VHH combined with human CD8beta and application thereof

Abstract

The invention relates to a VHH combined with human CD8beta and application of the VHH. The VHH provided by the invention comprises an HCDR1 as shown in SEQ ID NO: 12, an HCDR2 as shown in SEQ ID NO: 13 and an HCDR3 as shown in SEQ ID NO: 14. The VHH provided by the invention has good binding capacity with the human CD8 beta protein and / or cells expressing the human CD8 beta protein, and provides possibility for detection, diagnosis and / or treatment based on CD8 beta.

Description

Technical Field

[0001] The present invention belongs to the field of antibodies, and specifically relates to a VHH binding to human CD8β and an application thereof. Background Art

[0002] CD8 is a cell surface glycoprotein composed of two isoforms, α and β, which are encoded by different genes, and expressed as αα homodimers or αβ heterodimers. αβ heterodimers are more common. CD8 is expressed on the surface of some T cells to assist T cell receptors (TCR) in recognizing antigens and participating in the transduction of T cell activation signals. It is also called a co-receptor of TCR. T cells expressing CD8 (CD8+T cells) usually differentiate into cytotoxic T cells (CTLs) after activation, which can specifically kill target cells. CD8 molecules stabilize the T cell receptor-MHC-I interaction through their direct binding to the appropriate MHC molecules on the target cells, and trigger intracellular signal transduction through the phosphorylation of lymphocyte-specific protein tyrosine kinase (Lck) of CD3-related immunoreceptor tyrosine activation motifs (ITAMs) for activation.

[0003] For certain infectious diseases, immune diseases, and tumors, it is necessary to specifically activate or inhibit CTL cells without affecting other immune cells, so it is necessary to produce antibodies that can specifically bind to the CD8 molecule on the surface of CTL. In addition, at present, most anti-CD8 antibodies are anti-CD8α antibodies. However, CD8α is not only expressed on the surface of CTL cells, but also on the surface of NK cells. Compared with CD8α, the expression of CD8β on the surface of CTL cells is more specific. Therefore, in order to better target CTL cells, it is necessary to obtain anti-CD8β antibodies.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The present invention generally provides a VHH, a fusion protein, a nucleic acid molecule, a vector and a host cell that binds to human CD8β.

[0006] In a first aspect, the present invention provides a VHH that binds to human CD8β, the VHH comprising:

[0007] (1) HCDR1 as shown in SEQ ID NO: 12, HCDR2 as shown in SEQ ID NO: 13, and HCDR3 as shown in SEQ ID NO: 14;

[0008] (2) HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, and HCDR3 as shown in SEQ ID NO:11;

[0009] (3) HCDR1 as shown in SEQ ID NO: 15, HCDR2 as shown in SEQ ID NO: 16, and HCDR3 as shown in SEQ ID NO: 17;

[0010] (4) HCDR1 as shown in SEQ ID NO: 18, HCDR2 as shown in SEQ ID NO: 19, and HCDR3 as shown in SEQ ID NO: 20;

[0011] (5) HCDR1 as shown in SEQ ID NO:21, HCDR2 as shown in SEQ ID NO:22, and HCDR3 as shown in SEQ ID NO:23;

[0012] (6) HCDR1 as shown in SEQ ID NO:24, HCDR2 as shown in SEQ ID NO:25, and HCDR3 as shown in SEQ ID NO:26; or,

[0013] (7) HCDR1 as shown in SEQ ID NO:12, HCDR2 as shown in SEQ ID NO:22 and HCDR3 as shown in SEQ ID NO:27.

[0014] In some embodiments, the VHH comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 1-8.

[0015] In a second aspect, the present invention provides a fusion protein comprising the aforementioned VHH.

[0016] In some specific embodiments, the fusion protein further comprises an Fc subunit, and the Fc subunit is directly connected to the VHH via a peptide bond or via a linker.

