the present invention.

[0027] In another preferred embodiment, the multivalent anti-CD5 antibody or the antibody against multiple epitopes includes one or more anti-CD5 nanobodies.

[0028] In another preferred embodiment, the multivalent anti-CD5 antibody includes monomers, divalents (bivalent antibodies), tetravalents (tetravalent antibodies), and / or multivalents (multivalent antibodies).

[0029] In another preferred embodiment, the multivalent anti-CD5 antibody includes one or more VHH chains having an amino acid sequence as shown in any of SEQ ID NO: 1-10.

[0030] In another preferred embodiment, the multivalent anti-CD5 antibody includes two VHH chains having the amino acid sequence as shown in SEQ ID NO: 1-10.

[0031] In another preferred embodiment, the anti-CD5 multivalent antibody includes a humanized antibody, an animal-derived antibody, and a chimeric antibody.

[0032] In a third aspect of the present invention, there is provided a recombinant protein having:

[0033] (i) The anti-CD5 nanobody described in the first aspect of the present invention, or the anti-CD5 multivalent antibody described in the second aspect of the present invention, or an antibody against multiple epitopes; and

[0034] (ii) Optionally, a tag sequence for assisting expression and / or purification.

[0035] In another preferred embodiment, the tag sequence includes a Flag tag, an Fc tag, an HA tag, and a His tag.

[0036] In another preferred embodiment, the tag sequence is a His tag.

[0037] In another preferred embodiment, the recombinant protein specifically binds to the CD5 protein.

[0038] In a fourth aspect of the present invention, there is provided a targeted delivery vector for CD5-positive cells, the targeted delivery vector comprising: (1) a delivery vector; and (2) an antibody conjugated to the surface of the delivery vector;

[0039] wherein the antibody is the anti-CD5 nanobody described in the first aspect of the present invention, or the anti-CD5 multivalent antibody described in the second aspect of the present invention, or an antibody against multiple epitopes.

[0040] In another preferred embodiment, the delivery vector includes: lipid nanoparticles (LNP), polymer nanoparticles, nanomicelles, gold nanoparticles, liposomes.

[0041] In another preferred embodiment, the targeted delivery vector is an antibody-LNP conjugate.

[0042] In another preferred embodiment, the targeted delivery vector targets CD5-positive cells.

[0043] In another preferred embodiment, the CD5-positive cells are T cells, NK cells, or dendritic cells (DC).

[0044] In a fifth aspect of the present invention, there is provided a pharmaceutical composition containing:

[0045] (i) The targeted delivery vector described in the fourth aspect of the present invention;

[0046] (ii) A bioactive substance encapsulated in the targeted delivery vector; and

[0047] (iii) A pharmaceutically acceptable carrier, excipient or diluent.

[0048] In another preferred embodiment, the bioactive substance is selected from the group consisting of: nucleic acids, small molecule compounds, proteins, or combinations thereof.

[0049] In another preferred embodiment, the bioactive substance is a bioactive substance for in vivo generation of CAR-T cells and / or CAR-NK cell therapy.

[0050] In another preferred embodiment, the bioactive substance is a nucleic acid.

[0051] In another preferred embodiment, the nucleic acid is RNA, DNA, antisense nucleic acid, aptamer, ribozyme, immunostimulatory nucleic acid or PNA.

[0052] In another preferred embodiment, the RNA is linear RNA or circular RNA.

[0053] In another preferred embodiment, the RNA includes: mRNA, siRNA, saRNA, tRNA, snRNA, microRNA inhibitor, microRNA activator or shRNA.

[0054] In another preferred embodiment, the mRNA is mRNA encoding CAR.

[0055] 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, GD2.

[0056] In another preferred embodiment, the pharmaceutical composition is used for targeted delivery of the bioactive substance into cells.

[0057] In another preferred embodiment, the cell is a CD5-positive cell.

[0058] In another preferred embodiment, the cell is an immune cell, preferably a T cell.

[0059] In a sixth aspect of the present invention, an antibody-drug conjugate is provided, and the antibody-drug conjugate contains:

[0060] (a) The nanobody as described in the first aspect of the present invention, the multivalent antibody as described in the second aspect of the present invention or an antibody against multiple epitopes, the recombinant protein as described in the third aspect of the present invention; and

[0061] (b) A conjugate moiety conjugated to the antibody moiety, the conjugate moiety being selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

[0062] In another preferred embodiment, the antibody moiety is conjugated to the conjugate moiety via a chemical bond or a linker.

[0063] In a seventh aspect of the present invention, there is provided a chimeric antigen receptor (CAR), the CAR comprising an extracellular domain, the extracellular domain comprising 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.

[0064] In another preferred embodiment, the extracellular domain further comprises a signal peptide.

[0065] In another preferred embodiment, the extracellular domain further comprises other foreign proteins.

[0066] In another preferred embodiment, the CAR has the structure shown in formula (I):

[0067] L-Nb-H-TM-C-CD3ζ (I)

[0068] In the formula,

[0069] The "-" is a linker peptide or a peptide bond;

[0070] L is absent or a signal peptide sequence;

[0071] Nb is an antigen-binding domain that specifically binds to CD5;

[0072] H is absent or a hinge region;

[0073] TM is a transmembrane domain;

[0074] C is a co-stimulatory signal domain;

[0075] CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ (including wild-type, or its mutants / modifications).

