NKG2A antibody or antigen binding part thereof and application thereof

Abstract

The invention belongs to the field of nano antibodies, and relates to an NKG2A antibody or an antigen-binding part thereof and application of the NKG2A antibody or the antigen-binding part thereof. The NKG2A antibody or an antigen binding part of the NKG2A antibody is composed of a heavy chain variable structural domain, and the amino acid sequence of a heavy chain CDR1 is X1YTSX2TNFF (SEQ ID NO: 39); the amino acid sequence of the heavy chain CDR2 is ISX3TGX4YT (SEQ ID NO: 40); and the amino acid sequence of the heavy chain CDR3 is AAGX5RX6GDNWEX7EX8SWFY (SEQ ID NO: 41). Compared with the prior art, the invention has the beneficial effects that the NKG2A antibody or the antigen binding part thereof is screened by using a biological genetic engineering technology, and the affinity of the antibody is better.

Description

Technical Field

[0001] This invention relates to antibodies or antigen-binding portions thereof capable of binding to NKG2A, pharmaceutical compositions containing such NKG2A antibodies or antigen-binding portions as active ingredients, and their use in pharmaceutical therapy. Background Technology

[0002] Natural killer (NK) cells are a crucial type of lymphocyte in the body. Their surface expresses various activating and inhibitory receptors, which, upon binding to their corresponding ligands, collectively regulate NK cell function. This regulatory mechanism enables NK cells to efficiently recognize and kill abnormal cells in the body, such as senescent cells, tumor cells, and virus-infected cells, while simultaneously protecting normal cells from attack, playing a vital role in maintaining the stability of the body's internal environment.

[0003] The NKG2 receptor family is an important receptor family on the surface of NK cells, including members such as NKG2A, NKG2B, NKG2C, NKG2D, NKG2E, and NKG2F. NKG2A has been found to be a receptor for natural killer (NK) cells and CD8+ cells. + A key immune checkpoint for T cells. On the surface of human immune cells, the heterodimeric complex NKG2A-CD94, formed by the disulfide bond linking NKG2A and CD94 (NK cell surface membrane protein), is recognized by HLA-E, a non-classical histocompatibility complex class I (MHC I) molecule on target cells. While this molecule is normally expressed at low levels, HLA-E expression is elevated on the surface of most tumor cells, inducing a cascade of inhibitory signals that suppress the cytotoxic activity of NK cells and the secretion of cytokines. Certain viral infections, tumors, and immune diseases evade immune checkpointing through this pathway.

[0004] Currently, there are reports of monoclonal antibody drugs targeting NKG2A, mainly including Monalizumab from Innate / AstraZeneca and HY0102 from Huaiyue Biotechnology. However, no human anti-NKG2A nanobodies with high affinity, high selectivity, and high biological activity have been found clinically. Summary of the Invention

[0005] The purpose of this invention is to provide an NKG2A antibody or its antigen-binding portion and its uses.

[0006] The first aspect of the present invention provides an NKG2A antibody or its antigen-binding portion thereof, wherein the NKG2A antibody (which is a nanobody) or its antigen-binding portion thereof is composed of a heavy chain variable domain VHH, the heavy chain variable domain including heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3.

[0007] The amino acid sequence of the heavy chain CDR1 is: X1YTSX2TNFF (SEQ ID NO:39);

[0008] The amino acid sequence of the heavy chain CDR2 is: ISX3TGX4YT (SEQ ID NO:40);

[0009] The amino acid sequence of the heavy chain CDR3 is: AAGX5RX6GDNWEX7EX8SWFY (SEQ ID NO:41);

[0010] Where X1 is E, G or R; X2 is S or L; X3 is S or T; X4 is A or G; X5 is I or V; X6 is A or G; X7 is T or R; and X8 is A or T.

