Anti-CD5 single domain antibody and application thereof

Abstract

An anti-CD5 single domain antibody and an application thereof, which belong to the technical field of antibodies. Heavy chain complementarity determining regions of the provided anti-CD5 single domain antibody comprise CDR1, CDR2, and CDR3. The provided anti-CD5 single domain antibody can specifically bind to CD5, and can recognize an extracellular region D3 juxtamembrane domain of a CD5 molecule. Further provided is a humanized anti-CD5 single domain antibody, which reduces the immunogenicity of a molecule in the body, and the humanized CD5 single domain antibody can specifically recognize a CD5 molecule and has relatively high affinity. The present invention provides broad prospects for the development of drugs targeting CD5.

Description

Anti-CD5 single domain antibody and its application

[0001] The present invention claims priority to Chinese patent application No. 202311763267.1, filed with the Patent Office of China on December 20, 2023, entitled “Anti-CD5 Single Domain Antibodies and Their Applications,” the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present invention belongs to the technical field of antibodies, and in particular relates to an anti-CD5 single domain antibody and applications thereof. Background Art

[0003] CD5 is a large glycoprotein primarily found on T lymphocytes and to a lesser extent on B lymphocytes. It not only participates in innate immune responses but also regulates specific immune responses mediated by T and B lymphocytes. It influences the biological characteristics and functions of T and B lymphocytes through T lymphocyte receptor (TCR) and B lymphocyte receptor (BCR) signaling pathways. CD5 molecules not only assist T cells in regulating the various immune balances associated with autoimmune diseases, but also contribute to the development of autoantibodies in B cells to eliminate some autopathological antigens, thus contributing to the progression of various autoimmune diseases.

[0004] CD5 is a 67kDa type I transmembrane glycoprotein that structurally belongs to the ancient and highly conserved cysteine-rich scavenger receptor (SRCR) superfamily. SRCR superfamily molecules can mediate homotypic or heterotypic interactions, leading to pathogen-associated molecular pattern recognition. Cysteine-rich CD5 is mainly composed of an extracellular region consisting of 347 amino acid residues and an intracellular region consisting of 93 amino acid residues. [1] The extracellular region structure contains three SRCR domains (D1, D2 and D3) and a hydrophobic transmembrane region, which can act as a receptor to regulate T cell proliferation, making the CD5 molecule an ideal target for T cell malignancies. Among them, D3 is the proximal membrane domain [2] In studies on CAR-T cells targeting different antigens, it was shown that binding to the membrane epitopes of antigen molecules can enhance the immune function of CAR-T cells.

[0005] A single-domain antibody (sdAb) is a man-made antibody molecule, a naturally occurring heavy-chain antibody lacking a light chain, found in camelids such as alpacas and dromedaries, as well as cartilaginous fish such as sharks and rays. With a molecular weight only one-tenth that of a typical antibody, sdAbs are the smallest complete antigen-binding fragment, hence their nickname, nanobodies. The long CDR3 region of the sdAb heavy chain variable region forms a stable raised loop structure. The stable disulfide bonds within this structure allow for penetration into the interior of the antigen, resulting in a higher affinity for VHH antibodies compared to the concave topology formed by typical antibodies. The small molecular weight and long CDR3 of single-domain antibodies make sdAbs more flexible and allow them to carry targeted drugs into binding sites difficult for traditional antibodies to access. Single-domain antibodies also have improved solubility and higher hydrophilicity than traditional antibodies. Due to their higher number of hydrophilic amino acids, they are more thermally stable and retain biological activity even in the presence of proteases. Due to its structure, sdAb can be used to reveal hidden epitopes of traditional immunoglobulins, making it widely used in immunological research, diagnostic testing, medical and biological imaging, and therapeutic antibody development. [3] .

[0006] Currently, there are no marketed drugs targeting the CD5-D3 target. Therefore, there is an urgent need to develop a therapeutic single-domain antibody that has high affinity, specifically targets the CD5 molecule, and can recognize the D3 membrane-proximal domain of the CD5 molecule's extracellular region.

[0007] [1] He Xin, Xing Limin, Shao Zonghong. Research progress on CD5 and its role in autoimmune diseases[J]. Chinese Journal of Immunology, 2020, 36(14): 1766-1770.

[0008] [2]Consuegra-Fernández M,Aranda F, I,et al.CD5as a Target for Immune-Based Therapies.Crit Rev Immunol[J].2015;35(2):85-115.