[0076] In another preferred embodiment, the L is respectively selected from the signal peptides of the following proteins: CD28, 4-1BB, GM-CSF, CD3, CD8a, or a combination thereof.

[0077] In another preferred embodiment, the sequence of the Nb is as shown in any one of SEQ ID NO: 1-10.

[0078] In another preferred embodiment, the H is selected from the hinge regions of the following proteins: CD8, CD28, CD137, IgG, or a combination thereof.

[0079] In another preferred embodiment, 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 / modifications, or combinations thereof.

[0080] In an eighth aspect of the present invention, there is provided a polynucleotide encoding a protein selected from the group consisting of: the nanobody of the first aspect of the present invention, or the multivalent antibody or multi-epitope antibody of the second aspect of the present invention, the recombinant protein of the third aspect of the present invention, or the CAR of the seventh aspect of the present invention.

[0081] In a ninth aspect of the present invention, there is provided an expression vector containing the polynucleotide of the eighth aspect of the present invention.

[0082] In another preferred embodiment, the expression vector is selected from the group consisting of: DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof.

[0083] In another preferred embodiment, the expression vector is a lentiviral vector.

[0084] In a tenth aspect of the present invention, there is provided a host cell containing the expression vector of the ninth aspect of the present invention, or having the polynucleotide of the eighth aspect of the present invention integrated into its genome, or expressing the nanobody of the first aspect of the present invention, the multivalent antibody or multi-epitope antibody of the second aspect of the present invention, the recombinant protein of the third aspect of the present invention, the pharmaceutical composition of the fifth aspect of the present invention, or the CAR of the seventh aspect of the present invention.

[0085] In another preferred embodiment, the cell is an isolated cell, and / or the cell is a genetically engineered cell.

[0086] In another preferred embodiment, the cell is a mammalian cell.

[0087] In another preferred embodiment, the cell is a T cell.

[0088] In another preferred embodiment, the host cell is an engineered immune cell.

[0089] In another preferred embodiment, the engineered immune cell includes autologous or allogeneic αβ T cells, γδ T cells, NKT cells, NK cells, or combinations thereof.

[0090] In another preferred example, the engineered immune cells are CAR-T cells.

[0091] In the eleventh aspect of the present invention, a kit is provided, which 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.

[0092] In another preferred example, the kit is used for preparing immune cells expressing the CAR described in the seventh aspect of the present invention.

[0093] 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 cells expressing the CAR described in the seventh aspect of the present invention, for preparing a drug for preventing and / or treating CD5-related diseases or disorders.

[0094] In another preferred example, the drug contains a pharmaceutically acceptable carrier.

[0095] In another preferred example, the CD5-related diseases or disorders are selected from the group consisting of: immune-related diseases, tumors, or combinations thereof.

[0096] In the thirteenth aspect of the present invention, there is provided the 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 diseases.

[0097] In another preferred example, the diseases are diseases suitable for CAR-T therapy, including but not limited to: tumors, autoimmune diseases, or combinations thereof.

[0098] In another preferred example, the tumors include: hematological tumors, solid tumors, or combinations thereof.

[0099] In the fourteenth aspect of the present invention, a method for treating a disease is provided, which includes administering the pharmaceutical composition described in the fifth aspect of the present invention to a subject in need of treatment.

[0100] In another preferred example, the subject includes mammals, such as humans or mice.

[0101] In another preferred example, the diseases include diseases suitable for CAR-T therapy.

[0102] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here.

[0103] The beneficial effects of the present invention include:

[0104] (1) The anti-CD5 nanobody of the present invention has a high-affinity binding to CD5.

[0105] (2) The anti-CD5 nanobody of the present invention can specifically bind to positive cells expressing CD5, and does not bind to cells not expressing CD5, with high specificity.

[0106] (3) The anti-CD5 nanobody of the present invention can be efficiently endocytosed into T cells and enter the cell interior, and can be used as a targeting element in the delivery vector.

[0107] (4) The anti-CD5 nanobody of the present invention has a simple structure and is easy to prepare, and has broad application prospects in the field of tumor treatment, especially in vivo CAR therapy. Brief Description of the Drawings

[0108] Figure 1 The detection results of SDS-PAGE (A) and SEC-HPLC (B) of the antigen Mouse CD5-8XHis recombinant protein are shown.

[0109] Figure 2 The construction of the yeast display library is shown. A is the RNA agarose gel electrophoresis pattern; B is the agarose gel electrophoresis recovery of about 500 bp fragment of the RT-PCR one-step PCR product; C is the linearization of the yeast display vector pYDisplay.

[0110] Figure 3 The first round (A) and the second round (B) of sorting of the yeast display library are shown.

[0111] Figure 4 The ELISA detection results of the binding activity of the first group of antibodies to Mouse-CD5 are shown.

[0112] Figure 5 The FACS detection results of the binding activity of the first group of antibodies to EL4 are shown.

[0113] Figure 6 The FACS detection results of the binding activity of the second group of antibodies to EL4 are shown.

[0114] Figure 7 The detection results of the endocytosis experiment of the candidate antibody are shown.

[0115] Figure 8 The detection results of the efficiency of the candidate antibody conjugated with LNP transfected into murine T cells are shown. Detailed Embodiments

[0116] After extensive and in-depth research, the present 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 respectively. 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 for specific targeting of T cells in mice. The nanobodies of the present invention can be conjugated with a delivery vector to prepare a delivery vector with immunocyte targeting for research and application of in vivo CAR therapy and the like.

