Nanometer antibody specifically combined with CD161 and application thereof

Abstract

The invention belongs to the field of nano antibodies, and particularly relates to a nano antibody specifically combined with CD161 and application of the nano antibody. The nano antibody is specifically combined with the human CD161 protein at an EC50 value which is not greater than 0.5 nM; and the nano antibody, the CLEC2D and the human CD161 protein are competitively combined. Compared with the prior art, the method has the beneficial effects that the nano antibody specifically combined with CD161 is screened by using a biological genetic engineering technology, and the affinity and the specificity of the antibody are relatively good.

Description

Technical Field

[0001] This invention belongs to the field of nanobodies, specifically relating to a nanobody that can specifically bind to CD161 and its application as a pharmaceutical active ingredient. Background Technology

[0002] With the development of medicine, major breakthroughs have been made in the field of cancer treatment, especially the emergence of cancer immunotherapy represented by PD-1 / PD-L1 immune checkpoint inhibitors, which can be said to have completely changed the landscape of cancer treatment and brought hope to cancer patients.

[0003] A 2021 research paper published in the top international academic journal Cell, titled "Inhibitory CD161 receptor identified in glioma-infiltrating T cells by single-cell analysis," discovered a new potential target for the treatment of malignant brain tumors: CD161. The study showed that the CD161 receptor is activated by a molecule called CLEC2D on tumor cells and immunosuppressive cells in the brain. Activation of CD161 weakens the T cell response to tumor cells, thereby inhibiting the efficacy of immunotherapy.

[0004] The interaction between CLEC2D and CD161 can lead to various types of cancer. Therefore, blocking the CLEC2D-CD161 interaction provides a new therapeutic option for treating a variety of cancers.

[0005] US Patent Application No. US20210122826A1, filed on August 24, 2020, discloses a scheme for combining a monoclonal antibody with human CD161 and its uses. Research on various targets and related drugs has largely focused on monoclonal antibodies and their derivatives. Given the advantages of nanobodies—strong tissue penetration, easy identification of hidden sites, flexible expression methods, high stability, and simple and easily modifiable structures—CD161 inhibitors with higher affinity and other key drug properties, especially nanobodies, still require further in-depth research and development. Summary of the Invention

[0006] The purpose of this invention is to provide a nanobody that specifically binds to CD161 and its application.

[0007] A first aspect of the present invention provides a nanobody that specifically binds to CD161, wherein the nanobody specifically binds to human CD161 protein with an EC50 value of not more than 0.5 nM; wherein the nanobody and CLEC2D competitively bind to human CD161 protein.

[0008] A second aspect of the present invention discloses a nanobody that specifically binds to CD161, wherein the nanobody includes a heavy chain variable domain, and the heavy chain variable domain includes an amino acid sequence of heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3.

[0009] The heavy chain CDR1 contains any sequence selected from SEQ ID NO:32-SEQ ID NO:39 or a variant thereof with up to 5 amino acid substitutions;

[0010] The heavy chain CDR2 comprises any sequence selected from SEQ ID NO:40-SEQ ID NO:41 or a variant thereof with up to 5 amino acid substitutions;

[0011] The heavy chain CDR3 contains any sequence selected from SEQ ID NO:42-SEQ ID NO:45 or a variant thereof with up to 5 amino acid substitutions.

[0012] Preferably, the amino acid sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 are one of the following (1)-(10):

[0013] (1) CDR1 shown in SEQ ID NO:32, CDR2 shown in SEQ ID NO:41, and CDR3 shown in SEQ ID NO:45;

[0014] (2) CDR1 shown in SEQ ID NO:33, CDR2 shown in SEQ ID NO:41, and CDR3 shown in SEQ ID NO:45;

[0015] (3) CDR1 shown in SEQ ID NO:34, CDR2 shown in SEQ ID NO:41, and CDR3 shown in SEQ ID NO:45;

[0016] (4) CDR1 shown in SEQ ID NO:35, CDR2 shown in SEQ ID NO:40, and CDR3 shown in SEQ ID NO:42;

[0017] (5) CDR1 shown in SEQ ID NO:36, CDR2 shown in SEQ ID NO:41, and CDR3 shown in SEQ ID NO:45;

[0018] (6) CDR1 shown in SEQ ID NO:37, CDR2 shown in SEQ ID NO:41, and CDR3 shown in SEQ ID NO:45;

[0019] (7) CDR1 shown in SEQ ID NO:38, CDR2 shown in SEQ ID NO:41, and CDR3 shown in SEQ ID NO:45;

[0020] (8) CDR1 shown in SEQ ID NO:38, CDR2 shown in SEQ ID NO:41, and CDR3 shown in SEQ ID NO:43;

[0021] (9) CDR1 shown in SEQ ID NO:38, CDR2 shown in SEQ ID NO:41, and CDR3 shown in SEQ ID NO:44;

[0022] (10) CDR1 shown in SEQ ID NO:39, CDR2 shown in SEQ ID NO:41, and CDR3 shown in SEQ ID NO:45.