[0009] [3]Hamers-Casterman C, Atarhouch T, Muyldermans S, et al. Naturally occurring antibodies devoid of light chains[J]. Nature.1993 Jun 3;363(6428):446-448. Summary of the Invention

[0010] To address the above-mentioned deficiencies, the present invention provides an anti-CD5 single-domain antibody and its application. The anti-CD5 single-domain antibody provided by the present invention can specifically bind to CD5 and recognize the D3 juxtamembrane domain of the extracellular region of the CD5 molecule. The present invention also provides a humanized anti-CD5 single-domain antibody that reduces the immunogenicity of the molecule in vivo. The humanized CD5 single-domain antibody can specifically recognize the CD5 molecule with high affinity, providing broad prospects for the development of drugs targeting CD5.

[0011] the term:

[0012] In the present invention, the term "amino acid" includes natural amino acids, synthetic amino acids, and amino acid analogs and amino acid mimetics that function in a manner similar to natural amino acids. Natural amino acids are amino acids encoded by the genetic code. Amino acid analogs refer to amino acids having the same basic chemical structure as naturally occurring amino acids. Amino acids may be referred to herein by their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0013] In the present invention, the term "nucleotide sequence" refers to the order of bases in DNA or RNA, that is, the order of A, T, G, C in DNA, or the order of A, U, G, C in mRNA, and also includes the order of bases in rRNA, tRNA, and mRNA.

[0014] In the present invention, heavy chains refer to the two longer, relatively larger, identical heavy chains (H chains) in an antibody; light chains refer to the two shorter, relatively smaller, identical light chains (L chains) in an antibody.

[0015] In the present invention, the variable region refers to the region of the immunoglobulin light chain and heavy chain near the N-terminus where the amino acid sequence changes greatly.

[0016] In the present invention, the term "complementarity determining region" or "CDR" generally refers to the complementarity determining region within the variable region of an antigen-binding fragment. In the present invention, the heavy chain variable region has three CDRs, which are designated HCDR1, HCDR2, and HCDR3 for each variable region.

[0017] In the present invention, the term "FR" generally refers to the more highly conserved portions of antibody variable domains, which are called the framework regions.

[0018] In the present invention, the term "single domain antibody" or "VHH" refers to a type of antibody that lacks the light chain of the antibody and only has the variable region of the heavy chain.

[0019] In the present invention, the term "chimeric antigen receptor" or "CAR" generally refers to a group of polypeptides, which are generally two kinds in the simplest embodiment, which, when in immune effector cells, provide cell specificity for target cells (usually cancer cells) and generate intracellular signals. In some embodiments, CAR includes at least one extracellular antigen binding domain (such as VHH, scFv or a portion thereof), a transmembrane domain and a cytoplasmic signaling domain (also referred to herein as "intracellular signaling domain"), which includes a functional signaling domain derived from a stimulatory molecule and / or a costimulatory molecule as defined below.

[0020] In this invention, the term "human natural phage display antibody library screening" refers to the screening of a human natural phage display antibody library using a target protein or overexpressing cell line as an antigen. Based on the screening results, a sufficient number of monoclonal clones are selected for primary screening. Positive clones are sequenced and sequence diversity analyzed to identify sequence-specific antibody clones, after which antibody sample preparation is performed. Throughout this specification, "human natural phage display antibody library" and "human natural library" are used interchangeably.

[0021] In the present invention, the term "functional variant" generally refers to an amino acid sequence that has substantially the same function as the functional variant and has at least 85% sequence identity therewith.

[0022] As used herein, the term "natural killer cell" or "NK cell" generally refers to a type of cytotoxic lymphocyte in the immune system.

[0023] In the present invention, the term "expression" generally refers to the transcription and / or translation of a specific nucleotide sequence.

[0024] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term encompasses vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0025] The term "humanized" antibody refers to an antibody in which the constant region portion (i.e., CH and CL regions) or all of the constant regions of the antibody are encoded by human antibody genes. Humanized antibodies can greatly reduce the immune side effects caused by heterologous antibodies to the human body. Humanized antibodies include chimeric antibodies, modified antibodies, and fully humanized antibodies. It should be understood that those skilled in the art can prepare suitable humanized forms of the single-domain antibodies of the present invention according to actual needs, which is within the scope of the present invention.