[0117] Based on this, the present invention was completed.

[0118] Term

[0119] To make the present disclosure easier to understand, certain terms are first defined. As used in this application, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0120] The term "about" can refer to a value or a component within an acceptable error range of a specific value or component determined by a person of ordinary skill in the art, which will depend in part on how the value or component 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.).

[0121] As used herein, the terms "comprising" or "including (containing)" can be open-ended, semi-closed and closed. In other words, the terms also include "consisting essentially of...", or "consisting of...".

[0122] 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 where identical residues occur. Generally, this is expressed as a percentage. Methods for measuring sequence identity of nucleotide sequences are well known to those skilled in the art.

[0123] Antibody

[0124] As used herein, the term "antibody" refers to an immunoglobulin, which is a four-peptide chain structure formed by two identical heavy chains and two identical light chains linked by interchain disulfide bonds.

[0125] As used herein, the terms "single domain antibody (VHH)" and "nanobody" have the same meaning, referring to the variable region of the antibody heavy chain, and constructing a single domain antibody (VHH) consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete function. Usually, after obtaining an antibody that is naturally lacking the light chain and the first constant region of the heavy chain (CH1), 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.

[0126] 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 an antigen (e.g., CD5). It has been shown that fragments of full-length antibodies can be used to perform the antigen-binding function of antibodies. Examples of binding fragments included in the term "antigen-binding fragment of an antibody" include;

[0127] (i) Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains;

[0128] (ii) F(ab') 2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bridge on the hinge region;

[0129] (iii) Fd fragment consisting of the VH and CH1 domains;

[0130] (iv) Fv fragment consisting of the VH and VL domains of a single arm of the antibody.

[0131] Fv antibodies contain the variable region of the antibody heavy chain and the variable region of the light chain, but no constant region, and are the smallest antibody fragments with all antigen-binding sites. Generally, Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structure required for antigen binding.

[0132] The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody that mainly contribute to antigen binding. One of the most commonly used definitions of the six CDRs was provided by Kabat E.A. et al., (1991) Sequences of proteins of immunological interest. NIH Publication, No. 91-3242).

[0133] The term "epitope" or "antigenic determinant" refers to the site on an antigen to which an immunoglobulin or antibody specifically binds (e.g., a specific site on the CD5 molecule). Epitopes usually include 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.

[0134] The terms "specific binding", "selective binding", "selectively bind" and "specifically bind" refer to the binding of an antibody to an epitope on a predetermined antigen. Generally, an antibody binds with an affinity (KD) of about less than 10 -7 M, such as about less than 10 -8 M, 10 -9 M or 10 -10 M or less.

[0135] As used herein, the term "epitope" refers to a discontinuous three-dimensional spatial site on an antigen that is recognized by an antibody or antigen-binding fragment of the present invention.

[0136] The present invention includes not only intact antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Accordingly, the present invention also includes fragments, derivatives and analogs of said antibodies.

[0137] In the present invention, the antibodies of the present invention also include their conservative variants, which refer to polypeptides 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 having similar or close properties compared with the amino acid sequence of the antibodies of the present invention. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table 1 below.

[0138] Table 1

[0139]

[0140] Anti-CD5 nanobody

[0141] As used herein, the term "CD5" generally refers to a native or recombinant CD5 protein, including human CD5 protein and non-human homologs of CD5, including murine CD5.

[0142] As used herein, the terms "nanobodies of the present invention", "single-domain antibodies of the present invention", "anti-CD5 nanobodies of the present invention" are used interchangeably and all refer to the highly specific and highly affinity nanobodies of the present invention against CD5.

[0143] Compared with traditional monoclonal antibodies, the nanobodies of the present invention have a small molecular mass, strong penetrability, high sensitivity, high specificity and good stability; when it enters the body, it can efficiently penetrate cells to quickly capture antigens to achieve the targeting purpose.

[0144] In the present invention, the anti-CD5 nanobodies include monomers, divalents (bivalent antibodies), tetravalents (tetravalent antibodies), and / or multivalents (multivalent antibodies).

[0145] In a preferred embodiment 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 NO: 1-10; preferably selected from one or more of SEQ ID NO: 1-7.

[0146] The preferred nanobody names, VHH sequence numbers and corresponding CDR region sequence numbers of the present invention are shown in Table 2:

[0147] Table 2

[0148]

[0149] Targeted delivery vector

[0150] The present invention also provides a targeted delivery vector using the anti-CD5 nanobody as a targeting element, which can achieve targeted delivery to CD5-positive cells and is used for applications such as in vivo CAR-T therapy.

[0151] Specifically, the targeted delivery vector of the present invention comprises: (1) a delivery vector; and (2) an antibody conjugated to the surface of the delivery vector; wherein the antibody is the anti-CD5 nanobody, a multivalent antibody against CD5 or an antibody against multiple epitopes of the present invention.

[0152] As used herein, the term "delivery vector" refers to a tool or system that can effectively deliver drugs, genes or other bioactive substances to specific target cells. In the present invention, the type of the delivery vector is not limited and includes: lipid-based vectors (such as lipid nanoparticles LNP), polymer-based vectors (such as cationic polymer nanoparticles), inorganic carriers (such as gold nanoparticles), etc.