[0023] In one embodiment, in any one or more CDRs of the heavy chain CDR1, CDR2, and CDR3, one to five arbitrary amino acid residues may be substituted with their conserved amino acids. Specifically, in the heavy chain CDR1, one to five amino acid residues may be substituted with their conserved amino acids; in the heavy chain CDR2, one to five amino acid residues may be substituted with their conserved amino acids; and in the heavy chain CDR3, one to five amino acid residues may be substituted with their conserved amino acids.

[0024] The term "CD161-specific nanobody" in this invention includes not only complete nanobodies but also fragments, derivatives, and analogs of said CD161-specific nanobodies. As used herein, the terms "fragment," "derivative," and "analyte" have the same meaning and refer to polypeptides that substantially retain the same biological function or activity as the antibodies of this invention. The polypeptide fragments, derivatives, or analogs of this invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence, secretion sequence, or sequence used to purify this polypeptide, or a proteogenic sequence, or a fusion protein formed with an Fc tag). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0025] In a preferred embodiment, the sequence of the CD161-specifically binding nanobody further includes a framework region (the nanobody structure is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4); the framework region FR includes the amino acid sequences of FR1, FR2, FR3, and FR4; the amino acid sequences of the framework region FR are as follows:

[0026] FR1 or a variant of FR1 shown in any of SEQ ID NO:21-25, wherein the variant of FR1 contains substitutions of up to 5 amino acids in the FR1;

[0027] The FR2 or a variant of FR2 shown in any one of SEQ ID NO:26-27, wherein the variant of FR2 contains substitutions of up to 5 amino acids in the FR2;

[0028] FR3 or a variant of FR3 shown in any of SEQ ID NO:28-29, wherein the variant of FR3 contains substitutions of up to 5 amino acids in the FR3;

[0029] The FR4 or a variant thereof shown in any one of SEQ ID NO:30-31, wherein the variant thereof contains substitutions of up to 5 amino acids.

[0030] A third aspect of the present invention is to provide a nanobody that specifically binds to CD161, wherein the amino acid sequence of the nanobody has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology with any one of the sequences in SEQ ID NO:1-10.

[0031] As used herein, the term "sequence homology" refers to the degree to which two (nucleotide or amino acid) sequences have identical residues at the same positions in an alignment, and is typically expressed as a percentage. Preferably, homology is determined over the overall length of the sequences being compared. Thus, two copies having completely identical sequences have 100% homology.

[0032] In a preferred embodiment, the amino acid sequence of the CD161-specifically binding nanobody can be a variant of any one of SEQ ID NO: 1-10, wherein the FR1, FR2, FR3, or FR4 sequences contain up to 5 amino acid substitutions compared to the original FR.

[0033] A fourth aspect of the present invention is to provide an Fc fusion antibody or humanized antibody that specifically binds to CD161 of any of the foregoing.

[0034] Fc fusion antibodies are novel proteins produced by fusing the Fc fragment of a target antibody with a biologically active functional protein molecule.

[0035] "Humanized antibodies" refer to antibodies obtained by fusing the heavy chain variable domain of a target antibody (such as an animal antibody) with the constant region of a human antibody, or by transplanting the complementarity-determining region (CDR1-3 sequence) of a target antibody into the variable region of a human antibody, or by mutating the target antibody according to the characteristics of the human antibody backbone region (FR1-4). Humanized antibodies can be prepared by synthesis or site-directed mutagenesis.

[0036] A fifth aspect of the present invention discloses a recombinant polypeptide comprising the aforementioned nanobody that specifically binds to CD161; the recombinant polypeptide includes, but is not limited to, antibody-immunoconjugates, bispecific molecules, chimeric antigen receptors (CARs), multispecific molecules, multi-epitope antibodies, or multivalent antibodies.

[0037] For example, the recombinant polypeptide can be a nanobody containing any combination of CDRs (1)-(10) as described above, or a nanobody having the sequences shown in SEQ ID NO. 1-10, or a nanobody having at least 80% homology with those in SEQ ID NO. 1-10, or a multi-epitope antibody, bispecific antibody, multispecific antibody, or multivalent antibody; for example, the multi-epitope antibody can be composed of more than one sequence in SEQ ID NO. 1-10; the multivalent antibody can be composed of SEQ ID NO. 1-10. One of the sequences in IDNO.1-10 is repeated several times to form a multispecific antibody, including but not limited to trispecific antibodies and tetraspecific antibodies; the recombinant polypeptide can also be a conventional antibody or its functional fragment, wherein the variable domain of the heavy chain of the antibody is composed of the aforementioned CD161-specifically binding nanobody; preferably, the functional fragment is the Fab, Fab', (Fab')2, Fv, scFv or sdFv structure of the conventional antibody; the CD161-specifically binding nanobody can also be a component of antibody-drug conjugates (ADCs) and chimeric antigen receptors (CARs); in addition, the recombinant protein can be a fragment, derivative or analog of the aforementioned nanobody.

[0038] A sixth aspect of the present invention is to provide a nucleotide molecule that encodes the aforementioned nanobody that specifically binds to CD161.

[0039] Specifically, its nucleotide sequence has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with any one of SEQ ID NO: 11-20, and the nanobody it encodes is capable of specifically binding to human CD161 protein.