[0011] A second aspect of the present invention provides an NKG2A antibody or its antigen-binding portion thereof, wherein the NKG2A antibody or its antigen-binding portion is composed of a heavy chain variable domain VHH, the heavy chain variable domain including heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3; the amino acid sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 are one of the following (1)-(5):

[0012] (1) CDR1 shown in SEQ ID NO:30, CDR2 shown in SEQ ID NO:34, and CDR3 shown in SEQ ID NO:36;

[0013] (2) CDR1 shown in SEQ ID NO:29, CDR2 shown in SEQ ID NO:32, and CDR3 shown in SEQ ID NO:38;

[0014] (3) CDR1 shown in SEQ ID NO:30, CDR2 shown in SEQ ID NO:34, and CDR3 shown in SEQ ID NO:35;

[0015] (4) CDR1 shown in SEQ ID NO:31, CDR2 shown in SEQ ID NO:33, and CDR3 shown in SEQ ID NO:37;

[0016] (5) CDR1 shown in SEQ ID NO:31, CDR2 shown in SEQ ID NO:32, and CDR3 shown in SEQ ID NO:38.

[0017] 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.

[0018] The term "antigen-binding portion" in this invention includes fragments, derivatives, and analogs of the NKG2A nanobody. As used herein, the terms "fragment," "derivative," and "analyte" have the same meaning and refer to a polypeptide that substantially retains the same biological function or activity as the antibody of this invention. The polypeptide fragments, derivatives, or analogs of this invention may be (i) a polypeptide capable of specifically binding to a separation complementarity-determining region (CDR) of an antigen, or (ii) a polypeptide having one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) substituted, where such substituted amino acid residues may or may not be encoded by the genetic code, or (iii) a polypeptide having a substituent group in one or more amino acid residues, or (iv) a polypeptide 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 (v) a polypeptide formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence or secretion sequence, or a 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. The antigen-binding component also includes immunoconjugates of NKG2A antibodies, bispecific molecules, chimeric antigen receptors (CARs), multispecific molecules, multi-epitope antibodies, or multivalent antibodies.

[0019] In a preferred embodiment, the sequence of the NKG2A antibody or its antigen-binding portion further includes a frame region FR; the frame region FR includes the amino acid sequences of FR1, FR2, FR3, and FR4; the amino acid sequences of the frame region FR are as follows:

[0020] The FR1 or a variant of FR1 shown in any of SEQ ID NO:17-22, wherein the variant of FR1 contains substitutions of up to 5 amino acids in the FR1;

[0021] The FR2 or a variant thereof shown in any one of SEQ ID NO:23-24, wherein the variant thereof contains a substitution of up to 5 amino acids in the FR2;

[0022] The FR3 or a variant of FR3 shown in any one of SEQ ID NO:25-27, wherein the variant of FR3 contains substitutions of up to 5 amino acids in the FR3;

[0023] The FR4 or a variant thereof shown in SEQ ID NO:28, wherein the variant thereof contains substitutions of up to 5 amino acids.

[0024] A third aspect of the present invention is to provide an NKG2A antibody or its antigen-binding portion thereof, wherein the amino acid sequence of the NKG2A antibody or its antigen-binding portion 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 the sequences in SEQ ID NO:1-8, and wherein it is capable of specifically binding to the NKG2A protein.

[0025] 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.

[0026] In a preferred embodiment, compared with any one of SEQ ID NO: 1-8, at least one amino acid residue in the FR1, FR2, FR3 or FR4 sequence of the NKG2A antibody or its antigen-binding portion is replaced by a conserved amino acid.

[0027] A fourth aspect of the present invention is to provide an Fc fusion antibody of any of the aforementioned NKG2A antibodies or its antigen-binding portion. The term "Fc fusion antibody" refers to a novel protein produced by fusing the Fc fragment of a target antibody with a biologically active functional protein molecule using genetic engineering techniques.

[0028] The fifth aspect of this invention discloses antibody-immunoconjugates, bispecific molecules, chimeric antigen receptor CARs, multispecific molecules, multi-epitope antibodies, or multivalent antibodies comprising the aforementioned NKG2A antibody or its antigen-binding portion. For example, the multi-epitope antibody may be composed of more than one sequence from SEQ ID NO. 1-8; the multivalent antibody may be composed of a series of repetitions of one of the sequences from SEQ ID NO. 1-8; the bispecific molecule may simultaneously target two different epitopes of NKG2A or simultaneously target NKG2A and another antigen molecule; the multispecific molecule includes, but is not limited to, trispecific antibodies and tetraspecific antibodies; the NKG2A antibody or its antigen-binding portion may also be used as a component of an immunodrug conjugate (ADC) or a chimeric antigen receptor CAR.