[0017] In some specific embodiments, the fusion protein comprises a first peptide and a second peptide, wherein:

[0018] The first peptide includes, from N-terminus to C-terminus: the aforementioned VHH and Fc, wherein the VHH and Fc are directly connected via a peptide bond or via a linker;

[0019] The second peptide includes, from N-terminus to C-terminus: the aforementioned VHH and Fc, wherein the VHH and Fc are directly connected via a peptide bond or via a linker.

[0020] In some specific embodiments, the fusion protein comprises a first peptide and a second peptide, wherein:

[0021] The first peptide includes, from N-terminus to C-terminus: a PDL2 extracellular domain and an Fc, wherein the PDL2 extracellular domain and the Fc are directly connected via a peptide bond or via a linker;

[0022] The second peptide includes, from N-terminus to C-terminus: the PDL2 extracellular domain, the aforementioned VHH, IL2 and Fc, and the PDL2 extracellular domain, VHH, IL2 and Fc are directly connected via a peptide bond or via a linker.

[0023] In some embodiments, the fusion protein comprises a first peptide and a second peptide, wherein:

[0024] The first peptide comprises, from N-terminus to C-terminus: the aforementioned VHH and Fc, wherein the VHH and Fc are directly connected via a peptide bond or via a linker;

[0025] The second peptide includes, from N-terminus to C-terminus: the aforementioned VHH, IL2 and Fc, wherein the VHH, IL2 and Fc are directly connected via a peptide bond or via a linker.

[0026] In a third aspect, the present invention provides a nucleic acid molecule encoding the aforementioned VHH or the aforementioned fusion protein.

[0027] In a fourth aspect, the present invention provides a vector comprising the aforementioned nucleic acid molecule.

[0028] In a fifth aspect, the present invention provides a host cell, comprising the aforementioned vector.

[0029] Beneficial effects:

[0030] The present invention provides a novel anti-human CD8β VHH and application thereof. The VHH of the present invention has good binding ability with human CD8β protein and / or cells expressing human CD8β protein. Meanwhile, compared with traditional antibodies, VHH has the advantages of small molecular weight, strong tissue penetration, good stability and low immunogenicity. The VHH of the present invention provides the possibility for detection, diagnosis and / or treatment based on CD8β. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0032] Figure 1 The output phage and ELSIA results of CD8β antigen after three rounds of library screening in Example 2;

[0033] Figure 2 This is a graph showing the binding curve of the antibody expressed in Example 3 of the present invention to the CD8β-His protein by ELISA;

[0034] Figure 3 This is a flow cytometry binding curve diagram of the antibody expressed in Example 3 of the present invention and the overexpressing cell line Jurkat161-CD8β cells;

[0035] Figure 4 This is the flow cytometry analysis of the reaction between the antibody expressed in Example 6 of the present invention and PBMC and the preferential activation of immune cells. DETAILED DESCRIPTION

[0036] Unless otherwise defined, all technical terms, symbols and other scientific terms or expressions used in the present invention are intended to have the meanings commonly understood by those skilled in the art to which the present invention belongs. In some cases, terms with commonly understood meanings are defined in the present invention for clarity and / or for ease of reference, and the inclusion of such definitions in the present invention is not necessarily interpreted as indicating a substantial difference from what is commonly understood in the art. Many of the techniques and procedures described or referred to in the present invention are well understood by those skilled in the art and are commonly employed by those skilled in the art using conventional methods.

[0037] Definition of terms

[0038] As used herein, the singular forms "a or an" and "the" include plural referents unless otherwise stated.

[0039] Unless otherwise stated, the use of "A and / or B" herein includes all of the following alternatives: "A", "B", "A or B" and "A and B".

[0040] Unless otherwise specified, the use of numbers or letters to present headings (e.g., (1), (2), (3), etc., or (a), (b), (c), etc.) in the present invention is only for ease of reading and should not be construed as limiting the order of elements or method steps.