[0040] A seventh aspect of the present invention is to provide a carrier comprising the aforementioned nucleotide molecules.

[0041] In a preferred embodiment, the vector used is RJK-V4-hFC, but other general-purpose expression vectors can also be selected as needed.

[0042] An eighth aspect of the invention is to provide a host cell that can express the aforementioned CD161-specific nanobodies, Fc fusion antibodies, humanized antibodies, or recombinant peptides. Preferably, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

[0043] In another preferred embodiment, the host cell includes prokaryotic or eukaryotic cells, including bacteria and fungi.

[0044] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, mammalian cells, bacteriophages, or combinations thereof.

[0045] In another preferred embodiment, the prokaryotic cells are selected from the group consisting of Escherichia coli, Bacillus subtilis, lactic acid bacteria, Streptomyces, Proteus mirabilis, or combinations thereof.

[0046] In another preferred embodiment, the eukaryotic cells are selected from the group consisting of Pichia pastoris, Saccharomyces cerevisiae, Schizosomalidomiae, Trichoderma, or combinations thereof.

[0047] In another preferred embodiment, the eukaryotic cells are selected from the group consisting of insect cells such as armyworms, plant cells such as tobacco, BHK cells, CHO cells, COS cells, myeloma cells, or combinations thereof.

[0048] In another preferred embodiment, the host cell is a suspension ExpiCHO-S cell.

[0049] In another preferred embodiment, the host cell is a suspension 293F cell.

[0050] A ninth aspect of the present invention provides a method for preparing a nanobody that specifically binds to CD161, comprising culturing host cells capable of expressing a nanobody that specifically binds to CD161, expressing the nanobody that specifically binds to CD161, and separating and purifying it.

[0051] A tenth aspect of the invention is to provide a pharmaceutical composition comprising the aforementioned CD161-specifically binding nanobody and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally determined based on the isoelectric point of the antibody (the pH of the aqueous carrier medium must deviate from the isoelectric point of the antibody by approximately 2). The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intravenous, transdermal (direct application or patching to the affected area).

[0052] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the aforementioned CD161-specifically binding nanobody and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared using conventional methods with physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions.

[0053] The eleventh aspect of the present invention is to provide a diagnostic or detection kit comprising the aforementioned nanobody that specifically binds to CD161.

[0054] In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, buffer, etc. (refer to the prior art). Since human peripheral blood contains CD161 protein, related detection or diagnosis can be performed.

[0055] A twelfth aspect of the present invention is to provide a therapeutic agent for treating or preventing a disease comprising the aforementioned CD161-specific nanobody as an active ingredient. The disease is a condition related to abnormal CD161 expression.

[0056] The thirteenth aspect of the present invention is to provide the use of the aforementioned CD161-specifically binding nanobody or the aforementioned pharmaceutical composition in the preparation of a medicament for treating a disease.

[0057] In a preferred embodiment, the diseases include, but are not limited to, head and neck squamous cell carcinoma; skin squamous cell carcinoma; Burkitt lymphoma, colorectal cancer, diffuse large B-cell lymphoma, small bowel cancer, Hodgkin lymphoma, triple-negative breast cancer, esophageal cancer, non-small cell lung cancer, T-cell lymphoma, HR-positive breast cancer, follicular lymphoma, hematologic malignancies, melanoma, lung cancer, glioma, liver cancer, and autoimmune diseases.

[0058] The fourteenth aspect of the present invention discloses a method for treating a disease in a subject of need, comprising the step of administering to the subject of need an effective amount of the aforementioned CD161-specifically binding nanobody or the aforementioned pharmaceutical composition. Diseases include, but are not limited to, head and neck squamous cell carcinoma; skin squamous cell carcinoma; Burkitt lymphoma, colorectal cancer, diffuse large B-cell lymphoma, small bowel cancer, Hodgkin lymphoma, triple-negative breast cancer, esophageal cancer, non-small cell lung cancer, T-cell lymphoma, HR-positive breast cancer, follicular lymphoma, hematologic malignancies, melanoma, lung cancer, glioma, liver cancer, and autoimmune diseases.

[0059] By administering an effective amount of CD161-specific nanobodies to those in need, it is possible to inhibit tumor progression or shrink tumors, reduce the severity of autoimmune diseases, inhibit or block the interaction between CD161 and CLEC2D, and further activate immune cells (such as T cells).

[0060] Compared with the prior art, the beneficial effects of the present invention are:

[0061] The nanobody of the present invention specifically binds to human CD161 protein, and binds specifically to human CD161 protein with an EC50 value of no more than 0.5 nM (preferably no more than 0.4 nM, 0.3 nM, or 0.2 nM), exhibiting strong specificity and binding force.

[0062] Nanobodies have a simple structure and are easy to modify. They can be easily tandemly through genetic engineering to obtain antibodies with multiple epitopes, multiple valences, and multiple specificities. Furthermore, they have very low immunogenicity and do not produce a strong immune response without humanization. Attached Figure Description

[0063] To more clearly illustrate the technical solution of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0064] Figure 1 Library enrichment for screening antibodies targeting CD161;

[0065] Figure 2 The antigen-antibody binding pattern is shown in Figure 6B6.