[0029] A sixth aspect of the present invention is to provide a nucleotide molecule encoding the aforementioned NKG2A antibody or its antigen-binding portion, preferably having a nucleotide sequence having 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 SEQ ID NO: 9-16.

[0030] A seventh aspect of the present invention is to provide an expression vector comprising the aforementioned nucleotide molecules.

[0031] In a preferred embodiment, the expression vector used is RJK-V4-3, but other general-purpose expression vectors can also be selected as needed.

[0032] An eighth aspect of the present invention is to provide a host cell that can express the aforementioned NKG2A antibody or its antigen-binding portion, or the aforementioned Fc fusion antibody. Preferably, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

[0033] In another preferred embodiment, the host cell includes prokaryotic or eukaryotic cells, including bacteria and fungi.

[0034] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, mammalian cells, bacteriophages, or combinations thereof.

[0035] 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.

[0036] In another preferred embodiment, the eukaryotic cells are selected from the group consisting of Pichia pastoris, Saccharomyces cerevisiae, Schizosomalidomiae, Trichoderma, or combinations thereof.

[0037] 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.

[0038] In another preferred embodiment, the host cell is a suspension ExpiCHO-S cell.

[0039] In another preferred embodiment, the host cell is a suspension 293F cell.

[0040] A ninth aspect of the invention is to provide a pharmaceutical composition comprising the aforementioned NKG2A antibody or its antigen-binding portion and a pharmaceutically acceptable excipient. 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 administration, transdermal administration (direct application or patching to the affected area).

[0041] 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 NKG2A antibody or its antigen-binding moiety, 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.

[0042] A tenth aspect of the present invention is to provide a disease treatment agent comprising the aforementioned NKG2A antibody or its antigen-binding portion, or the aforementioned pharmaceutical composition. The disease is cancer, an autoimmune disease, or a viral infection.

[0043] The eleventh aspect of the present invention discloses a method for generating an NKG2A antibody or an antigen-binding portion thereof, comprising providing the aforementioned host cell, culturing the host cell under conditions suitable for expressing the antibody or antigen-binding portion, and isolating and purifying the obtained antibody or antigen-binding portion.

[0044] The twelfth aspect of the present invention is to provide a method for detecting the presence of NKG2A protein in a sample, wherein the sample is brought into contact with the aforementioned NKG2A antibody or its antigen-binding portion under conditions that allow the formation of an antigen-antibody complex between the antibody or its antigen-binding portion and the NKG2A protein, and the formation of the antigen-antibody complex is detected.

[0045] In a preferred embodiment, the sample includes, but is not limited to, biological fluids (e.g., serum, lymph, blood), cell samples, or tissue samples (e.g., tumor tissue).

[0046] The thirteenth aspect of the present invention is to provide the use of the aforementioned NKG2A antibody or its antigen-binding portion or the aforementioned pharmaceutical composition in the preparation of a medicament for treating a disease.

[0047] In a preferred embodiment, the disease includes cancer, an autoimmune disease, or a viral infection.

[0048] Cancers include, but are not limited to, squamous cell carcinoma of the head and neck, non-small cell lung cancer, colorectal cancer, small cell lung cancer, MSI-H or dMMR solid tumors, non-muscle-invasive bladder cancer, breast cancer, chronic lymphocytic leukemia, esophageal squamous cell carcinoma, gastroesophageal junction cancer, ovarian cancer, gastric cancer, gastroesophageal junction lung cancer, liver cancer, head and neck cancer, urothelial carcinoma, pancreatic cancer, lung cancer, gynecological tumors, esophageal cancer, acute myeloid leukemia, acute lymphoblastic leukemia, myelodysplastic syndrome, multiple myeloma, chronic myeloid leukemia, myeloproliferative neoplasms, Hodgkin lymphoma, or non-Hodgkin lymphoma.