[0026] The technical solution of the present invention includes:

[0027] In one aspect, the present invention provides an anti-CD5 single domain antibody, comprising a heavy chain variable region, wherein the heavy chain variable region comprises complementarity determining regions CDR1, CDR2, and CDR3;

[0028] The CDR1 comprises the amino acid sequence shown in SEQ ID NO: 1;

[0029] SEQ ID NO: 1: SYAMG.

[0030] The CDR2 comprises the amino acid sequence shown in SEQ ID NO: 2;

[0031] SEQ ID NO: 2: GAIHKSGGDTYYADSVK.

[0032] The CDR3 comprises the amino acid sequence shown in SEQ ID NO: 3;

[0033] SEQ ID NO: 3: CAAGDGTDTWDEYDY.

[0034] Specifically, the amino acid sequence of the anti-CD5 single-domain antibody includes an amino acid sequence having at least 80% homology with the amino acid sequence shown in SEQ ID NO: 4.

[0035] SEQ ID NO: 4:

[0036] In another aspect, the present invention provides a nucleotide encoding the above-mentioned anti-CD5 single domain antibody.

[0037] Specifically, the nucleotide sequence is as shown in SEQ ID NO: 5; or a nucleotide sequence that is at least 80% homologous to the sequence shown in SEQ ID NO: 5.

[0038] SEQ ID NO: 5:

[0039] In another aspect, the present invention provides an expression vector comprising the above-mentioned nucleotide sequence.

[0040] Specifically, the expression vector is a prokaryotic cell expression vector or a eukaryotic cell expression vector.

[0041] Preferably, the expression vector is pComb3xss vector.

[0042] In yet another aspect, the present invention provides a host cell comprising the above-mentioned expression vector.

[0043] Specifically, the host cell is a prokaryotic expression cell or a eukaryotic expression cell.

[0044] Preferably, the host cell is Escherichia coli.

[0045] In yet another aspect, the present invention provides a humanized anti-CD5 single-domain antibody, which is obtained by humanizing the anti-CD5 single-domain antibody according to any one of claims 1 to 2.

[0046] Specifically, the amino acid sequence of the humanized anti-CD5 single domain antibody is shown in SEQ ID NO: 6; or an amino acid sequence that is at least 80% homologous to the sequence shown in SEQ ID NO: 6.

[0047] SEQ ID NO: 6:

[0048] In yet another aspect, the present invention provides a nucleotide sequence encoding a humanized anti-CD5 single domain antibody.

[0049] Specifically, the nucleotide sequence is as shown in SEQ ID NO: 7; or a nucleotide sequence that is at least 80% homologous to the sequence shown in SEQ ID NO: 7.

[0050] SEQ ID NO: 7:

[0051] In another aspect, the present invention provides an expression vector comprising the above nucleotide sequence.

[0052] Specifically, the expression vector is a prokaryotic cell expression vector or a eukaryotic cell expression vector.

[0053] Preferably, the expression vector is IG17479-1 01H-PTT5-hFc vector.

[0054] In another aspect, the present invention provides a host cell comprising the above expression vector.

[0055] Specifically, the host cell is a prokaryotic expression cell or a eukaryotic expression cell.

[0056] Preferably, the host cell is a 293TS cell.

[0057] In another aspect, the present invention provides use of the above-mentioned anti-CD5 single domain antibody in the preparation of a drug.

[0058] Specifically, the application includes the above-mentioned anti-CD5 single-domain antibody; nucleotides; expression vectors and / or host cells.

[0059] Specifically, the application includes application in the preparation of drugs for combating cancer or autoimmune diseases.

[0060] More specifically, the cancer comprises a T-cell malignancy or a B-cell malignancy.

[0061] Preferably, the T-cell malignancy comprises acute lymphoblastic leukemia, T-cell large granular lymphocytic leukemia, adult T-cell leukemia / lymphoma, T-cell prolymphocytic leukemia or peripheral T-cell lymphoma.

[0062] Preferably, the B-cell malignancy comprises non-Hodgkin's lymphoma or chronic lymphocytic leukemia.

[0063] More specifically, the autoimmune disease includes rheumatoid arthritis or graft-versus-host disease.

[0064] The beneficial effects of the present invention are:

[0065] (1) The anti-CD5 single-domain antibody provided by the present invention can specifically bind to CD5 with high affinity.

[0066] (2) The single-domain antibody against CD5 provided by the present invention can recognize the D3 membrane-proximal domain of the extracellular region of the CD5 molecule and enhance the immune function of CAR-T cells.