[0066] Figure 3 The antigen-antibody binding patterns (12D1, 13B10);

[0067] Figure 4 The antigen-antibody binding patterns (13E2, 14D6);

[0068] Figure 5 This is the antigen-antibody binding pattern (16D10);

[0069] Figure 6 This is an antigen-antibody binding pattern (18D5);

[0070] Figure 7 The antigen-antibody binding patterns are shown in the diagrams (20B8, 20F11, 20H5).

[0071] Figure 8 This is an antigen-antibody binding map (Tab1, hIgG). Detailed Implementation

[0072] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0073] As used in this article, a “nanobody” (sdAb, also known as a single-domain antibody or VHH) contains a single complementarity-determining region (a single CDR1, a single CDR2, and a single CDR3). Examples of nanobodies include antibodies containing only heavy chain variable domains (which naturally do not contain light chains), nanobodies derived from conventional antibodies, and engineered antibodies.

[0074] Nanobodies can be derived from any species, including mice, humans, camels, llamas, goats, rabbits, and cattle. For example, naturally occurring VHH molecules can be derived from antibodies provided by camelid species such as camels, dromedaries, alpacas, llamas, and guanacos. Like complete antibodies, nanobodies can selectively bind to specific antigens. Nanobodies can contain only variable domains of immunoglobulin chains, which have CDR1, CDR2, and CDR3, as well as a framework region.

[0075] In some embodiments, the invention can also achieve the objective of replacing only one or a few amino acids compared to the sequences in SEQ ID NO. 1-10, for example, by substituting 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conserved amino acid substitutions. Such conserved amino acid substitutions can generally be described as the substitution of an amino acid residue by another amino acid residue having a similar chemical structure, and this substitution has little or no effect on the function, activity, or other biological properties of the polypeptide. Such conserved amino acid substitutions are common in the art.

[0076] The preferred host cell of the present invention is a bacterial cell, a fungal cell, or a mammalian cell.

[0077] This invention involves preparing a target protein or a truncated form of the target protein using genetic engineering technology. The obtained antigen protein is then used to immunize alpacas. After multiple immunizations, peripheral blood lymphocytes or spleen cells of the alpacas are obtained. The variable region coding sequence of the camel-derived antibody is recombined into a phage display vector through genetic engineering. Specific nanobodies against the antigen protein are screened using phage display technology, and their ability to bind to the antigen is further tested.

[0078] The above technical solution will now be broken down and explained in detail, and described with specific embodiments:

[0079] Example 1: Preparation of recombinant extracellular domain protein of human CD161:

[0080] The recombinant human CD161 extracellular domain protein used in this invention was purchased from the Chinese company ACRO. The purchased protein has the accession number NM_002258.2 in the NCBI database, and the amino acid sequence encoded by this sequence has the accession number NP_002249.1. The expressed region is the extracellular terminal of the CD161 protein (i.e., amino acids 67 to 225), fused with a 6*His tag, and the protein purity reaches over 90%, for use in animal immunization.

[0081] Example 2: Construction of a nanobody library specifically binding to CD161 protein:

[0082] The human CD161 protein from Example 1 was mixed with an equal volume of Freund's adjuvant and used to immunize an alpaca without an immune background. The immunization protocol was as follows: immunization was performed every 2 weeks for a total of 4 consecutive immunizations. The first immunization used 500 μg of CD161 protein mixed with an equal volume of Freund's complete adjuvant. The remaining 3 immunizations used 250 μg of CD161 protein mixed with an equal volume of Freund's incomplete adjuvant. This immunization process was designed to concentrate stimulation of the alpaca to produce antibodies against CD161 protein.

[0083] After animal immunization, 50 mL of whole blood was drawn from alpacas and PBMCs (peripheral blood mononuclear cells) were isolated. RNA was extracted from the cells. cDNA was synthesized using the extracted total RNA, and VHH (antibody heavy chain variable domain) was amplified using nested PCR with the cDNA as a template.

[0084] Then, the pMECS vector and VHH fragment were digested with restriction endonucleases, and the digested fragments were ligated to the vector. The ligated fragments were electroporated into competent TG1 cells to construct a phage display library of CD161 protein, and the library size was determined to be approximately 1 × 10⁻⁶. 9 Meanwhile, the insertion rate of the target fragment in the library was detected by colony PCR.

[0085] The results showed that after PCR amplification of 30 colonies randomly selected from the library, 29 clones were able to amplify bands of the predicted size, and 1 clone amplified an incorrect band. Therefore, the correct insertion rate was 29÷30×100%≈97%.

[0086] Example 3: Screening of nanobodies that specifically bind to CD161 protein:

[0087] Take 200 μL of the recombinant TG1 cells from Example 2 and culture them in 2×TY medium. During the culture, add 40 μL of helper phage VCSM13 to infect the TG1 cells and culture overnight to amplify the phage. The next day, precipitate the phage with PEG / NaCl and collect the phage by centrifugation.