[0049] The autoimmune diseases mentioned include, but are not limited to, rheumatoid arthritis, graft-versus-host disease, autoimmune hemolytic anemia, pernicious anemia, polyarteritis nodosa, systemic lupus erythematosus, Wegener's granulomatosis, autoimmune hepatitis, Behcet's disease, Crohn's disease, primary biliary cirrhosis, scleroderma, ulcerative colitis, Sjögren's syndrome, type I diabetes, uveitis, Graves' disease, thyroiditis, myocarditis, rheumatic fever, scleroderma, ankylosing spondylitis, glomerulonephritis, sarcoidosis, dermatomyositis, myasthenia gravis, polymyositis, Guillain-Barré syndrome, multiple sclerosis, alopecia areata, pemphigus / pemphigoid, psoriasis, and vitiligo.

[0050] The fourteenth aspect of the present invention is the use of the aforementioned NKG2A antibody or its antigen-binding portion or the aforementioned pharmaceutical composition in enhancing the immune activity of a patient, treating the patient's cancer, or treating the patient's autoimmune disease.

[0051] In a preferred embodiment, enhancing the patient's immune activity primarily refers to enhancing the immune activity of the patient's NK cells. Cancer and autoimmune diseases are as described above.

[0052] Compared with the prior art, the beneficial effects of the present invention are:

[0053] The NKG2A nanobody of the present invention specifically targets the NKG2A protein with the correct spatial structure. The nanobody obtained by the present invention has simple multi-combination antibody modification. It can be obtained by simple tandem through genetic engineering to obtain multivalent and multispecific antibodies. Moreover, it has very low immunogenicity and does not produce a strong immune response without humanization. Attached Figure Description

[0054] 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.

[0055] Figure 1 Library enrichment for screening antibodies targeting NKG2A;

[0056] Figure 2 The antigen-antibody binding patterns are shown in Figures 1A2, 1A8, and 1D5.

[0057] Figure 3 The antigen-antibody binding patterns are shown (1H5, 1H7, 2D11, 1F10).

[0058] Figure 4 This is an antigen-antibody binding map (3A4, Tab1, hIgG). Detailed Implementation

[0059] 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.

[0060] 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.

[0061] 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, 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.

[0062] In some embodiments, the inventive objective can also be achieved by substituting only one or a few amino acids compared to the sequences in SEQ ID NO. 1-8, for example, by including 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.

[0063] The preferred host cell of the present invention is a bacterial cell, a fungal cell, or a mammalian cell.

[0064] This invention involves preparing a target protein and a truncated form of the target protein using genetic engineering technology. The obtained antigen protein is then used to immunize Bactrian camels in Alashan, Inner Mongolia. After multiple immunizations, peripheral blood lymphocytes or spleen cells of the camels are obtained. The variable region coding sequence of the camel-derived antibody is recombined into a phage display vector through genetic engineering. Specific antibodies against the antigen protein are screened using phage display technology, and their ability to bind to the antigen is further tested.

[0065] The above technical solution will now be broken down and explained in detail, and described with specific embodiments:

[0066] Example 1: Preparation of recombinant human NKG2A extracellular domain protein:

[0067] The human recombinant extracellular domain protein used in this invention was expressed and purified by the company itself. The specific design scheme of the expression vector for the human recombinant NKG2A protein is as follows:

[0068] (1) The coding sequence of NKG2A was obtained by searching in NCBI. Its accession number is NM_002259.4, and the accession number of the amino acid sequence encoded by this sequence is NP_002250.1.

[0069] (2) The nucleotide sequence encoding the extracellular terminus (i.e., amino acids 94-233) of NKG2A was fused with the C-terminal 6*His sequence and cloned into the vector pcDNA3.4 using gene synthesis. The constructed vector was subjected to Sanger sequencing, and after comparison with the original sequence and confirmation that it was correct, the recombinant plasmid was extracted in large quantities, endotoxin was removed, and it was transfected into suspension 293F cells for expression and purification of the target protein. The purity reached more than 90%, which met the requirements for animal immunization.