[0067] (3) The present invention also provides a humanized anti-CD5 single domain antibody that reduces the immunogenicity of the molecule in vivo. The humanized CD5 single domain antibody can specifically recognize the CD5 molecule with high affinity. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 is the electrophoresis diagram of the single domain antibody gene in the first round of PCR.

[0069] Figure 2 is the electrophoresis diagram of the second round of PCR single domain antibody gene.

[0070] FIG3 is a FACS assay showing the binding reaction of CD5 single domain antibody to CD5 positive Jurkat cells.

[0071] FIG4 shows the binding reaction of 12C single domain antibody to CD5 positive Jurkat cells and CD5 negative Raji cells detected by FACS.

[0072] FIG5 is a FACS assay showing the binding reaction of 12C single domain antibody to 293TS-CD5D3 cells.

[0073] FIG6 shows the binding and dissociation curves and fitting diagram of the 12C single domain antibody and the CD5 truncated protein.

[0074] FIG7 is a test of the affinity level of 12C single domain antibody binding to target cells.

[0075] FIG8 shows the binding reaction of humanized 12C-huVHH antibody to CD5-positive Jurkat cells, CD5-overexpressing 293T-CD5 cells, and CD5-negative Raji cells detected by FACS.

[0076] FIG9 is a test of the affinity level of humanized 12C-huVHH antibody binding to target cells. DETAILED DESCRIPTION

[0077] The present invention is described below with reference to specific examples. The following examples are not intended to limit the present invention but are merely intended to illustrate the present invention so that the technical solutions of the present invention are more easily understood and grasped. The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0078] The experimental materials used in the present invention are shown in Table 1:

[0079] Table 1 Experimental materials

[0080] Example 1 Construction of anti-CD5 antigen-specific antibody library

[0081] 1. CD5 antigen immunization of alpacas

[0082] Alpaca immunization and serum titer testing uses human CD5 (Arg25-Pro372)-His. A healthy adult alpaca was selected for immunization, with multiple subcutaneous injections of the antigen at the nape of the neck for a total of six immunizations. The absorption of the injection site mass was monitored to confirm the correct immunization. Immunization intervals were two weeks. After the fifth immunization, serum was collected and the antigen titer was determined. When the titer reached 10,000-fold or higher (ELISA method), approximately 100 mL of whole blood was collected, lymphocytes were isolated, and stored at 80°C until further use.

[0083] 2. Construction of phage antibody library and screening of CD5 single domain antibodies

[0084] (1) Using the QIAGEN kit according to the instructions, RNA was extracted from the lysed lymphocytes and purified;

[0085] (2) Using PrimeScript TM II 1st Strand cDNA Synthesis Kit converts RNA into cDNA and constructs a cDNA library;

[0086] (3) Using nested PCR to clone the target gene;

[0087] The nested PCR method was used to amplify the antibody heavy chain variable region (VHH) gene fragment in two rounds of PCR using two sets of primers;

[0088] 1) Perform the first round of PCR amplification:

[0089] Construct PCR primers:

[0090] Table 2 Downstream primers for the first round of PCR amplification

[0091] Table 3 Upstream primers for the first round of PCR amplification

[0092] Table 4 Primer sets for the first round of PCR amplification

[0093] In the table, F and R are amplification primers, where F represents the forward primer and R represents the reverse primer.

[0094] The first round of PCR amplification can obtain a common heavy chain antibody gene fragment greater than 700bp, a heavy chain antibody gene fragment with a light chain deletion between 500bp-700bp, and an antibody heavy chain variable region (VHH) target gene fragment of 400bp-500bp. Through gel electrophoresis analysis, the gene fragment of 500bp-700bp and the gene fragment of 400bp-500bp were screened out for recovery. The electrophoresis results are shown in Figure 1. The PCR product of the 1# group of primers showed two bright bands, one of which was greater than 700bp, representing common antibody DNA, and the other band was between 500bp-700bp in size, representing heavy chain antibody DNA. The PCR products of the 2# and 3# groups of primers were heavy chain antibody variable region fragments (VHH), with a size between 400bp-500bp. The heavy chain antibody gene fragment with a light chain deletion between 500bp-700bp and the antibody heavy chain variable region (VHH) target gene fragment between 400bp-500bp were cut and recovered.