[0041] Unless otherwise specified, when the present invention uses terms such as "first" and "second" to describe polypeptides, it is only used to distinguish more than one polypeptide and should not be understood as other limitations such as a specific order or direction, nor does it imply that the more than one polypeptides are the same or different.

[0042] In the present invention, "variable region" refers to the domain of the amino terminal of the heavy chain or light chain of an antibody that recognizes and binds to an antigen. The composition and arrangement of the amino acids in this segment determine the specificity of the antibody in recognizing an antigen. In the present invention, "VHH (variable domain of heavy chain of heavy-chain antibody)" or "VHH domain" refers to the variable domain of a "heavy chain antibody" (i.e., "antibody lacking a light chain") (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)). The term "VHH domain" is used to distinguish the variable domain from the heavy chain variable domain present in conventional 4-chain antibodies (which is referred to herein as "VH domain") and the light chain variable domain present in conventional 4-chain antibodies (which is referred to herein as "VL domain"). The VHH domain specifically binds an epitope without the need for additional antigen binding domains (this is in contrast to either the VH or VL domains in conventional 4-chain antibodies, where the epitope is recognized by the VL domain together with the VH domain).

[0043] In the present invention, "complementarity determining region" or "CDR region" or "CDR" is a region in the variable domain of an antibody that is highly variable in sequence and forms a structurally determined loop ("hypervariable loop") and / or contains antigen contact residues ("antigen contact points"). CDR is primarily responsible for binding to antigen epitopes. The CDRs of the heavy chain and light chain are generally referred to as CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus. The CDRs located in the variable domain of the heavy chain of an antibody are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located in the variable domain of the light chain of an antibody are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment schemes, including, for example, Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (on the World Wide Web at imgt.cines.fr / ), and Northern CDR definitions based on affinity propagation clustering using a large number of crystal structures. Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above ways.

[0044] In the present invention, "Fc" is used to define the carboxyl terminal region of at least a portion of the constant region of the immunoglobulin heavy chain. The term includes native sequence Fc regions and variant Fc regions. In some embodiments, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminal of the heavy chain. However, the antibody produced by the host cell can undergo post-translational cleavage of one or more (particularly one or two) amino acids from the carboxyl terminal of the heavy chain. Therefore, the antibody produced by the host cell by expressing a specific nucleic acid molecule encoding a full-length heavy chain can include a full-length heavy chain, or the antibody can include a cleaved variant of the full-length heavy chain. This may be the case where the last two carboxyl terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, EU numbering). Therefore, the carboxyl terminal lysine (Lys447) or the carboxyl terminal glycine (Gly446) and lysine (Lys447) in the Fc region may be present or may not be present. Unless otherwise indicated in the present disclosure, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described by Kabat et al. (Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, National Institutes of Health, Bethesda, Maryland, 1991).

[0045] In the present invention, "identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When the positions in the two compared sequences are occupied by the same base or amino acid monomer subunit, for example, if every position of the two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage of identity between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100. For example, when the sequences are optimally aligned, if 6 out of 10 positions in the two sequences are matched or homologous, then the two sequences are 60% identical; if 95 out of 100 positions in the two sequences are matched or homologous, then the two sequences are 95% identical. In general, comparison is performed when the two sequences are aligned to obtain the maximum percentage of identity.

[0046] In the present invention, IL2 includes both wild-type IL2 and mutant IL2. In addition, IL2 of the present invention includes not only human IL2, but also IL2 from other species. Similarly, "PDL2" of the present invention includes wild-type and mutant types, including human PDL2 and PDL2 from other species.

[0047] In the present invention, "linker" refers to a connecting unit connecting two polypeptide fragments, which is usually flexible, and the use of the linker will not cause the original function of the protein domain to be lost. In this article, the linkers appearing in the same structure can be the same or different. The linker can be a peptide linker, which contains one or more amino acids, typically about 1-30, 2-24 or 3-15 amino acids. The linkers used in this article can be the same or different.