[0070] Example 2: Construction of a nanobody library targeting the NKG2A protein:

[0071] The purified human recombinant NKG2A protein obtained in Example 1 was mixed with an equal volume of Freund's adjuvant and used to immunize a Bactrian camel from Alashan, Inner Mongolia. The immunization protocol was as follows: immunization was performed weekly for a total of 7 weeks. The first immunization used an equal volume mixture of 600 μg NKG2A protein and Freund's complete adjuvant. The remaining six immunizations used an equal volume mixture of 300 μg NKG2A protein and Freund's incomplete adjuvant. This immunization process was designed to concentrate the stimulation of the camel to produce antibodies against NKG2A protein.

[0072] After animal immunization, 150 mL of whole blood was drawn from camels 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 region) was amplified using nested PCR with the cDNA as a template.

[0073] 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 NKG2A protein, and the library size was measured. The library size was approximately 1 × 10⁻⁶. 9 Meanwhile, the insertion rate of the target fragment in the library was detected by colony PCR.

[0074] The results showed that after PCR amplification of 30 colonies randomly selected from the library, 28 clones were able to amplify bands of the predicted size, while 2 clones amplified bands incorrectly. Therefore, the correct insertion rate was 28 ÷ 30 × 100% ≈ 93.3%.

[0075] Example 3: Screening of nanobodies targeting the NKG2A protein:

[0076] 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.

[0077] 500 ng of NKG2A 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.

[0078] Phages that specifically bind to NKG2A 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.

[0079] When the enrichment factor reaches 10 times or more, the enrichment effect is as follows: Figure 1 As shown.

[0080] Figure 1 In 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.

[0081] Example 4: Screening for specific positive clones against NKG2A using phage enzyme-linked immunosorbent assay (ELISA):

[0082] Nanobodies against NKG2A protein were screened according to the screening method in Example 3 above. The phage enrichment factor against NKG2A 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.

[0083] Crude antibody was obtained using the osmotic burst method. NKG2A 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.

[0084] 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.

[0085] 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 NKG2A protein were obtained.

[0086] 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.

[0087] The CDR and FR sequences of the eight nanobodies screened are shown in Tables 1-7, and the amino acid and nucleotide sequences of the eight nanobodies are shown in Tables 8 and 9, respectively.

[0088] Table 1. CDR1 sequences of 8 antibodies

[0089]

[0090] Table 2. CDR2 sequences of 8 antibodies

[0091]

[0092] Table 3. CDR3 sequences of 8 antibodies

[0093]

[0094] Table 4. FR1 sequences of 8 antibodies

[0095]

[0096] Table 5. FR2 sequences of 8 antibodies

[0097]

[0098] Table 6. FR3 sequences of 8 antibodies

[0099]

[0100] Table 7. FR4 sequences of 8 antibodies

[0101]

[0102] Table 8. Amino acid sequences of the eight antibodies

[0103]

[0104] Table 9. Nucleotide sequences of eight antibodies

[0105]

[0106]

[0107] Example 5: Purification and expression of NKG2A protein-specific nanobodies in host bacterium *Escherichia coli*

[0108] The plasmids (pMECS-VHH) of different clones obtained from the 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.

[0109] 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.

[0110] The antibody was purified by nickel column affinity chromatography.

[0111] Example 6: Construction of a eukaryotic expression vector for an Fc fusion antibody of an anti-NKG2A nanobody

[0112] (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;

[0113] (2) The above nucleotide sequence was synthesized into the vector RJK-V4-3 designed and modified by our company through sequence synthesis to obtain a recombinant eukaryotic expression vector. The modification method of the vector is as described in Example 10.

[0114] (3) Transform the recombinant eukaryotic expression vector constructed in step (2) into DH5α Escherichia coli, culture it for plasmid extraction, and remove endotoxins;

[0115] (4) The extracted plasmids were then sequenced and identified.

[0116] (5) After confirming the recombinant vector, prepare it for subsequent eukaryotic cell transfection and expression. After expressing the Fc protein of VHH using the method in Example 7 or 8, purify the antibody using the method in Example 9.