[0088] 500 ng of CD161 protein diluted in 100 mM pH 8.3 NaHCO3 was coupled onto an ELISA plate and incubated overnight at 4°C. A negative control well (NaHCO3) was also included. The next day, 200 μL of 3% skim milk was added, and the plate was blocked at room temperature for 2 h. After blocking, 100 μL of the amplified phage library (approximately 2 × 10⁻⁶) was added. 11 (1 phage particle), incubate at room temperature for 1 hour; after 1 hour, wash 15 times with PBS + 0.05% Tween-20 to remove unbound phage.

[0089] Phages that specifically bind to CD161 protein were dissociated using trypsin at a final concentration of 0.25 mg / mL and then used to infect E. coli TG1 cells in the logarithmic growth phase. The cells were cultured at 37°C for 1 h to produce and collect phages for the next round of screening. The same screening process was repeated twice to gradually enrich the cells.

[0090] When the enrichment factor reaches 10 times or more, the enrichment effect is as follows: Figure 1 As shown.

[0091] Figure 1In this context, P / N = the number of monoclonal bacteria grown from phages eluted from positive wells in the biopanning process after infecting TG1 bacteria / the number of monoclonal bacteria grown from phages eluted from negative wells after infecting TG1 bacteria. This parameter gradually increases after enrichment occurs. I / E = the total number of phages added to positive wells in each round of the biopanning process / the total number of phages eluted from positive wells in each round of the biopanning process. This parameter gradually approaches 1 after enrichment occurs.

[0092] Example 4: Screening for CD161-specific positive clones using phage enzyme-linked immunosorbent assay (ELISA):

[0093] Nanobodies that specifically bind to CD161 protein were screened according to the screening method in Example 3 above. The phage enrichment factor against CD161 protein reached more than 10. After screening, single colonies were selected from the positive clones and inoculated into 96-well plates of 2×TY medium containing 100 μg / mL ampicillin. A blank control was set up. After incubation at 37°C to the logarithmic phase, IPTG was added to a final concentration of 1 mM and incubated overnight at 28°C.

[0094] Crude antibody was obtained using the osmotic burst method. CD161 recombinant protein was released into 100 mM NaHCO3 (pH 8.3), and 100 ng of protein was coated overnight at 4°C in an ELISA plate. 100 μL of the obtained crude antibody extract was transferred to an ELISA plate containing the antigen and incubated at room temperature for 1 h. Unbound antibody was washed away with PBST, and 100 μL of Mouse Anti-HA tag Antibody (HRP) (Mouse anti-HA horseradish peroxidase labeled antibody, ThermoFisher) diluted 1:2000 was added. The plate was incubated at room temperature for 1 h. Unbound antibody was washed away with PBST, and horseradish peroxidase chromogenic solution was added. The reaction was carried out at 37°C for 15 min, and then stop solution was added. The absorbance was read at 450 nm using an ELISA reader.

[0095] When the OD value of the sample well is more than 5 times that of the control well, it is determined to be a positive clone well. The bacteria in the positive clone well are transferred to LB medium containing 100 μg / mL ampicillin for plasmid extraction and sequencing.

[0096] The gene sequences of each clone were analyzed using the sequence alignment software VectorNTI. Clones with the same CDR1, CDR2, CDR3 and FR sequences were considered as the same clone, while clones with different sequences were considered as different clones. Finally, nanobodies specifically targeting the CD161 protein were obtained.

[0097] The amino acid sequence of the antibody is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, constituting the entire VHH. The obtained recombinant nanobody plasmid can be expressed in prokaryotic or eukaryotic systems to ultimately obtain the nanobody protein.

[0098] The CDR and FR sequences of the 10 nanobodies are shown in Tables 1-7, and the amino acid and nucleotide sequences of the 10 nanobodies are shown in Tables 8 and 9, respectively.

[0099] Table 1. CDR1 sequences of 10 antibodies

[0100]

[0101] Table 2. CDR2 sequences of 10 antibodies

[0102]

[0103] Table 3. CDR3 sequences of 10 antibodies

[0104]

[0105] Table 4. FR1 sequences of 10 antibodies

[0106]

[0107] Table 5. FR2 sequences of 10 antibodies

[0108]

[0109]

[0110] Table 6. FR3 sequences of 10 antibodies

[0111]

[0112] Table 7. FR4 sequences of 10 antibodies

[0113]

[0114] Table 8. Amino acid sequences of 10 antibodies

[0115]

[0116]

[0117] Table 9. Nucleotide sequences of 10 antibodies

[0118]

[0119]

[0120] Example 5: Expression and purification of CD161-specifically binding nanobodies in host bacterium *Escherichia coli*

[0121] The plasmids (pMECS-VHH) of different clones obtained from sequencing analysis in Example 4 were electroporated into Escherichia coli HB2151 and plated on LB+amp+glucose culture plates containing ampicillin and glucose, and incubated overnight at 37°C. Single colonies were selected and inoculated into 5 mL of LB culture medium containing ampicillin and incubated overnight on a shaker at 37°C.

[0122] Inoculate 1 mL of overnight cultured bacteria into 330 mL of TB culture medium and incubate at 37°C in a shaker. When the OD600nm value reaches 0.6-0.9, add 1 M IPTG and incubate overnight at 28°C in a shaker. Centrifuge to collect E. coli and obtain crude antibody extract using the osmotic rupture method.

