A VHH that binds to human CD8β and its applications

Abstract

The present invention relates to a VHH that binds to human CD8β and its applications. The VHH of the present invention comprises an HCDR1 shown in SEQ ID NO:12, an HCDR2 shown in SEQ ID NO:13, and an HCDR3 shown in SEQ ID NO:14. The VHH of the present invention has good binding ability to human CD8β protein and / or cells expressing human CD8β protein, providing possibilities for CD8β-based detection, diagnosis, and / or treatment.

Description

Technical Field

[0001] The present invention belongs to the field of antibodies, and particularly relates to a VHH that binds to human CD8β and its applications. Background Art

[0002] CD8 is a cell surface glycoprotein composed of two isotypes, α and β (encoded by different genes), and is expressed as an αα homodimer or an αβ heterodimer. The αβ heterodimer is more common. CD8 is expressed on the surface of some T cells and is used to assist the T cell receptor (TCR) in recognizing antigens and participating in the transduction of T cell activation signals. It is also known as a co-receptor of TCR. T cells expressing CD8 (CD8+ T cells) usually differentiate into cytotoxic T cells (CTLs) after activation and can specifically kill target cells. The CD8 molecule stabilizes the interaction between the T cell receptor and MHC-I by directly binding to the appropriate MHC molecule on the target cell, and initiates intracellular signal transduction by phosphorylating lymphocyte-specific protein tyrosine kinase (Lck) of the CD3-related immunoreceptor tyrosine activation motif (ITAM) for activation.

[0003] For certain infectious diseases, immune diseases, tumors, etc., it is necessary to specifically activate or inhibit CTL cells without affecting other immune cells, and thus it is necessary to produce antibodies that can specifically bind to the CD8 molecule on the surface of CTLs. In addition, currently, most anti-CD8 antibodies are anti-CD8α antibodies. However, CD8α is expressed not only on the surface of CTL cells but also on the surface of NK cells. Compared with CD8α, CD8β is more specifically expressed on the surface of CTL cells. 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 specifically 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 bind to human CD8β.

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

[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 specific embodiments, the VHH comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NO: 1-8.

[0015] In a second aspect, the present invention provides a fusion protein, which comprises 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 by a peptide bond or through a linker.

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

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

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

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

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

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

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

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

[0025] The second peptide, from the N-terminus to the C-terminus, comprises: the aforementioned VHH, IL2 and Fc, and the VHH, IL2 and Fc are directly connected by a peptide bond or connected by 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] Advantageous effects:

[0030] The present invention provides a new anti-human CD8β VHH and its application. The VHH of the present invention has good binding ability to human CD8β protein and / or cells expressing human CD8β protein. At the same time, compared with traditional antibodies, VHH has the advantages of small molecular weight, strong tissue penetrability, good stability and low immunogenicity. The VHH of the present invention provides the possibility for CD8β-based detection, diagnosis and / or treatment. Description of the drawings

[0031] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

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

[0033] Figure 2 ELISA binding curve of the antibody expressed in Example 3 of the present invention and CD8β-His protein;

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

[0035] Figure 4 Flow cytometry result analysis of the antibody expressed in Example 6 of the present invention reacting with PBMC and preferentially activating immune cells. Detailed implementation manners

[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 pertains. In some cases, terms with commonly understood meanings are defined in the present invention for the purpose of clarity and / or for ease of reference, and the inclusion of such definitions in the present invention should not be construed as indicating a substantial difference from what is commonly understood in the art. Many of the techniques and procedures described or mentioned in the present invention are well understood by those skilled in the art and are generally employed by those skilled in the art using conventional methods.

[0037] Term definitions

[0038] Unless otherwise specified, the singular forms "a or an" and "the" in the present invention include plural referents.

[0039] Unless otherwise specified, the present invention uses "A and / or B" to include all of the following alternative forms: "A", "B", "A or B", and "A and B".

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

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

[0042] In the present invention, the "variable region" refers to the amino-terminal domain of the heavy or light chain of an antibody that recognizes and binds an antigen, and the amino acid composition and arrangement in this region determine the specificity of the antibody for recognizing the 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., an "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 said variable domain from the heavy-chain variable domain present in a conventional 4-chain antibody (which is referred to herein as the "VH domain") and the light-chain variable domain present in a conventional 4-chain antibody (which is referred to herein as the "VL domain"). The VHH domain specifically binds an epitope without the need for other antigen-binding domains (in contrast to the VH or VL domains in a conventional 4-chain antibody, where the epitope is recognized by the VL domain together with the VH domain).