[0117] Example 7: Expression of anti-NKG2A protein nanobodies in suspension ExpiCHO-S cells

[0118] (1) Three days before transfection, use 2.5×10 5 After passage and expansion of ExpiCHO-STM cells at / mL, the calculated desired cell volume was transferred to a 500mL shake flask containing 120mL (final volume) of fresh, preheated ExpiCHOTM expression medium to achieve a cell concentration of approximately 4×10⁶ cells / mL. 6 -6×10 6 live cells / mL;

[0119] (2) One day before transfection, the ExpiCHO-STM cells were diluted to a concentration of 3.5 × 10⁻⁶. 6 Live cells / mL, incubate cells overnight;

[0120] (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;

[0121] (4) Dilute the cells to 6 × 10⁶ using fresh ExpiCHO™ expression medium preheated to 37°C. 6 viable cells / mL. The calculated desired cell volume was transferred to a 500 mL shake flask containing 100 mL (final volume) of fresh, preheated ExpiCHO™ expression medium;

[0122] (5) Gently invert to mix the ExpiFectamine™ CHO reagent, dilute the ExpiFectamine™ CHO reagent with 3.7 mL of OptiPRO™ medium, and swirl or mix.

[0123] (6) Dilute the plasmid DNA with 4 mL of refrigerated OptiPRO™ medium and swirl to mix.

[0124] (7) Incubate the ExpiFectamine CHO / plasmid DNA (the plasmid DNA is the Fc fusion antibody eukaryotic expression vector of the anti-NKG2A nanobody prepared in Example 6) complex at room temperature for 1-5 minutes, and then gently add it to the prepared cell suspension, gently shaking the flask during the addition process.

[0125] (8) The cells were cultured with shaking in humidified air at 37°C and 8% CO2.

[0126] (9) On day 1 after transfection (18-22 hours later), add 600ul ExpiFectamineTMCHO Enhancer and 24mL ExpiCHO feed.

[0127] (10) Collect the supernatant about 8 days after transfection (when cell viability is less than 70%).

[0128] Example 8: Expression of anti-NKG2A protein nanobodies in suspension 293F cells

[0129] Recombinant nanobody expression experimental procedure (taking a 500mL shake flask as an example):

[0130] (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.

[0131] (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.

[0132] (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.

[0133] (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 anti-NKG2A 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.

[0134] (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.

[0135] (6) Culture the cells at 37°C, 5% CO2, and 120 rpm with shaking.

[0136] (7) Add 5 mL of OPM-CHO PFF05 feed at 24 h and 72 h after transfection.

[0137] (8) Collect the supernatant about 7 days after transfection (when cell viability is less than 70%).

[0138] Example 9: Purification of nanobodies against NKG2A protein

[0139] (1) The protein expression supernatant obtained in Example 7 or 8 was filtered with a 0.45 μm disposable filter to remove insoluble impurities;

[0140] (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.

[0141] (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.

[0142] (4) Wash the impurity proteins bound to the column with low-salt and high-salt buffers;

[0143] (5) Use a low pH buffer to systemically bind the target protein on the column;

[0144] (6) Quickly add the eluent to a Tris-HCl solution with a pH of 9.0 to neutralize it;

[0145] (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.

[0146] Example 10: Construction of the eukaryotic expression vector RJK-V4-3 for nanobodies

[0147] The aforementioned universal target vector for nanobodies, RJK-V4-3, 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 IgG4 heavy chain coding sequence. Specifically, this vector contains the CH2 and CH3 hinge regions of the IgG4 heavy chain. The specific modification scheme is as follows:

[0148] (1) Select the restriction enzyme sites XbaI and AgeI on pcDNA3.4;

[0149] (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.

[0150] (3) The above fragment was amplified by PCR using a pair of primers with XbaI and AgeI restriction sites respectively;

[0151] (4) The recombinant DNA fragments in pcDNA3.4 and (3) were digested with restriction endonucleases XbaI and AgeI, respectively;

[0152] (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.

[0153] Example 11: Expression and purification of a tool antibody (Tab) targeting human NKG2A

[0154] In this study, Tab1 is monalizumab. 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: FreeStyle293Expression 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.

[0155] The Tab obtained by this method has a concentration of not less than 2 mg / ml and a purity greater than 95%.

[0156] Example 12: Determination of Antibody-Antigen Binding Dose-Reaction Curve

[0157] This embodiment uses the standard enzyme-linked immunosorbent assay (ELISA) procedure.