[0123] The antibody was purified by nickel column affinity chromatography.

[0124] Example 6: Construction of a eukaryotic expression vector for an Fc fusion antibody of a nanobody specifically binding to CD161

[0125] (1) Subcloning the target sequence obtained in Example 4 into a eukaryotic expression vector: The antibody screened in Example 4 was obtained by Sanger sequencing to obtain its nucleotide sequence;

[0126] (2) The above nucleotide sequence was synthesized into the vector RJK-V4-hFC designed and modified by our company by sequence synthesis to obtain a recombinant eukaryotic expression vector. The modification method of the vector is as described in Example 10.

[0127] (3) Transform the recombinant eukaryotic expression vector constructed in step (2) into DH5α Escherichia coli, culture it for plasmid extraction, and remove endotoxins;

[0128] (4) The extracted plasmids were then sequenced and identified.

[0129] (5) After confirming the recombinant vector, prepare it for subsequent eukaryotic cell transfection and expression. After expressing the Fc protein of VHH by the method of Example 7 or 8, purify the above antibody by the method of Example 9.

[0130] Example 7: Expression of nanobodies that specifically bind to CD161 protein in suspended ExpiCHO-S cells

[0131] (1) Three days before transfection, use 2.5×10 5 / mL cell passage and expansion culture ExpiCHO-S TMCells, the calculated desired cell volume, were transferred to 120 mL (final volume) of fresh, preheated ExpiCHO. TM In a 500 mL shake flask of expression medium, the cell concentration was increased to approximately 4 × 10⁻⁶ cells / mL. 6 -6×10 6 live cells / mL;

[0132] (2) One day before transfection, ExpiCHO-S TM Cells were diluted to a concentration of 3.5 × 10⁻⁶. 6 Live cells / mL, incubate cells overnight;

[0133] (3) On the day of transfection, measure cell density and percentage of viable cells. Cell density should reach approximately 7 × 10⁻⁶ cells / day before transfection. 6 -10×10 6 live cells / mL;

[0134] (4) Use fresh ExpiCHO preheated to 37°C TM The expression medium was used to dilute the cells to 6 × 10⁶. 6 viable cells / mL. The calculated desired cell volume was transferred to 100 mL (final volume) of fresh, preheated ExpiCHO. TM In a 500 mL shake flask of expression medium;

[0135] (5) Gently invert to mix ExpiFectamine TM CHO reagent, using 3.7 mL OptiPRO TM Culture medium for diluting ExpiFectamine TM CHO reagent, vortex or mix well;

[0136] (6) Use 4 mL of refrigerated OptiPRO TM Dilute the plasmid DNA in the culture medium and vortex to mix.

[0137] (7) Incubate the ExpiFectamine CHO / plasmid DNA (the plasmid DNA is the Fc fusion antibody eukaryotic expression vector of the nanobody that specifically binds to CD161 prepared in Example 6) complex at room temperature for 1-5 minutes, and then gently add it to the prepared cell suspension while gently shaking the flask during the addition process.

[0138] (8) The cells were cultured with shaking in humidified air at 37°C and 8% CO2.

[0139] (9) Add 600ul of ExpiFectamine on the first day after transfection (18-22 hours later). TM CHO Enhancer and 24mLExpiCHO feed.

[0140] (10) Collect the supernatant about 8 days after transfection (when cell viability is less than 70%).

[0141] Example 8: Expression of nanobodies that specifically bind to CD161 protein in suspension 293F cells

[0142] Recombinant nanobody expression experimental procedure (taking a 500mL shake flask as an example):

[0143] (1) Three days before transfection, use 2.5×10 5 After passage and expansion of 293F cells at / mL, the calculated desired cell volume was transferred to a 500mL shake flask containing 120mL (final volume) of fresh, preheated OPM-293CD05 Medium to achieve a cell concentration of approximately 2×10⁻⁶. 6 -3×10 6 Live cells / mL.

[0144] (2) On the day of transfection, measure cell density and percentage of viable cells. Cell density should reach approximately 2 × 10⁻⁶ cells / day before transfection. 6 -3×10 6 Live cells / mL.

[0145] (3) Dilute the cells to 1×10⁻⁵ using preheated OPM-293CD05 Medium. 6 1 live cells / mL. Calculate the required cell volume and transfer it to a 500 mL shake flask containing 100 mL (final volume) of fresh, preheated culture medium.

[0146] (4) Dilute PEI (1 mg / mL) reagent with 4 mL Opti-MEM medium, and mix by swirling or pipetting; dilute plasmid DNA (the plasmid DNA is the eukaryotic expression vector of the Fc fusion antibody of the CD161-specific nanobody prepared in Example 6) with 4 mL Opt-MEM medium, mix by swirling, and filter with a 0.22 μm filter. Incubate at room temperature for 5 min.

[0147] (5) Add the diluted PEI reagent to the diluted DNA and mix by inverting. Incubate the PEI / plasmid DNA complex at room temperature for 15-20 minutes, then gently add it to the prepared cell suspension while gently shaking the flask during the addition process.