[0048] In the present invention, "nucleic acid molecule" refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is operably linked to the coding sequence.

[0049] In the present invention, "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. In one embodiment, the vector is a "plasmid", which refers to a circular double-stranded DNA loop into which other DNA segments can be connected. In another embodiment, the vector is a viral vector, in which other DNA segments can be connected to the viral genome. The vector disclosed herein can be autonomously replicated in the host cell introduced into them (e.g., bacterial vectors and additional mammalian vectors with a bacterial origin of replication) or can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating with the host genome (e.g., non-additional mammalian vectors).

[0050] In the present invention, "host cell" refers to a cell into which a vector has been introduced. Host cells may include microorganisms (e.g., bacteria), plants, or animal cells. Easily transformed bacteria include members of the family Enterobacteriaceae, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese Hamster Ovary cell line) and NS0 cells.

[0051] The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. If specific conditions are not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0052] Example 1. Construction of artificially synthesized nanobody (VHH antibody) library

[0053] By comparing the nucleotide sequences and protein structures of 321 non-repetitive nanoantibodies (VHH antibodies) in the PDB (Protein Data Bank) database, the mutation strategy of the CDRs of the artificially synthesized nanoantibody library was determined, among which the lengths of CDR3 (Kabat) were three types: 12, 15 and 19 amino acids. The commercially available nanoantibody Caplacizumab (DrugBank ID: DB06081) was selected as the skeleton to synthesize the full nucleotide sequence of the monovalent VHH of the nanoantibody Caplacizumab. After cloning the artificially synthesized full nucleotide sequence into the HP153 phage vector, the single-stranded DNA of the HP153-Caplacizumab VHH phage vector was extracted, and the extracted single-stranded DNA was used as a template to mutate the nucleotides in the CDRs region of the nanoantibody Caplacizumab-VHH using the Kunkel Mutagenesis method, and obtain the double-stranded DNA library of the mutated nanoantibody. The double-stranded DNA was electroporated into competent E. coli SS320 cells pre-infected with M13KO7 helper phage. The phage supernatant was collected after overnight culture, and a diversity of 6.15×10 10 The artificial synthetic nanoantibody library NanoIW_1.0 lib (Longqi Biotechnology (Shanghai) Co., Ltd.) was used as a seed library for antibody sequence screening.

[0054] Example 2: Screening of anti-CD8β antibodies using NanoIW_1.0 lib

[0055] Recombinant human CD8β protein (Beijing Sino Biological, 11031-H02H) was used as an antigen for sorting of NanoIW_1.0 lib library (Longqi Biotechnology (Shanghai) Co., Ltd.). 1 ml of antigen CD8β protein was coated with PBS (pH=7.4) buffer solution, and the concentration of CD8β protein was 10 μg / ml (first round), 5 μg / ml (second round), and 2 μg / ml (third round) at 4°C overnight; the next day, 2 ml of PBS buffer solution containing 10% skimmed milk powder was used to block the immunotube; 1 ml of blocked phage was added to the immunotube and incubated at room temperature for 2 h; PBST was used to wash 10 times (first round), 30 times (second round) or 50 times (third round); 800 μL of Gly-HCl buffer solution with pH=2.2 was added for elution, and 400 μL of Tris-HCl buffer with pH=8.0 was added for neutralization immediately; then added to 20 ml of E. coli with an OD of about 0.8 in the logarithmic growth phase. In TG1, mix well and let stand at 37°C for 1h; take out 500μL to determine the phage titer and glycerol preservation; add 20:1 M13K07 helper phage to the remaining bacterial solution, then mix well and let stand at 37°C for 30min; add IPTG, Amp and kanamycin at a concentration of 1:1000, 200rpm, 30°C, and shake and culture overnight; collect the supernatant, precipitate the phage with PEG / NaCl solution, and resuspend in 1.5ml PBS buffer; the resuspended phage is used for the next round of enrichment screening.