[0153] The preferred vector is a lipid nanoparticle. As used herein, the terms "lipid nanoparticle", "lipid nanoparticle" or "LNP" can be used interchangeably and refer to particles with nanoscale dimensions self-assembled from biocompatible lipid molecules, with a diameter of about 5 to 500 nm. LNPs usually consist of a variety of functional lipid molecules, including but not limited to: ionizable lipids, which electrostatically bind to the loaded substances; helper lipids, which regulate the stability and release rate of LNPs and enhance biocompatibility; polyethylene glycolylated lipids (PEG-lipids), which prolong the circulation time of LNPs in the blood and reduce the recognition and clearance by the reticuloendothelial system.

[0154] In the present invention, the components and preparation methods of LNPs are not limited. Common components include: ionizable lipids, DSPC (distearoylphosphatidylcholine), DSPE-PEG (distearoylphosphatidylethanolamine-polyethylene glycol), DMG-PEG (dimyristoyl glycerol-polyethylene glycol), and cholesterol. Ionizable lipids are positively charged under acidic conditions and can bind to negatively charged nucleic acid molecules, assisting phospholipids such as DSPC, DOPC, POPC, DOPE, etc., cholesterol, and PEG lipids to form stable lipid nanoparticles.

[0155] The delivery carrier of the present invention is used to load or encapsulate "cargo", and the cargo is usually a bioactive substance with therapeutic effects. As used herein, the term "bioactive substance" includes but is not limited to: RNA (mRNA, rRNA, circRNA, siRNA, saRNA, tRNA, snRNA, antagomir, microRNA inhibitor, microRNA activator, or shRNA, etc.), DNA (including plasmids), natural and synthetic oligonucleotides (including antisense oligonucleotides, interfering RNAs, and small interfering RNAs), nucleoproteins, peptides, nucleic acids, ribozymes, aptamers, immunostimulatory nucleic acids or PNAs, DNA-containing nucleoproteins, etc. In some embodiments, the bioactive substance can be mixed with an adjuvant.

[0156] In the present invention, the bioactive substance can be mRNA for generating a target polypeptide or protein in vivo. In one embodiment of the present invention, the bioactive substance is mRNA for generating CAR in vivo.

[0157] As used herein, the term "CAR" refers to a chimeric antigen receptor, and its basic structure includes an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain. The extracellular antigen recognition domain is usually composed of a monoclonal antibody fragment or a derivative of an antibody-like molecule, and can specifically recognize and bind to a specific antigen on the surface of target cells, such as cancer-associated antigens on the surface of tumor cells, etc. This property enables CAR-T cells to accurately find target cells. The transmembrane domain plays a role in connecting the extracellular antigen recognition domain and the intracellular signaling domain, ensuring that signals can be transmitted from the extracellular to the intracellular, triggering subsequent cellular reactions. 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 functions of T cells, causing T cells to proliferate, release cytotoxic factors, etc., and ultimately leading to the death of target cells.

[0158] The target of the CAR targeting of the present invention is not restricted and can be any target related to diseases known in the art. Common targets include: CD19, BCMA, CD20, CD22, CD33, CD38, CD70, CD123, HER2, MSLN, EGFR, CLDN18.2, GPC3, GD2, but not limited thereto. The structure of the CAR of the present invention is not restricted and can be a first-generation, second-generation, third-generation or fourth-generation CAR. The design or selection of the CAR can be routinely mastered by those skilled in the art.

[0159] The targeting 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 interior through endocytosis. As used herein, the term "endocytosis" refers to the process by which, after an antibody binds to a specific antigen on the cell surface, the cell's intrinsic mechanism causes the cell to wrap and internalize the antigen and the antibody bound thereto into the cell interior. When 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 local invagination of the cell membrane and ultimately enclosing the antigen and antibody in a vesicle, separating from the cell surface and entering the cytoplasm. When the anti-CD5 antibody is conjugated to the delivery vector, the delivery vector and its internal cargo can be delivered into the target cell interior through endocytosis. In a preferred embodiment, the target cells of the present invention are CD5-positive cells, especially T cells and NK cells.

[0160] Antibody-drug conjugate (ADC)

[0161] The present invention also provides an antibody-drug conjugate (ADC) based on the nanobody of the present invention.

[0162] Typically, the antibody-drug conjugate includes the antibody and an effector molecule, and the antibody is conjugated to the effector molecule, preferably by chemical conjugation. Among them, the effector molecule is preferably a drug with therapeutic activity. In addition, the effector molecule can be one or more of a toxic protein, a chemotherapeutic drug, a small molecule drug or a radionuclide.

[0163] Certain residues on the antibody (such as Cys or Lys, etc.) are used to connect with a variety of functional groups, including imaging reagents (such as chromophores and fluorescent groups), diagnostic reagents (such as MRI contrast agents and radioisotopes), stabilizers (such as ethylene glycol polymers) and therapeutic agents. The antibody can be conjugated to a functional agent to form an antibody-functional agent conjugate. The functional agent (such as a drug, a detection reagent, a stabilizer) is conjugated (covalently linked) to the antibody. The functional agent can be directly or indirectly connected to the antibody through a linker.

[0164] The drug can be any cytotoxic, cell growth-inhibiting or immunosuppressive drug. In an embodiment, the linker connects the antibody and the drug, and the drug has a functional group that can bond with the linker. For example, the drug can have an amino group, a carboxyl group, a mercapto group, a hydroxyl group, or a keto group that can bond with the linker. In the case where the drug is directly linked to the linker, the drug has a reactive functional group before being linked to the antibody.