[0095] 2) Perform the second round of amplification:

[0096] Primer construction:

[0097] Table 5 Second round PCR amplification downstream primers

[0098] Table 6 Upstream primers for the second round of PCR amplification

[0099] Amplification of the antibody heavy chain variable region (VHH) target gene. Using the recovered complete heavy chain antibody and its heavy chain variable region gene fragment as a template, a second round of PCR amplification using antibody heavy chain variable region (VHH) specific primers was performed to obtain the antibody heavy chain variable region (VHH) target gene (400bp-500bp). The electrophoresis results are shown in Figure 2. A bright band can be seen, and the antibody heavy chain variable region (VHH) target gene is between 400bp-500bp. That is, the bright band is mixed with multiple antibody heavy chain variable region (VHH) target genes between 400bp-500bp. In the second round of PCR amplification, gel electrophoresis can be used to screen out the antibody heavy chain variable region (VHH) target gene (i.e., the gene fragment between 400bp-500bp).

[0100] (4) Construction of target gene library

[0101] The antibody heavy chain variable region (VHH) target gene fragment obtained above and the phage vector pComb3xss were double-enzymed with BamHI and XhoI endonucleases. After the digestion was completed, the antibody heavy chain variable region (VHH) target gene fragment was ligated to the pComb3xss vector using a ligase to construct a recombinant plasmid. The constructed recombinant plasmid was electroporated into TG1 competent cells to enable fusion expression of the antibody heavy chain variable region (VHH) target gene fragment and the pComb3xss vector. The reservoir size was calculated by gradient dilution plating to be 1.58×10 8 To test the insertion rate of the library, 48 clones were randomly selected for colony PCR. The results showed that the insertion rate reached 94%.

[0102] (5) Expand training

[0103] Phage-containing bacterial seeds stored at -80°C were dissolved at room temperature, mixed, and 500 μL was added to 70 mL of 2YT medium. Helper phage M13KO7 was added for infection. The culture was carried out overnight, centrifuged, and the supernatant was collected and mixed with 20% PEG-2.5M NaCl (the phage was in the supernatant). The precipitate was collected by centrifugation, resuspended in PBS and glycerol, and stored at 80°C for later use.

[0104] Example 2 Screening of specific phage

[0105] Since there are many heavy chain antibody variable region (VHH) fragments amplified by nested PCR, and not all of these gene fragments are Anti CD5 target fragments, after these heavy chain antibody variable region (VHH) fragments are transferred into phage, the target phage needs to be purified. The steps of purifying the target phage are as follows:

[0106] 1. Dilute CD5 protein to 100 μg / mL with coating solution, and coat with 150 μL / well. Incubate at room temperature for 2 hours, then incubate at 4°C overnight.

[0107] 2. Aspirate the coating solution, add 300 μL / well of blocking solution, and incubate at 37°C for 2 hours;

[0108] 3. Aspirate the blocking solution and add phage antibody library (5×10 11 -1×10 12 ), and let stand at room temperature for 2 h;

[0109] 4. Wash the sieve holes 10 times with PBST (containing 0.05% Tween 20) and PBS for 2 minutes each time to wash away unbound phages;

[0110] 5. Add TEA to the sieve to elute the phage, pipette and mix evenly, and let it stand at room temperature for 10 minutes;

[0111] 6. Pipet and resuspend the suspension evenly, then add it into pre-cooled 1M Tris HCl and mix well. Then measure the titer.

[0112] 7. Amplify and purify the amplified phage.

[0113] The above steps 1 to 7 were repeated for 4 rounds, and the phage prepared in step 7 was used as the phage added to the microwells in step 3 of the next round (the first three rounds were full-length CD5 protein (Arg25-Pro372)-His, and the fourth round was CD5 truncated protein (Ser276-Gln368)-Fc; the coating concentration for the first screening was 100 μg / mL, the coating concentration for the second screening round was 10 μg / mL, the coating concentration for the third screening round was 1 μg / mL, and the coating concentration for the fourth screening round was 10 μg / mL). As the number of screening rounds increased, the coating solution concentration decreased step by step, but the eluted phage increased, that is, highly enriched CD5-specific phage was obtained.

[0114] Example 3 Screening of specific positive monoclones

[0115] 1. PCR amplification of the enriched CD5-specific phage to obtain a specific CD5 single-domain antibody gene (PCR product with restriction endonuclease BbsI and BamHI sites);

[0116] The primers used for the PCR amplification are shown in Table 7-Table 8:

[0117] Table 7 PCR amplification downstream primers

[0118] In the table, F and R are amplification primers, where F represents the forward primer and R represents the reverse primer.