[0056] After each round of panning, the phage supernatant collected after elution was taken for phage pool ELISA detection. The specific method is as follows: 1μg / ml human CD8β protein (Beijing Yiqiao Shenzhou, 11031-H02H) was coated on a 96-well ELISA plate and incubated at 4°C overnight. Then use 5% skim milk powder to block nonspecific binding sites. After thorough washing, take the phage supernatant collected after each round of panning and elution, dilute it in half as the first well, and then dilute it 3 times, a total of 8 gradients, add it to the 96-well plate, and incubate at 37°C for 2 hours. After thorough washing, add Anti-M13 Antibody (HRP) (Beijing Yiqiao Shenzhou, 11973-MM05T-H), act at 37°C for 30 minutes, add TMB for color development after thorough washing, act at room temperature for 5-10 minutes, and finally terminate the reaction with stop solution, and measure the OD value of each well at 450nm. The results are as follows Figure 1 As shown, after 3 rounds of panning, significant enrichment of phages binding to human CD8β was observed.

[0057] Table 1 CD8β nanobody selection conditions and results

[0058]

[0059] The above-mentioned glycerol-preserved bacterial solution was applied to a bacterial plate containing Amp antibiotics. The next day, a single clone was selected and cultured in a 96-deep-well plate. When E. coli TG1 grew to the logarithmic growth phase (OD of about 0.8), 20:1 M13K07 helper phage was added to the bacterial solution, mixed and allowed to stand at 37°C for 1 hour, and then IPTG, Amp and kanamycin were added at a concentration of 1:1000, 200 rpm, 30°C, and shaken and cultured overnight; the supernatant was collected by centrifugation to obtain the supernatant of the monoclonal phage. ELISA was used to identify the monoclonal phages selected by panning: 1 μg / ml human CD8β protein (Novoprotein, C581), human CD8α protein (Beijing Sino Biological, 10980-H02H) and Neg-His (PD1-His) protein (Beijing Sino Biological, 10377-H08H) were coated on 96-well ELISA plates, respectively, and incubated at 4°C overnight. Then, non-specific binding sites were blocked with 5% skim milk powder. After thorough washing, the monoclonal phage supernatant was added to the 96-well plate and incubated at 37°C for 2 hours. After thorough washing, Anti-M13Antibody (HRP) (Beijing Sino Biological, 11973-MM05T-H) was added and incubated at 37°C for 30 minutes. After thorough washing, TMB was added for color development. After 5-10 minutes at room temperature, the reaction was terminated with stop solution, and the OD value of each well was measured at 450nm.

[0060] As shown in Table 2, 8 phage antibody clones that can specifically bind to human CD8β were obtained by phage monoclonal ELISA, namely 8B01-A8, 8B01-C1, 8B01-D4, 8B01-F3, 8B01-F11, 8B02-A4, 8B02-D2, and 8B02-E12. The variable region sequences and CDRs sequences were obtained by sequencing and bioinformatics analysis (see Tables 3-4).

[0061] Table 2 CD8β monoclonal phage ELISA results

[0062]

[0063] Table 3 VHH sequence information

[0064]

[0065] Table 4 CDR region sequence information

[0066]

[0067] Example 3. Expression and purification of anti-CD8β VHH fusion protein

[0068] The anti-CD8β VHH fusion protein was constructed, expressed and purified using the VHH obtained by screening in Example 2: As shown in Table 5, a fusion protein fused with the anti-CD8β VHH described in Example 2 was designed, and the nucleic acid fragment encoding the fusion protein was loaded on an expression vector by molecular biological technology. After correct sequencing, the plasmid was electroporated into CHO-K1 cells, stable transfected cells were screened, and stable transfected cells were cultured to express the anti-CD8β VHH fusion protein, and the fusion protein was purified by Protein A purification column using the Fc subunit of the fusion protein for later use.