[0165] Useful drug classes include, for example, anti-tubulin drugs, DNA minor groove-binding reagents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, etc. In the present invention, the drug-linker can be used to form an ADC in a single step. In other embodiments, a bifunctional linker compound can be used to form an ADC in a two-step or multi-step method. For example, a cysteine residue reacts with the reactive portion of the linker in the first step, and in a subsequent step, the functional group on the linker reacts with the drug to form an ADC.

[0166] Applications

[0167] The present invention provides the application of the anti-CD5 nanobody for preparing a composition for treating diseases. In a preferred example, the composition is a pharmaceutical composition, which contains the above-mentioned anti-CD5 nanobody or derivative, including a fusion protein, an ADC or a corresponding CAR-T cell, 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 usually about 5-8, preferably about 6-8, although the pH value can vary depending on the nature of the substance to be formulated and the disease to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or topical administration.

[0168] The anti-CD5 nanobody and its derivative of the present invention can be directly used to bind to the CD5 protein molecule, and thus can be used for the prevention and treatment of 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.

[0169] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001 - 99 wt%, preferably 0.01 - 90 wt%, more preferably 0.1 - 80 wt%) of the nanobody (or its conjugate, derivative, etc.) of the present invention as described above, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solution, glucose, water, glycerol, ethanol, and their combinations. The pharmaceutical preparation should match the administration route. The pharmaceutical composition of the present invention can be made into an injectable form, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injectables and solutions should be manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram per kilogram of body weight per day - about 5 milligrams per kilogram of body weight. In addition, the polypeptide of the present invention can also be used together with other therapeutic agents.

[0170] When using the 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 per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight. Preferably, the dosage is about 10 micrograms per kilogram of body weight - about 20 milligrams per kilogram of body weight. Of course, the specific dosage should also consider factors such as the administration route and the patient's health condition, which are within the scope of the skills of a skilled physician.

[0171] The antibody described in the present invention can also be used as a targeting element to prepare a targeted delivery carrier for delivering bioactive substances such as drugs to CD5-positive cells for gene therapy or cell therapy. In a preferred embodiment, the bioactive substance to be delivered can be a nucleic acid molecule encoding a CAR, such as mRNA encoding a CAR, especially circular mRNA, for generating chimeric antigen receptor T cell immunotherapy (in vivo CAR-T) in vivo, etc.

[0172] Different from the traditional CAR-T therapy, the in vivo CAR-T therapy does not require extracting T cells from the patient's body for in vitro modification and amplification, but directly converts T cells into CAR-T cells with specific targeting in the patient's body. This process can be achieved by introducing the gene encoding CAR into CD5-positive cells using the targeted delivery carrier of the present invention. The delivery carrier containing the anti-CD5 nanobody of the present invention is particularly suitable for studying the T cell targeting system in normal mice for screening and studying the in vivo efficacy, metabolism, and distribution characteristics of CAR therapy in vivo.

[0173] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and fractions are weight percentages and weight fractions.

[0174] Example 1: Screening of Anti-CD5 Antibodies

[0175] 1. Alpaca Immunization

[0176] Two alpacas were immunized with Mouse CD5-8XHis recombinant protein as the antigen. The SDS-PAGE and SEC-HPLC detection results of Mouse CD5-8XHis recombinant protein are shown in Figure 1 , showing that the protein purity > 95%, which can be used for immunization and screening.

[0177] The specific immunization protocol is as shown in Table 3 below, where the adjuvant is Gerbu:

[0178] Table 3 Alpaca Immunization Protocol

[0179]

[0180] 2. Serum Titer Detection

[0181] After the immunization was completed, 5 mL of alpaca peripheral blood was collected. After separating the serum, ELISA immune titer detection was carried out. The method is as follows: The enzyme-linked immunosorbent assay (ELISA) plate was coated with 1 μg / ml of Mouse-CD5-His at 4 °C overnight; the serum was diluted in a gradient, with the initial dilution factor of 1:1K and a 5-fold gradient dilution for a total of 7 points. 100 μl / well was added to the ELISA plate and incubated. After washing the plate, Goat antillama IgG(H+L) diluted 1:50000 was added as the secondary antibody solution. After incubation, the termination solution was added, the plate was washed, developed, and detected with an ELISA reader.

[0182] The results are shown in Table 4 and Table 5. The results show that the immune titers of both alpacas reached above 1:64K, meeting the requirements for library construction.

[0183] Table 4

[0184]

[0185] Table 5

[0186]

[0187] Meanwhile, the binding activity of serum with EL4 cells positive for CD5 was detected by FACS, and MC38 cells that do not express CD5 were used as control cells, and Rat-anti-mCD5 (SEQ ID NO: 40) was used as a positive control antibody, and its secondary antibody was Anti-rat-lgG-FITC.

[0188] The test results are shown in Table 6.

[0189] Table 6

[0190]

[0191] The FACS results showed that the serum after the third immunization could bind to EL4 cells, and blood collection could be arranged for library construction.

[0192] 3. Construction of antibody display library

[0193] PBMC cells were isolated from the peripheral blood of alpacas after the above immunization, RNA was extracted, reverse transcribed into cDNA, the VHH sequence was amplified from the sample, and cloned into a yeast display vector, and electrotransformed into competent EYB100 cells to construct a single-domain antibody yeast display library, as Figure 2 shown.