[0119] Table 8 PCR amplification upstream primers

[0120] 2. The PCR product and pSJF2 vector prepared in step 1 were treated with restriction endonucleases BbsI and BamHI, respectively, and ligated and recombined using T4 ligase to obtain the plasmid sdAb pSJF2 that can be efficiently expressed in Escherichia coli;

[0121] 3. Randomly pick multiple single colonies from the agar plate with growing colonies and then inoculate them into a 96-well deep-well culture plate containing 2YT liquid medium containing Amp;

[0122] 4. After 4 hours of culture, single clones were inoculated one by one on numbered LB solid plates containing Amp separated by small grids;

[0123] 5. Add IPTG to the deep-well culture plate to induce the cells to a final concentration of 0.5 mM;

[0124] 6. After overnight culture, harvest the protein-expressing bacterial supernatant;

[0125] 7. Perform ELISA test using CD5 antigen and select the Anti CD5 positive clone ELISA test results.

[0126] Table 9 ELISA test results

[0127] The ELISA test results are shown in Table 9: consistent with the 96-well deep-well plate, the first column AH is the vertical column number, the first row 1-12 is the horizontal row number, and the clones are numbered and located (A1-H12) using the horizontal and vertical numbers as coordinates. The clones in the deep-well plate are compared with the OD 450nm The assay readouts were matched; 94 clones were inoculated per plate (G12 and H12 were left uninoculated as negative controls). All but 9A and 7H were negative clones. Positive clones were sequenced and the sequencing results analyzed to obtain the heavy chain variable region (VHH) gene sequences of the positive candidate antibodies.

[0128] Example 4 Expression and purification of CD5 single domain antibody in host Escherichia coli

[0129] After obtaining the above-mentioned positive monoclonal clone, it needs to be expressed to obtain the CD5 single-domain antibody. The subsequent process is mainly through E. coli expression and then purification to obtain the desired CD5 single-domain antibody. The specific operation process is as follows:

[0130] (1) Based on the diversity analysis of ELISA-positive sequences, the seven representative positive clones (1A, 1F, 2G, 6C, 8G, 9H, and 12C) selected in Example 3 were inoculated into 4 mL of LB culture medium containing ampicillin and cultured in a shaking incubator at 37°C overnight.

[0131] (2) Transfer 1 mL of the overnight culture into 100 mL of LB medium containing ampicillin and culture at 37°C, 230 rpm, on a shaker until the OD value reaches 0.4-0.6; add 1.0 mM IPTG and continue to culture overnight, then centrifuge and harvest the bacteria; lyse the bacteria by hypertonic method, centrifuge, and collect the soluble single-domain antibody protein in the supernatant;

[0132] (4) The protein with a purity of more than 95% was obtained by Ni+ ion affinity chromatography.

[0133] Example 5 Flow cytometry assay to detect the binding of CD5 single domain antibody to human CD5 expressing cells

[0134] In order to evaluate the binding activity of the CD5 candidate antibodies on Jurkat cells, a T-line acute lymphoblastic leukemia cell line expressing CD5 molecules, the FACS method was used to detect the binding activity of the candidate antibodies to the CD5 protein on the cells. The results are shown in Figure 3.

[0135] As can be seen from Figure 3, clones 1A, 1F, 2G, 6C, 8G, and 9H do not bind to or bind very weakly to the positive Jurkat cells; the single-domain antibody candidate molecule with clone number 12C has the strongest binding activity on Jurkat cells, a T-lineage acute lymphoblastic leukemia cell line expressing CD5 molecules.

[0136] Example 6 Flow cytometry assay to detect the specificity of 12C single domain antibody

[0137] In order to further analyze the specificity of the primary screening clone 12C single domain antibody and determine the best candidate clone, FACS was used to detect the binding reactivity of 12C antibody to CD5-positive Jurkat cells and CD5-negative Raji cells.

[0138] The results are shown in FIG4 . 12C single domain antibody can bind to CD5-positive Jurkat cells, but does not bind to CD5-negative Raji cells, indicating that the 12C single domain antibody is a specific clone.