[0069] Table 5 Anti-CD8 VHH fusion protein information

[0070]

[0071] Example 4. ELISA detection of binding activity of anti-CD8β VHH fusion protein to CD8β protein

[0072] The binding activity of anti-CD8β VHH fusion protein (2715, 2716, 2718, 2719, 2721, 2723, 2724, 2727) and CD8β-His protein was detected by ELISA method. The specific process is as follows: 2μg / ml human CD8β-His antigen (Novoprotein, C581) was coated in a 96-well ELISA plate at 4°C overnight. After blocking with 5% skim milk powder the next day, the anti-CD8β VHH fusion protein as described above was added (50nM initial concentration, 5-fold gradient dilution, a total of 7 wells, and the last well was PBS). Incubate for 1 hour, and use goat anti-human IgG HRP (Abcam, ab97225) as the secondary antibody at 1:8000. Incubate at 37°C for 30 minutes, add TMB for color development after thorough washing, and act at room temperature for 5-10 minutes. Finally, the reaction is terminated with stop solution, and the OD value of each well is measured at 450nm. The test results are as follows Figure 2 And as shown in Table 6.

[0073] from Figure 2 As shown in Table 6, fusion protein 2716 has the strongest ability to bind to human CD8β protein, followed by 2721 and 2718. 2715, 2719, 2723, 2724 and 2727 all show affinity to human CD8β protein.

[0074] Table 6 EC50 values ​​of binding of CD8β antibody to human CD8β-His protein

[0075]

[0076] Example 5. Detection of the binding activity of anti-CD8β VHH fusion protein to cell surface CD8β

[0077] The binding activity of the cell surface target antigen (CD8β) and the anti-CD8β VHH fusion protein (2715, 2716, 2718, 2719, 2721, 2723) described in Example 3 was detected by flow cytometry fluorescence sorting technology (FACS) using a CytoFlex S flow cytometer (purchased from Beckman Co.) and PBS containing 0.1% BSA as a buffer. The specific process is as follows:

[0078] Use buffer to prepare the concentration to 1x10 6 cells / ml of target cells (i.e., Jurkat-hCD8β overexpressing cell line) were added to a 96-well U-shaped plate, 50 μl per well; anti-CD8β VHH fusion protein (100 nM initial concentration, 5-fold gradient dilution, 7 wells in total, and PBS was used for the last well) was added, and the prepared antibodies of different concentrations were added to the already plated target cells at a volume of 50 μl / well and mixed; incubated in a refrigerator at 4°C for 1 hour; 150 μl of buffer was added to each well, centrifuged at 300g for 5 minutes, and the cells were shaken loose after discarding the supernatant; and the washing was repeated once.

[0079] Use buffer to prepare goat F(ab')2 anti-human IgG-Fc (DyLight® 650) (ab98593) fluorescent secondary antibody at 1:200, add 50μl to each well and mix, incubate in 4℃ refrigerator for 30 minutes; add 200μl buffer to each well, centrifuge at 300g for 5 minutes, discard the supernatant and shake the cells loose; repeat the wash once; add 60μl buffer to each well and mix, then detect with flow cytometry. The analysis results of the flow affinity binding experiment are shown in the figure. Figure 4 shown.

[0080] from Figure 3 It can be seen that 2716 and 2715 have high average fluorescence intensity when binding to the overexpression cell line Jurkat161-hCD8β cells. 2721, 2718, 2719 and 2723 all bind to Jurkat161-hCD8β cells.

[0081] Example 6. Anti-CD8β VHH / IL2 fusion protein and PBMC activity experiment

[0082] Construction, expression and purification of anti-CD8β VHH / IL2 fusion protein: After the binding activity was verified by ELISA and FACS, the screened anti-CD8β VHH (8B01-C1, 8B01-F11 and 8B02-D2) and the negative control VHH fragment sequence were used to construct fusion proteins (see Table 7 for details) for constructing anti-CD8β VHH / IL2 fusion proteins (see Table 7 for details). The nucleic acid sequence encoding the fusion protein was loaded on an expression vector by molecular biology techniques. After the sequencing was correct, the plasmid was electroporated into CHO-K1 cells, stable cells were screened, and stable cells were cultured to express the fusion proteins shown in Table 7, and the fusion proteins were purified by using the Fc subunit of the fusion protein through a Protein A purification column for later use.