[0194] 4. Panning of antibody display library

[0195] The Mouse CD5-8XHis recombinant protein was biotinylated. After induction of the antibody display library, first, magnetic sorting enrichment was performed with biotin-protein, and the results of the first and second rounds of sorting are as Figure 3 shown. The enriched display library was further sorted by a sorting flow cytometer to obtain monoclonal cells that could recognize the target protein, and the candidate clones were sequenced, and the amino acid sequences of the candidate single-domain antibodies obtained by sequencing were aligned.

[0196] 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, 3-G8; the second group contained 4 antibodies: 6-C7, 6-E10, 6-G7, 8-C6.

[0197] The candidate antibody sequences are as follows, with the CDR region sequences underlined.

[0198] > 3-F1 (SEQ ID NO: 1)

[0199] MGWSCIILFLVATATGVHSKAQPAMAQVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIG WFRQAPGKEREGVS CMSSSGGSTNYADSVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA VYSIGDPGAYFCSGHIPYDY WGQGTQVTVSSEPKTPKPGHEG

[0200] >6-C7 (SEQ ID NO: 2)

[0201] MGWSCIILFLVATATGVHSKAQPAMAQLQLVESGGGLVQPGGSLRLSCAGSGFSLD DYAIA WFRRAPGKEREGVS CISSNGRATNYADFVRG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA VYSLGDPGAYFCSGYVPYDY WGQGTQVTVSSEPKTPKPGHEG

[0202] >4-E6 (SEQ ID NO: 3)

[0203] MGWSCIILFLVATATGVHSKAQPAMAQLQLVESGGGLVQPGGSLRLSCTASESISN IYGMN WYRQAPGKEREWVA YITTKGGQYYTDSVKG RFTIARDDAKNTMYLQMANLKPEDTAVYYCST DQGD WGQGTQVIVSSEPKTPKPGHEG

[0204] >6-G7 (SEQ ID NO: 4)

[0205] MGWSCIILFLVATATGVHSKAQPAMAQVQLVESGGGLVQPGGSLRLSCVASGPFFA ISIMR WYRQAPGKQRELVA AITNDGSTNYADSVED RFSISRDNAKKTLYLQMNDLKPEDTAVYYCNE ASPDWTNIKAYDF WGQGTQVRVSSEPKTPKPGHEG

[0206] >3-C1 (SEQ ID NO: 5)

[0207] MGWSCIILFLVATATGVHSKAQPAMAQVQLVESGGGLVQPGGSLRLSCAASGFTLD YYAIG WFRQAPGKEREGVS CMSSSGGSTNYADSVKGRFTISRDNAKNTVYLFMNCLKPEDTAVYYCAA VYSIGDLGAYFCSGYVPYDY WGQGTQVAVSSEPKTPKPGHEG

[0208] >4-C8 (SEQ ID NO: 6)

[0209] MGWSCIILFLVATATGVHSKAQPAMAQLQLVESGGGLAQPGGSLRISCAASLNIFA DTNLH WYRQAPGNQREWVA TMVPSGVAMYADPVRG RFSVSSDSAKKTAFLQMNSLRPEDTAVYYCAT PQGN WGQGTQVTVSSEPKTPKPGHEG

[0210] >5-F10 (SEQ ID NO: 7)

[0211] MGWSCIILFLVATATGVHSKAQPAMAQVQLVESGGGSVQPGGSLKLSCAASGSIFS INAMN WYRQAPGKERELVA AISSGGSTYYADSVKG RFTISRDNAKNSLYLQMNSLKPEDTAMYYCAS PDGA WGQGTQVTVSSEPKTPKPGHEG

[0212] >4-D3 (SEQ ID NO: 8)

[0213] MGWSCIILFLVATATGVHSKAQPAMAQVQLVESGGGLVQPGGSLTLSCVASGFTFS SYAMS WYRQAPGKEREMVA FISNTGGNTYYPDSMKG RFTISRDNAKSTVYLQMNSLKPEDTAVYRCAC VNGV WGQGTQVTVPSEPKTPKPGHEG

[0214] >5-D3 (SEQ ID NO: 9)

[0215] MGWSCIILFLVATATGVHSKAQPAMAQVQLVESGGGLVQAGGSLRLSCAASGRTFS INAMN WYRQAPGKERELVA AISSGGSTYYADSVKG RFTISRDNAKNTLYLQMNSLKPEDTAMYYCAA PQGDWGQGTQVTVSSEPKTPKPGHEG

[0216] >4-F12 (SEQ ID NO: 10)

[0217] MGWSCIILFLVATATGVHSKAQPAMAQVQLVESGGGLVQPGGSLTLSCVAPRGFIQ SNSIN WYHEAPGKEREWVA WIAPNGYTNYADAVLH HFTISRDNSKNTIYLEMDDLKPEDTGMYRCAS PSGI WGQGTQVTVSSEPKTPKPGHEG

[0218] >8-C6 (SEQ ID NO: 11)

[0219] MGWSCIILFLVATATGVHSKAQPAMAQLQLVESGGGLVQAGGSLKLSCAASKNIFN FATMA WYRQAPGKERELVA LITFRGGTNYADSVKG RFTISRDNARNMVYMQMNSLKPEDTAVYYCNQ KLGTRDD WGQGTQVTVSSEPKTPKPGHEG

[0220] >6-E10 (SEQ ID NO: 12)