[0148] (6) Culture the cells at 37°C, 5% CO2, and 120 rpm with shaking.

[0149] (7) Add 5 mL of OPM-CHO PFF05 feed at 24 h and 72 h after transfection.

[0150] (8) Collect the supernatant about 7 days after transfection (when cell viability is less than 70%).

[0151] Example 9: Purification of nanobodies that specifically bind to CD161 protein

[0152] (1) The protein expression supernatant obtained in Example 7 or 8 was filtered with a 0.45 μm disposable filter to remove insoluble impurities;

[0153] (2) The above filtrate was purified by affinity chromatography using a protein purifier. The agarose packing material coupled with Protein A was used to purify the filtrate by utilizing the ability of human Fc to bind to Protein A.

[0154] (3) Pass the filtrate through a ProteinA pre-packed column at a flow rate of 1 mL / min. In this step, the target protein in the filtrate will bind to the packing material.

[0155] (4) Wash the impurity proteins bound to the column with low-salt and high-salt buffers;

[0156] (5) Use a low pH buffer to systemically bind the target protein on the column;

[0157] (6) Quickly add the eluent to a Tris-HCl solution with a pH of 9.0 to neutralize it;

[0158] (7) After dialysis of the above-neutralized protein solution, perform SDS-PAGE analysis to confirm that the protein purity is above 95% and the concentration is above 0.5 mg / mL, and then store it at low temperature for later use.

[0159] Example 10: Construction of the eukaryotic expression vector RJK-V4-hFC for nanobodies

[0160] The aforementioned universal target vector for nanobodies, RJK-V4-hFC, is a modified version of Invitrogen's commercial vector pCDNA3.4 (vector data link: https: / / assets.thermofisher.com / TFS-Assets / LSG / manuals / pcdna3_4_topo_ta_cloning_kit_man.pdf) by incorporating the Fc region of the human IgG1 heavy chain coding sequence. Specifically, this vector contains the CH2 and CH3 hinge regions of the IgG1 heavy chain. The specific modification scheme is as follows:

[0161] (1) Select the restriction enzyme sites XbaI and AgeI on pcDNA3.4;

[0162] (2) Multiple cloning sites (MCS) and 6×His tags were introduced at the 5' and 3' ends of the Fc fragment coding sequence, respectively, by overlapping PCR.

[0163] (3) The above fragment was amplified by PCR using a pair of primers with XbaI and AgeI restriction sites respectively;

[0164] (4) The recombinant DNA fragments in pcDNA3.4 and (3) were digested with restriction endonucleases XbaI and AgeI, respectively;

[0165] (5) The digested vector and the insert fragment were ligated with T4 ligase, and then the ligation product was transformed into E. coli, amplified, sequenced and verified to obtain the recombinant plasmid.

[0166] Example 11: Expression and purification of a tool antibody (Tab) targeting human CD161

[0167] In this study, Tab1 is KW1.3.12 (US20210122826A1). The searched sequence was commissioned to General Biosystems (Anhui) Co., Ltd. for codon optimization in a mammalian cell expression system and cloned into the pcDNA3.1 vector. After antibiotic selection, plasmid-positive bacteria were selected for amplification, and plasmids were extracted using a plasmid extraction kit (Macherey Nagel, Cat#740412.50). 100 μg of plasmid (40 μg heavy chain + 60 μg light chain) was added per 100 mL of cells, and PEI was used to transiently express the plasmid in 293F cells (medium: FreeStyle 293 Expression medium, Thermo, Cat#12338026+F-68, Thermo, Cat#24040032). 6–24 h after transfection, 5% volume of 10% Peptone (Sigma, Cat#P0521-100G) was added, and the cells were cultured in 8% CO2 at 130 rpm for approximately 7–8 days. When cell viability decreased to 50%, the expression supernatant was collected and purified using a gravity column with Protein A (GE, Cat#17-5438-02). After dialysis with PBS, the concentration was determined using Nanodrop, purity was identified by SEC, and binding capacity was verified by indirect ELISA.

[0168] The Tab obtained by this method has a concentration of not less than 2 mg / ml and a purity greater than 95%.

[0169] Example 12: Determination of Antibody-Antigen Binding Dose-Reaction Curve

[0170] This embodiment uses the standard enzyme-linked immunosorbent assay (ELISA) procedure.

[0171] (1) Coat 50 μL of 1 μg / mL human CD161 protein and incubate overnight at 4°C.

[0172] (2) Wash the plate; add 200 μL of 5% milk and seal at 37°C for 2 hours.

[0173] (3) VHH-hFc was diluted to 2 μg / mL, and then the antibody was serially diluted 5-fold to a total of 8 concentration gradients. Here, VHH-hFc refers to the Fc fusion antibody of the CD161 protein-specific nanobody prepared in Example 8 (expressed in 293F cells), which was purified in Example 9. In addition, hIgG and Tab controls were set up respectively; Tab1 was prepared in Example 11.

[0174] (4) Wash the plate; add 50 μL of the nanobody obtained by dilution in step (3), double the wells, and incubate at 37°C for 1 h.

[0175] (5) Wash the plate; add 50 μL of HRP-Goat anti hIgG secondary antibody and incubate at 37°C for 30 min.