[0083] Table 7 Anti-CD8β / IL2 fusion protein sequence information

[0084]

[0085] Verify whether the anti-CD8β VHH / IL2 fusion protein described in Table 7 specifically activates CD8+ T cells: Purchase fresh PBMC (Heyousheng Biotechnology), Donor ID: E2404156175W, detect the cell viability to be 96.60%, centrifuge at 500g for 5 minutes. The harvested PBMC was resuspended and mixed in RPMI1640+10% FBS (anti-CD3 (OKT3) 1ug / mL) medium to prepare 1×10 6 cells / ml suspension, and then added to a 96-well culture plate, 100ul / well; then the fusion protein described in Table 7 was diluted to 0.5nM and added to the above-laid PBMC, 100ul / well. The PBMC was cultured in a 37°C carbon dioxide incubator for 6 and 11 days, and then the PBMC was taken out for flow cytometry analysis of PBMC cell subsets. CD8+T and CD4+T cells were gated by flow cytometry with direct labeled antibodies.

[0086] from Figure 4 It can be seen that on the 11th day of culture, anti-CD8β VHH / IL2 fusion proteins 2900, 2905 and 2908 had a significant biased activation effect on CD8+T cells compared with the control.

[0087] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A VHH that binds to human CD8β, characterized in that The VHHs include: HCDR1 as shown in SEQ ID NO:12, HCDR2 as shown in SEQ ID NO:13 and HCDR3 as shown in SEQ ID NO:

14.

2. The VHH according to claim 1, characterized in that The VHH comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 2 or 5.

3. A fusion protein, characterized in that The fusion protein comprises the VHH according to claim 1 or 2.

4. The fusion protein according to claim 3, characterized in that The fusion protein further comprises an Fc subunit, and the Fc subunit is directly linked to the VHH via a peptide bond or via a linker.

5. The fusion protein according to claim 3, characterized in that The fusion protein comprises a first peptide and a second peptide, wherein: The first peptide comprises, from N-terminus to C-terminus: the VHH and Fc of claim 1 or 2, wherein the VHH and Fc are directly connected via a peptide bond or via a linker; The second peptide comprises, from N-terminus to C-terminus: the VHH and Fc according to claim 1 or 2, wherein the VHH and Fc are directly connected via a peptide bond or via a linker.

6. The fusion protein according to claim 3, characterized in that The fusion protein comprises a first peptide and a second peptide, wherein: The first peptide includes, from N-terminus to C-terminus: a PDL2 extracellular domain and an Fc, wherein the PDL2 extracellular domain and the Fc are directly connected via a peptide bond or via a linker; The second peptide comprises, from N-terminus to C-terminus, the PDL2 extracellular domain, the VHH according to claim 1 or 2, IL2 and Fc, wherein the PDL2 extracellular domain, VHH, IL2 and Fc are directly connected via a peptide bond or via a linker.

7. The fusion protein according to claim 3, characterized in that The fusion protein comprises a first peptide and a second peptide, wherein: The first peptide comprises, from N-terminus to C-terminus: the VHH and Fc of claim 1 or 2, wherein the VHH and Fc are directly connected via a peptide bond or via a linker; The second peptide comprises, from N-terminus to C-terminus: the VHH, IL2 and Fc of claim 1 or 2, wherein the VHH, IL2 and Fc are directly connected via a peptide bond or via a linker.

8. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the VHH according to claim 1 or 2 or the fusion protein according to any one of claims 3-7.

9. A carrier, characterized in that The vector comprises the nucleic acid molecule of claim 8.

10. A host cell, characterized in that The host cell comprises the vector according to claim 9.

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