[0043] In the present invention, "complementary 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 structurally defined loops ("hypervariable loops") and / or contains antigen - contacting residues ("antigen - contact points"). CDRs are mainly responsible for binding to antigen epitopes. The CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N - terminus. The CDRs located within the variable domain of the antibody heavy chain are called HCDR1, HCDR2, and HCDR3, while the CDRs located within the variable domain of the antibody light chain are called LCDR1, LCDR2, and LCDR3. In a given amino acid sequence of a light - chain variable region or a heavy - chain variable region, the precise amino - acid sequence boundaries of each CDR can be determined using any one or a combination of many well - known antibody CDR assignment schemes, including, for example: Chothia, based on the three - dimensional structure of the antibody 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), the international ImMunoGeneTics database (IMGT) (at imgt.cines.fr / on the World Wide Web), and the North CDR definition based on affinity - propagation clustering using a large number of crystal structures. Unless otherwise specified, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above - mentioned ways.

[0044] In the present invention, "Fc" is used to define the carboxy-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. 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 terminus of the heavy chain. However, antibodies produced by a host cell can undergo post-translational cleavage of one or more (particularly one or two) amino acids from the carboxyl terminus of the heavy chain. Thus, an antibody produced by a host cell by expressing a specific nucleic acid molecule encoding a full-length heavy chain can include the full-length heavy chain, or the antibody can include a cleaved variant of the full-length heavy chain. This can be the case where the last two carboxyl-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, EU numbering). Thus, the carboxyl-terminal lysine (Lys447) of the Fc region or the carboxyl-terminal glycine (Gly446) and lysine (Lys447) may or may not be present. Unless otherwise specified 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 sequence similarity between two polynucleotide sequences or between two polypeptides. When the positions in two compared sequences are occupied by the same base or amino acid monomer subunit, for example, if each position in 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 two sequences are optimally aligned, if 6 out of 10 positions in the two sequences match or are homologous, then the two sequences are 60% identical; if 95 out of 100 positions in the two sequences match or are homologous, then the two sequences are 95% identical. Generally, comparison is made when the maximum percentage of identity is obtained by aligning the two sequences.

[0046] In the present invention, IL2 includes both wild-type IL2 and mutant IL2. Additionally, 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 forms, including human PDL2 and PDL2 from other species.

[0047] In the present invention, a "linker" refers to a linking unit that connects two polypeptide fragments, usually having a certain flexibility, and the use of the linker does not cause the loss of the original function of the protein domain. In this text, the linkers present 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 applied in this text can be the same or different.

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

[0049] In the present invention, a "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid linked thereto. In one embodiment, the vector is a "plasmid", which refers to a circular double-stranded DNA loop to which additional DNA segments can be ligated. In another embodiment, the vector is a viral vector, into which additional DNA segments can be ligated into the viral genome. The vectors disclosed herein are capable of autonomous replication in host cells into which they have been introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) or can be integrated into the genome of the host cell after introduction into the host cell, and thus replicate with the host genome (e.g., non-episomal mammalian vectors).

[0050] In the present invention, a "host cell" refers to a cell into which a vector has been introduced. Host cells can include microorganisms (e.g., bacteria), plant, or animal cells. Bacteria that are easily transformable include members of the 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 examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0052] Example 1. Construction of an artificial synthetic nanobody (VHH antibody) library

[0053] By comparing the nucleotide sequences and protein structures of 321 non-redundant nanobody (VHH antibody) sequences in the Protein Data Bank (PDB), the mutation strategy of the CDRs of the artificial synthetic nanobody library was determined. Among them, the length of CDR3 (Kabat) was three types: 12, 15, and 19 amino acids. The commercially available nanobody Caplacizumab (DrugBank ID: DB06081) was selected as the backbone, and the full nucleotide sequence of the monovalent VHH of Caplacizumab was synthesized. 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. Using the extracted single-stranded DNA as a template, the nucleotides in the CDRs region of the nanobody Caplacizumab-VHH were mutated by the Kunkel Mutagenesis method, and a double-stranded DNA library of the mutated nanobody was obtained. The double-stranded DNA was electrotransformed into the competent Escherichia coli SS320 pre-infected with M13KO7 helper phage. After overnight culture, the phage supernatant was collected, and finally, an artificial synthetic nanobody library NanoIW_1.0 lib (Longqi Biotechnology (Shanghai) Co., Ltd.) with a diversity of 6.15×10 10 was constructed and used as a seed library for antibody sequence screening.