[0221] MGWSCIILFLVATATGVHSKAQPAMAQVQLVESGGGLVQPGGSLRLSCTASGMQFS IYDMN WYRLAPGKEREWVG SVSFDGTTFYADSVKG RFTISRDSAKSTMYLQMSSLKADDTAVYLCST LSGY WGQGTQVTVSLEPKTPKPGHEG

[0222] >3-G8 (SEQ ID NO: 13)

[0223] MGWSCIILFLVATATGVHSKAQPAMAEVQLVESWGGLVQAGGSLRLSCAASGSIFN IYGIN WYRQAPGKQREFIA GITSDGSTTYADSVKG RFTISRDNAKNTVYLQMNSLKPEDTAAYRCSD PRGI WGQWTQVTVSSEPKTPKPGHEG

[0224] Example 2: Antibody Function Detection

[0225] The functional activities of the two groups of antibodies screened above were detected. The positive control (PC) used was a murine anti-CD5 antibody, and its sequence is shown as follows:

[0226] MGWSCIILFLVATATGVHSNIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLRWMGWINTHTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCTRRGYDWYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFHHKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLDYEDMGIYYCQQYDESPWTFGGGTKLEMKEPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 40)

[0227] 1. Detection of the Function of the First Group of Antibodies

[0228] (1) Supernatant Detection

[0229] For the first group of antibodies, a vector was constructed to express and verify the antibody: A eukaryotic expression vector pcDNA34-Fc (Human IgG1Fc tag) was constructed and transiently transfected into 293F cells. After collecting the culture medium supernatant, the binding of the antibody in the transfected supernatant to the MouseCD5 recombinant protein was detected by ELISA. The enzyme-linked immunosorbent assay (ELISA) plate was coated with 1 μg / ml of Mouse-CD5-His at 4 °C overnight; after washing and blocking, 100 μl of the transfected supernatant was added to each well as the primary antibody, and after incubation, it was washed; Human IgG-Fc antibody diluted 1:1000 was used as the secondary antibody, 100 μl / well, and after incubation, it was washed, developed, and the OD value was read.

[0230] The ELISA results are shown in Table 7.

[0231] Table 7

[0232]

[0233] Meanwhile, FACS was used to detect the binding of the antibody in the transfection supernatant to EL4 and MC38. The detection results are shown in Table 8.

[0234] Table 8

[0235]

[0236] The above results showed that all the candidate antibodies tested could bind to EL4 cells expressing CD5, and 3-G8 had non-specific binding to MC38. Subsequently, the remaining 8 antibodies except 3-G8 were purified and tested.

[0237] (2)Detection of purified antibodies

[0238] The eukaryotic expression vector of the candidate antibody was transiently transfected into 293F cells. The recombinant antibody was separated and purified by affinity chromatography, and the purity of the recombinant antibody was analyzed by SDS-PAGE. After diluting the target recombinant protein to 1 μg / mL and coating the ELISA plate, the candidate was serially diluted for ELISA experiments. A curve was plotted with the candidate antibody concentration as the abscissa and OD450 as the ordinate. The results are as Figure 4 shown in Table 9.

[0239] Table 9

[0240]

[0241] The EC50 of the binding activity of the purified antibody to EL4 cells was detected by FACS. The results are as Figure 5 shown in Table 10.

[0242] Table 10

[0243]

[0244] Among them, the binding activities of antibodies 3-C1, 4-E6, 3-F1, and 4-C8 were equivalent to or better than those of the positive control antibody, showing excellent binding activities.

[0245] 2. Detection of the functions of the second group of antibodies

[0246] For the second group of antibodies, ELISA or FACS detection was directly performed after purification, and the detection method was the same as above.

[0247] ELISA was used to detect the binding activities of 6-C7, 6-E10, 6-G7, and 8-C6 to Mouse CD5. The results are shown in Table 11. The candidate antibodies 6-C7, 6-E10, and 6-G7 had strong binding activities.

[0248] Table 11

[0249]

[0250] The EC50 values of 6-C7, 6-G7 binding to EL4 were detected by FACS, and the results are as Figure 6 shown in Table 12. 6-C7 and 6-G7 have strong binding activities to EL4.

[0251] Table 12

[0252]

[0253] Example 3: Detection of candidate antibody endocytosis

[0254] In this example, the efficiency of candidate antibody endocytosis by EL4 cells was detected, and the detection steps are as follows:

[0255] Add 3×10 5 EL4 cells per well, add 10 μg / ml, 100 μl / well of candidate antibody, and incubate at 4°C for 30 min; then centrifuge at 4°C, 800 x g for 3 min, discard the supernatant containing the antibody, and wash the cells 3 times with pre-cooled 2% FBS. Divide the cells into 2 groups on average, and incubate at 4°C and 37°C for 120 min and 240 min respectively. After the incubation, immediately add ice-cold 2% FBS to terminate the endocytosis experiment, centrifuge at 4°C, 800 x g for 3 min, and wash the cells 3 times with pre-cooled 2% FBS; immediately add the secondary antibody PE-Goat anti-Human IgG Fc (diluted 1:1000), and incubate at 4°C for 30 min; centrifuge at 4°C, 800 x g for 3 min, discard the supernatant containing the antibody, and wash the cells 3 times with pre-cooled 2% FBS. Resuspend the cells with 200 μl of pre-cooled 2% FBS, prepare the corresponding number of 1.5 ml EP tubes, add 300 μl of pre-cooled 2% FBS to each EP tube, transfer the corresponding samples to the EP tubes respectively, and place them on ice for flow cytometry analysis.