[0139] Example 7 12C single-domain antibody recognizes the D3 membrane-proximal domain of the CD5 extracellular region (CD5-D3)

[0140] The CD5 molecule has three extracellular domains (D1, D2, and D3), of which D3 is the membrane-proximal domain with an amino acid sequence of Ser276-Gln368. The gene sequence of the human CD5 truncated protein (CD5-D3) was synthesized by gene synthesis, constructed into a vector, and introduced into Escherichia coli after ligation. The E. coli monoclone was picked and sequenced to obtain the correct plasmid clone, and the plasmid was extracted. 293TS cells in the logarithmic growth phase were transiently transfected with the transfection reagent PEI. The binding activity of the 12C single-domain antibody to 293TS-CD5D3 cells was detected by FACS. As shown in Figure 5, the 12C single-domain antibody was able to bind to the 293TS-CD5D3 cells, indicating that the 12C single-domain antibody can recognize the D3 membrane-proximal domain of the extracellular region of the CD5 molecule.

[0141] Example 8 Protein level affinity determination experiment

[0142] The binding affinity of CD5-VHH to human CD5 truncated proteins was measured using surface plasmon resonance (SPR). Human CD5 truncated proteins were coupled to a CM5 chip to capture CD5-VHH antibodies. Affinity testing was performed using a 12C single-domain antibody as the mobile phase. The results are shown in Figure 6.

[0143] Figure 6 shows the binding and dissociation curves and fitting plots of the 12C single-domain antibody for the CD5 truncated protein. The affinity values ​​for the 12C single-domain antibody and the antigen were calculated and analyzed. The affinity data are shown in Table 10. The results show that the 12C single-domain antibody is a high-affinity anti-human CD5 antibody, with an affinity KD value reaching the nm level.

[0144] Table 10 Antibody SPR detection results

[0145] Example 9 Expression, purification and cell-level affinity detection of CD5 single domain antibodies in eukaryotic cells

[0146] 1. Expression and purification of CD5 single domain antibody in eukaryotic cells

[0147] The single-domain antibody 12C-VHH sequence was inserted into the IG17479-1 01H-PTT5-hFc vector plasmid and expressed in a 293TS cell expression system. After one week of expression, the supernatant was collected and purified using Protein A GE. The protein was quantified using a Nanodrop assay and analyzed by SDS-PAGE.

[0148] 2. Cell-level affinity detection

[0149] The 12C-VHH antibody was serially diluted with FACS diluent and incubated with Jurkat cells. The detection antibody was anti-human IgG Fc PE. The EC value of the antibody was calculated by fitting the curve.50 The results are shown in Figure 7. Figure 7 shows that the 12C single domain antibody binds to CD5 on the surface of Jurkat cells. 50 The value is 10.53nM.

[0150] Example 10 Candidate VHH sequences

[0151] After characterization in the above multiple steps, a single-domain antibody 12C with high affinity and good specificity that recognizes the D3 membrane-proximal domain of the extracellular region of the CD5 molecule was obtained. The amino acid sequence of the 12C single-domain antibody is shown in SEQ ID NO: 4; its complementary determining regions CDR1, CDR2, and CDR3 are shown in Table 11; and its nucleotide sequence is shown in SEQ ID NO: 5.

[0152] Table 11 VHH Antibody CDR Region Information

[0153] Example 11 Antibody Humanization

[0154] To reduce the immunogenicity of the molecule in vivo, the present invention humanized the candidate molecule. The resulting humanized molecule is numbered 12C-huVHH. The amino acid sequence of 12C-huVHH is shown in SEQ ID NO: 6. After codon optimization for expression in human cells, the nucleotide sequence is shown in SEQ ID NO: 7.

[0155] Example 12 Expression and purification of humanized CD5 single domain antibody in eukaryotic cells

[0156] The humanized single-domain antibody 12C-huVHH sequence was inserted into the IG17479-1 01H-PTT5-hFc vector plasmid and expressed in a 293TS cell expression system. After one week of expression, the supernatant was harvested and purified by Protein AGE. The protein was quantified using a Nanodrop assay and analyzed by SDS-PAGE.

[0157] Example 13 Flow cytometry assay to detect the specificity of humanized 12C single domain antibody

[0158] To evaluate the impact of humanization on the specificity of the 12C single-domain antibody, the humanized derivatives were tested by FACS. The binding reactivity of 12C-huVHH to CD5-positive Jurkat cells, CD5-overexpressing 293T-CD5 cells, and CD5-negative Raji cells was assessed by flow cytometry (FACS), as shown in Figure 8.