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

[0055] Using recombinant human CD8β protein (Beijing Protein Innovation Co., Ltd., 11031-H02H) as an antigen for sorting the NanoIW_1.0 lib library (Longqi Biotechnology (Shanghai) Co., Ltd.). The enzyme-linked immunosorbent tubes were coated with 1 ml of antigen CD8β protein using PBS (pH = 7.4) buffer. The concentration of CD8β protein was 10 μg / ml (first round), 5 μg / ml (second round), and 2 μg / ml (third round), and incubated overnight at 4°C; the next day, the immunosorbent tubes were blocked with 2 ml of PBS buffer containing 10% skim milk powder; 1 ml of blocked phage was added to the immunosorbent tubes and incubated at room temperature for 2 h; washed with PBST 10 times (first round), 30 times (second round), or 50 times (third round); 800 μL of Gly-HCl buffer with pH = 2.2 was added for elution, and immediately 400 μL of Tris-HCl buffer with pH = 8.0 was added for neutralization; then added to 20 ml of E. coli TG1 in the logarithmic growth phase with an OD of approximately 0.8, mixed well and left standing at 37°C for 1 h; 500 μL was taken out to determine the phage titer and store bacteria with glycerol; the remaining bacterial solution was added with M13K07 helper phage at a ratio of 20:1, then mixed well and left standing at 37°C for 30 min; IPTG, Amp, and kanamycin were added at a concentration of 1:1000, shaken at 200 rpm at 30°C overnight; the supernatant was collected, and the phage was precipitated with PEG / NaCl solution and resuspended in 1.5 ml of PBS buffer; the resuspended phage was used for the next round of enrichment screening.

[0056] After each round of panning, the phage supernatant collected after elution was subjected to phage pool ELISA detection. The specific method is as follows. 1 μg / ml of human CD8β protein (Beijing Protein Innovation Co., Ltd., 11031-H02H) was coated on a 96-well ELISA plate and incubated overnight at 4°C. Then, non-specific binding sites were blocked with 5% skim milk powder. After thorough washing, the phage supernatant collected after elution in each round of panning was taken, diluted in half for the first well, and then diluted 3-fold for a total of 8 gradients and added to the 96-well plate, and incubated at 37°C for 2 h. After thorough washing, Anti-M13 Antibody (HRP) (Beijing Protein Innovation Co., Ltd., 11973-MM05T-H) was added and allowed to act at 37°C for 30 min. After thorough washing, TMB was added for color development and allowed to act at room temperature for 5 - 10 min, and finally the reaction was terminated with a stop solution, and the OD values of each well were measured at 450 nm. The results are as Figure 1 shown. After 3 rounds of panning, a significant enrichment of phages binding to human CD8β was observed.

[0057] Table 1 CD8β Nanobody Panning Conditions and Results

[0058]

[0059] Spread the bacteria solution preserved by panning glycerol on a bacterial plate containing Amp antibiotic. The next day, pick monoclonal colonies into a 96-deep well plate for culture. When E. coli TG1 grows to the logarithmic growth phase (OD is about 0.8), add the M13K07 helper phage at a ratio of 20:1 to the bacteria solution, mix well, and let it stand at 37°C for 1 hour. Then add IPTG, Amp, and kanamycin at a concentration of 1:1000, and incubate with shaking at 200 rpm and 30°C overnight. Centrifuge to collect the supernatant, which is the supernatant of the monoclonal phage. Use ELISA to identify the monoclonal phage selected by panning: Coat 1 μg / ml of human CD8β protein (Novoprotein, C581), human CD8α protein (Beijing Sino Biological Inc., 10980-H02H), and Neg-His (PD1-His) protein (Beijing Sino Biological Inc., 10377-H08H) on a 96-well ELISA plate respectively and incubate overnight at 4°C. Then block the non-specific binding sites with 5% skim milk powder. After thorough washing, add the supernatant of the monoclonal phage to the 96-well plate and incubate at 37°C for 2 hours. After thorough washing, add Anti-M13 Antibody (HRP) (Beijing Sino Biological Inc., 11973-MM05T-H), incubate at 37°C for 30 min. After thorough washing, add TMB for color development and let it act at room temperature for 5 - 10 min. Finally, terminate the reaction with the termination solution and measure the OD value of each well at 450 nm.