[0256] Calculate the internalization level of cell surface-bound antibody at a certain time point according to the following formula: MFI% = MFI of the sample incubated at 37°C / MFI of the control sample incubated at 4°C; internalization percentage = 100% - MFI%.

[0257] The experimental detection results are as Figure 7 shown in Table 13.

[0258] Table 13

[0259]

[0260] The results showed that candidate antibodies 6-C7, 3-F1, and 4-E6 all had relatively high internalization percentages, indicating that they could be well internalized by cells and could be used as targeting elements for cell-specific delivery.

[0261] Example 4: Efficiency of candidate antibody-conjugated LNP transfection of murine T cells

[0262] Taking antibody 3-F1 as an example, the targeting ability of the antibody-LNP conjugate to murine T cells was verified. The experimental steps are as follows:

[0263] 1. Preparation of targeted lipid nanoparticle-circular mRNA complex (tLNP-cmRNA)

[0264] The preparation of the T cell-targeted tLNP complex encapsulating circular mRNA (cmRNA) includes three main steps: LNP preparation, antibody-SH preparation, and antibody-LNP conjugation. The specific process is as follows:

[0265] LNP preparation:

[0266] ALC-0315: cholesterol: DSPC: ALC-0159: MAL-DSPE-PEG2000 (Aventis Pharma (Shanghai) Co., Ltd.) was dissolved in an ethanol solution at a molar ratio of 46.3:42.7:9.4:1.1:0.5, and circular RNA was dissolved in a 25 mM sodium citrate buffer at pH = 4.0. The two phases were rapidly 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, an ultrafiltration tube was used to ultrafilter to remove ethanol and replace it with a conjugation buffer (PBS + 10 mM EDTA, pH 7.6).

[0267] Antibody-SH preparation:

[0268] In the conjugation buffer, 3-F1 antibody was mixed with SATA at a molar ratio of antibody:SATA = 1:10 and reacted at room temperature for 30 min, and then ultrafiltered or dialyzed to the conjugation buffer. Deamidation solution (0.5 M hydroxylamine) was added to the antibody-SATA to a final concentration of 0.05 M and reacted at room temperature for 2 h. Then ultrafiltered or dialyzed to the conjugation buffer.

[0269] Antibody-LNP preparation:

[0270] Mix the antibody-SH and LNP at a molar ratio of antibody:maleimide (LNP) = 1:10, and react at room temperature for 1 h; after the reaction, add L-cysteine with a final concentration of 10 mM to block the unreacted maleimide groups for 30 min. Using PBS as the mobile phase, separate the Ab-LNP and the unreacted antibody-SH using a CL-4B gel column. Collect the antibody-LNP separation product, ultrafiltrate and concentrate it, and then dialyze it overnight at 4 °C in the preservation solution. After sterile filtration, quantify the final product with ribogreen, and simultaneously measure the particle size, PDI, and potential. Use a laser particle size analyzer (Zetasizer Nano-ZSP, Malvern) to detect the particle size, polydispersity index (PDI), and Zeta potential of the final product by dynamic light scattering (DLS), and use Quant-iT Ribogreen (ThermoFisher Scientific) to detect the content and encapsulation efficiency of circular RNA in the complex.

[0271] 2. Evaluation of the effect of tLNP-cmRNA on in vitro transfection of T cells

[0272] Prepare a tLNP-cmRNA complex (abbreviated as tLNP-cmCAR) containing circular mRNA encoding enhanced green fluorescent protein (EGFP) as the delivery cargo. Use PBS as the negative control group, and the experimental groups are respectively loaded with 0.4 μg (tLNP-low) and 2 μg (tLNP-high) of EGFP-cmRNA. Co-incubate the tLNP-cmCAR with activated murine T cells, and detect the proportion of EGFP-positive cells by flow cytometry after 24 hours.

[0273] The experimental results are as Figure 8 shown. No green fluorescence signal was detected in the control group PBS, while obvious green fluorescence signals were observed in the experimental groups where tLNP conjugated with 3-F1 was loaded with EGFP-cmRNA. Among them, the proportion of EGFP-positive cells in the high-dose group was as high as 79%.

[0274] This experiment proves that the antibody-LNP delivery system of the present invention can efficiently deliver circular mRNA into murine T cells and express the target protein.

[0275] All the documents mentioned in the present invention are cited herein as references, as if each document was individually cited as a reference. 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 appended claims of 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 CD5 multi-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 as described in claim 3, or an antibody against multiple CD5 epitopes.

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

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 conjugate, characterized in that: The antibody conjugate contains: (a) the Nanobody according to claim 1, the anti-CD5 multivalent antibody according to claim 3, or an antibody against multiple CD5 epitopes; and (b) a coupling moiety coupled to the antibody moiety, wherein the coupling moiety is a detectable label.

9. A polynucleotide, characterized in that The polynucleotide encodes a protein selected from the group consisting of the Nanobody of claim 1 or the anti-CD5 multivalent antibody of claim 3 or an antibody against multiple CD5 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 of claim 10, or the polynucleotide of claim 9 is integrated into its genome, or expresses the nanobody of claim 1, the anti-CD5 multivalent antibody of claim 3, or an antibody against multiple CD5 epitopes.

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