[0159] The results showed that the humanized 12C single-domain antibody could bind to CD5-positive Jurkat and 293T-CD5 cells, but not to CD5-negative Raji cells, indicating that humanized 12C can specifically recognize the CD5 molecule.

[0160] Example 14: Cellular affinity detection of humanized derivative molecules

[0161] To evaluate the effect of humanization on the binding of antibodies to human CD5-positive Jurkat cells, FACS was used to detect the humanized derivatives. A gradient of dilutions of the antibody was co-incubated with CD5-expressing Jurkat cells, and the detection antibody was anti-human IgG Fc PE. The EC value of the antibody was calculated by fitting the curve. 50 The results are shown in FIG9 , which show that the binding affinity of the humanized derivative molecule 12C-huVHH to CD5 on the surface of Jurkat cells was 9.76 nM.

[0162] In summary, the technical solution of the present invention is combined with genetic engineering methods to obtain a single-domain antibody that specifically recognizes the D3 membrane-proximal domain of the extracellular region of the CD5 molecule, which has good specificity and high affinity.

[0163] Comparative Example 1 is compared with the anti-CD5 single domain antibody in the prior art

[0164] Table 12 is compared with the anti-CD5 single domain antibodies in the prior art Note: NA in the table means not recorded.

[0165] The above results indicate that the anti-CD5 single-domain antibody provided by the present invention can specifically bind to CD5, has a strong binding ability to Jurkat, and can recognize the D3 membrane-proximal domain of the extracellular region of the CD5 molecule.

[0166] The above detailed description is a specific description of one feasible embodiment of the present invention and is not intended to limit the scope of the present invention. It should be noted that any equivalent implementation or modification that does not depart from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the scope of protection of the patent of this invention should be based on the attached requirements.

Claims

1. An anti-CD5 single domain antibody, characterized in that: The anti-CD5 single-domain antibody includes a heavy chain variable region, which includes complementary determining regions CDR1, CDR2 and CDR3; the CDR1 includes the amino acid sequence shown in SEQ ID NO: 1; the CDR2 includes the amino acid sequence shown in SEQ ID NO: 2; and the CDR3 includes the amino acid sequence shown in SEQ ID NO:

3.

2. The anti-CD5 single domain antibody according to claim 1, characterized in that The amino acid sequence of the anti-CD5 single domain antibody includes the amino acid sequence shown in SEQ ID NO:

4.

3. A nucleotide, characterized in that The nucleotide encodes the anti-CD5 single domain antibody according to any one of claims 1-2.

4. The nucleotide according to claim 3, characterized in that The nucleotide sequence is shown in SEQ ID NO:

5.

5. An expression vector, characterized in that: The expression vector comprises the nucleotide according to any one of claims 3-4.

6. A host cell, characterized in that The host cell comprises the expression vector according to claim 5.

7. The host cell according to claim 6, characterized in that The host cell is a prokaryotic expression cell or a eukaryotic expression cell.

8. A humanized anti-CD5 single domain antibody, characterized in that: The humanized anti-CD5 single domain antibody is obtained by humanizing the anti-CD5 single domain antibody according to any one of claims 1 to 2.

9. The humanized anti-CD5 single domain antibody according to claim 8, characterized in that: The amino acid sequence of the humanized anti-CD5 single domain antibody is shown in SEQ ID NO:

6.

10. A nucleotide sequence encoding a humanized anti-CD5 single domain antibody.

11. The nucleotide according to claim 10, characterized in that The nucleotide sequence of the nucleotide is shown in SEQ ID NO:

7.

12. An expression vector comprising the nucleotide sequence of any one of claims 10-11.

13. A host cell comprising the expression vector of claim 12.

14. Use of an anti-CD5 single domain antibody in the preparation of a drug.

15. The use according to claim 14, characterized in that: The drug comprises the anti-CD5 single domain antibody described in any one of claims 1-2, or the nucleotide described in any one of claims 3-4, or the expression vector described in claim 5, or the host cell described in any one of claims 6-7, or the humanized anti-CD5 single domain antibody described in any one of claims 8-9, the nucleotide described in any one of claims 10-11, or the expression vector described in claim 12, or the host cell described in claim 13.

16. The use according to claim 14, characterized in that The drugs include drugs for treating cancer or treating autoimmune diseases.

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