[0176] (6) Wash the plate (wash several times); add 50 μL of TMB that has been brought to room temperature beforehand, and react at room temperature in the dark for 15 min.

[0177] (7) Add 50 μL of stop solution (1N HCl) and save the microplate reader reading.

[0178] (8) Plot the curve and calculate EC50, where hIgG refers to the isotype control, an immunoglobulin molecule that does not bind to any target and is obtained through commercial purchase. The results are as follows: Figures 2-8 As shown.

[0179] Compared with hIgG and Tab1 controls, all nanobodies (6B6, 12D1, 13B10, 13E2, 14D6, 16D10, 18D5, 20B8, 20F11, 20H5) showed strong binding ability and affinity to human CD161 protein.

[0180] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A nanobody that specifically binds to CD161, characterized in that: The nanobody specifically binds to human CD161 protein with an EC50 value of no more than 0.5 nM; the nanobody and CLEC2D competitively bind to human CD161 protein.

2. A nanobody that specifically binds to CD161, characterized in that: The nanobody includes a heavy chain variable domain, which includes the amino acid sequences of heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3. The heavy chain CDR1 contains any sequence selected from SEQ ID NO:32-SEQ ID NO:39 or a variant thereof with up to 5 amino acid substitutions; The heavy chain CDR2 comprises any sequence selected from SEQ ID NO:40-SEQ ID NO:41 or a variant thereof with up to 5 amino acid substitutions; The heavy chain CDR3 contains any sequence selected from SEQ ID NO:42-SEQ ID NO:45 or a variant thereof with up to 5 amino acid substitutions.

3. The nanobody that specifically binds to CD161 according to claim 2, characterized in that: The amino acid sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 are one of the following (1)-(10): (1) CDR1 shown in SEQ ID NO:32, CDR2 shown in SEQ ID NO:41, and CDR3 shown in SEQ ID NO:45; (2) CDR1 shown in SEQ ID NO:33, CDR2 shown in SEQ ID NO:41, and CDR3 shown in SEQ ID NO:45; (3) CDR1 shown in SEQ ID NO:34, CDR2 shown in SEQ ID NO:41, and CDR3 shown in SEQ ID NO:45; (4) CDR1 shown in SEQ ID NO:35, CDR2 shown in SEQ ID NO:40, and CDR3 shown in SEQ ID NO:42; (5) CDR1 shown in SEQ ID NO:36, CDR2 shown in SEQ ID NO:41, and CDR3 shown in SEQ ID NO:45; (6) CDR1 shown in SEQ ID NO:37, CDR2 shown in SEQ ID NO:41, and CDR3 shown in SEQ ID NO:45; (7) CDR1 shown in SEQ ID NO:38, CDR2 shown in SEQ ID NO:41, and CDR3 shown in SEQ ID NO:45; (8) CDR1 shown in SEQ ID NO:38, CDR2 shown in SEQ ID NO:41, and CDR3 shown in SEQ ID NO:43; (9) CDR1 shown in SEQ ID NO:38, CDR2 shown in SEQ ID NO:41, and CDR3 shown in SEQ ID NO:44; (10) CDR1 shown in SEQ ID NO:39, CDR2 shown in SEQ ID NO:41, and CDR3 shown in SEQ ID NO:

45.

4. A nanobody that specifically binds to CD161, characterized in that: The amino acid sequence of the nanobody has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology with any one of the sequences in SEQ ID NO:1-10.

5. The Fc fusion antibody or humanized antibody of the nanobody that specifically binds to CD161 as described in any one of claims 1-4.

6. A recombinant polypeptide, characterized in that, The recombinant polypeptide comprises the CD161-specific nanobody as described in any one of claims 1-4; the recombinant polypeptide includes antibody-immunoconjugates, bispecific molecules, chimeric antigen receptors (CARs), multispecific molecules, multi-epitope antibodies, or multivalent antibodies.

7. A nucleotide molecule, characterized in that: It encodes the CD161-specific nanobody as described in any one of claims 1-4.

8. A carrier, characterized in that, Includes the nucleotide molecule as described in claim 7.

9. A host cell, characterized in that: It can express the CD161-specific nanobody as described in any one of claims 1-4, the Fc fusion antibody or humanized antibody as described in claim 5, or the recombinant polypeptide as described in claim 6.

10. A method for preparing a nanobody that specifically binds to CD161, characterized in that: Host cells that can express nanobodies that specifically bind to CD161 are cultured, and the nanobodies that specifically bind to CD161 are expressed and isolated.

11. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises a CD161-specifically binding nanobody selected from any one of claims 1-4, and a pharmaceutically acceptable carrier.

12. A diagnostic or testing kit, characterized in that, Nanobodies comprising the specific binding to CD161 of any one of claims 1-4.

13. Use of the CD161-specifically binding nanobody according to any one of claims 1-4 or the pharmaceutical composition according to claim 11 in the preparation of a medicament for treating a disease.

14. A method for treating a disease in a person in need, characterized in that, The steps include administering an effective amount of the CD161-specifically binding nanobody of any one of claims 1-4 or the pharmaceutical composition of claim 11.

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