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

[0061] Table 2 Results of CD8β monoclonal phage ELISA

[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] Constructing, expressing, and purifying anti-CD8β VHH fusion protein using the VHH screened in Example 2: As shown in Table 5, design a fusion protein fused with the anti-CD8β VHH described in Example 2. Through molecular biotechnology, load the nucleic acid fragment encoding this fusion protein onto an expression vector. After correct sequencing, electrotransfer the plasmid into CHO-K1 cells, screen for stable transfected cells, culture the stable transfected cells to express the anti-CD8β VHH fusion protein, and purify the fusion protein using the Fc subunit of the fusion protein through a Protein A purification column for standby.

[0069] Table 5 Information of anti-CD8 VHH fusion protein

[0070]

[0071] Example 4. ELISA Detection of the Binding Activity between anti-CD8β VHH Fusion Protein and CD8β Protein

[0072] Detect the binding activity between anti-CD8β VHH fusion proteins (2715, 2716, 2718, 2719, 2721, 2723, 2724, 2727) and CD8β-His protein by ELISA method. The specific process is as follows: Coat 2 μg / ml of human CD8β-His antigen (Novoprotein, C581) overnight at 4°C in a 96-well ELISA plate. After blocking with 5% skim milk the next day, add the above-mentioned anti-CD8β VHH fusion protein (initial concentration of 50 nM, 5-fold serial dilution, a total of 7 wells, and the last well with PBS), incubate for 1 hour. Use goat anti-human IgG HRP (Abcam, ab97225) as the secondary antibody at a ratio of 1:8000. Incubate at 37°C for 30 min, wash thoroughly, add TMB for color development, and react at room temperature for 5 - 10 min. Finally, terminate the reaction with the stop solution and measure the OD value of each well at 450 nm. The detection results are as Figure 2 and Table 6 show.

[0073] From Figure 2 and Table 6, it can be seen that the 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 for human CD8β protein.

[0074] Table 6 Binding EC50 Values of CD8β Antibodies 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] By flow cytometry fluorescence-activated cell sorting (FACS), using a CytoFlex S flow cytometer (purchased from Beckman Coulter), PBS containing 0.1% BSA was used as a buffer to detect the binding activity of the cell surface target antigen (CD8β) to the anti-CD8β VHH fusion proteins (2715, 2716, 2718, 2719, 2721, 2723) described in Example 3. The specific procedure is as follows:

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

[0079] Goat F(ab') 2 anti-human IgG-Fc (DyLight® 650) (ab98593) fluorescent secondary antibody was prepared at a ratio of 1:200 using the buffer, 50 μl of which was added to each well and mixed well with the cells, incubated in a 4°C refrigerator for 30 minutes; 200 μl of buffer was added to each well, centrifuged at 300 g for 5 minutes, and the supernatant was discarded, then the cells were shaken loose; washed once more; 60 μl of buffer was added to each well and mixed well, and then detected using a flow cytometer. The analysis results of the flow cytometry affinity binding experiment are as Figure 4 shown.

[0080] From Figure 3 it can be seen that 2716 and 2715 have high mean fluorescence intensities when binding to the overexpressing cell line Jurkat161-hCD8β cells. 2721, 2718, 2719, and 2723 all bind to Jurkat161-hCD8β cells.

[0081] Example 6. Activity experiment of anti-CD8β VHH / IL2 fusion protein with PBMC

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

[0083] Table 7 Sequence information of anti-CD8β / IL2 fusion protein

[0084]

[0085] Verify whether the anti-CD8β VHH / IL2 fusion protein described in Table 7 specifically activates CD8+ T cells: Fresh PBMCs (Heyousheng Biotechnology) were purchased, Donor ID: E2404156175W, and the cell viability was detected to be 96.60%. Centrifuge at 500g for 5 minutes. The harvested PBMCs were resuspended and mixed evenly with RPMI1640 + 10% FBS (anti-CD3 (OKT3) 1 μg / mL) medium to prepare a 1×10 6 cells / ml suspension, and then added to a 96-well culture plate, 100 μl / well; then the fusion protein described in Table 7 was diluted to 0.5 nM and added to the above-prepared PBMCs, 100 μl / well. Incubate statically in a 37°C carbon dioxide incubator for 6 days and 11 days, and then take out the PBMCs for flow cytometry to analyze the PBMC cell subsets. CD8+ T and CD4+ T cells were gated out through flow cytometry direct-labeled antibodies.

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

[0087] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to 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% identical to SEQ ID NO: 2 or 5.

3. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the VHH according to claim 1 or 2.

4. A carrier, characterized in that The vector comprises the nucleic acid molecule according to claim 3.

5. A host cell, characterized in that The host cell comprises the vector according to claim 4.

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