[0158] (1) Coat 50 μL of 1 μg / mL human NKG2A & CD94 (ACRO Cot#NC4H5-253 Lot 3343-209XF1-TX) overnight at 4℃.

[0159] (2) Wash the plate; add 200 μL of 5% milk and seal at 37°C for 2 hours.

[0160] (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 anti-NKG2A protein nanobody prepared in Example 8 (expressed in 293F cells) purified in Example 9. In addition, hIgG and Tab controls were set up respectively; Tab1 was prepared in Example 11;

[0161] (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.

[0162] (5) Wash the plate; add 50 μL of HRP-Goat anti hIgG secondary antibody and incubate at 37°C for 30 min.

[0163] (6) Wash the plate (wash several times); add 50 μL of TMB that has been brought back to room temperature, and react at room temperature in the dark for 15 min.

[0164] (7) Add 50 μL of stop solution (1N HCl) and save the microplate reader reading.

[0165] (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 2-4 As shown, the eight nanobodies of this invention exhibit strong affinity for the NKG2A protein.

[0166] 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. An NKG2A antibody or its antigen-binding moiety, characterized in that: The NKG2A antibody or its antigen-binding portion is composed of a heavy chain variable domain VHH, which includes heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3. The amino acid sequence of the heavy chain CDR1 is: X1YTSX2TNFF (SEQ ID NO:39); The amino acid sequence of the heavy chain CDR2 is: ISX3TGX4YT (SEQ ID NO:40); The amino acid sequence of the heavy chain CDR3 is: AAGX5RX6GDNWEX7EX8SWFY (SEQ ID NO:41); Where X1 is E, G or R; X2 is S or L; X3 is S or T; X4 is A or G; X5 is I or V; X6 is A or G; X7 is T or R; and X8 is A or T.

2. An NKG2A antibody or its antigen-binding moiety, characterized in that: The NKG2A antibody or its antigen-binding portion is composed of heavy chain variable domains, which include heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3; the amino acid sequences of heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 are one of the following (1)-(5): (1) CDR1 shown in SEQ ID NO:30, CDR2 shown in SEQ ID NO:34, and CDR3 shown in SEQ ID NO:36; (2) CDR1 shown in SEQ ID NO:29, CDR2 shown in SEQ ID NO:32, and CDR3 shown in SEQ ID NO:38; (3) CDR1 shown in SEQ ID NO:30, CDR2 shown in SEQ ID NO:34, and CDR3 shown in SEQ ID NO:35; (4) CDR1 shown in SEQ ID NO:31, CDR2 shown in SEQ ID NO:33, and CDR3 shown in SEQ ID NO:37; (5) CDR1 shown in SEQ ID NO:31, CDR2 shown in SEQ ID NO:32, and CDR3 shown in SEQ ID NO:

38.

3. An NKG2A antibody or its antigen-binding moiety, characterized in that: The amino acid sequence of the NKG2A antibody or its antigen-binding portion has at least 80% homology with any one of the sequences in SEQ ID NO:1-8.

4. The NKG2A antibody according to any one of claims 1-3, or the Fc fusion antibody of the antigen-binding portion thereof.

5. An antibody immunoconjugate, bispecific molecule, chimeric antigen receptor (CAR), multispecific molecule, multi-epitope antibody, or multivalent antibody comprising the NKG2A antibody or its antigen-binding portion as described in any one of claims 1-3.

6. A nucleotide molecule encoding the NKG2A antibody or its antigen-binding moiety according to any one of claims 1-3, characterized in that: Its nucleotide sequence has at least 80% homology with any one of SEQ ID NO: 9-16.

7. An expression vector, characterized in that: It comprises the nucleotide molecule as described in claim 6.

8. A host cell, characterized in that: It can express the NKG2A antibody or its antigen-binding portion as described in any one of claims 1-3 or the Fc fusion antibody as described in claim 4.

9. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises an NKG2A antibody or its antigen-binding moiety selected from any one of claims 1-3, and a pharmaceutically acceptable excipient.

10. A disease treatment agent, characterized in that: The pharmaceutical composition comprising the NKG2A antibody or its antigen-binding portion as described in any one of claims 1-3, or the pharmaceutical composition as described in claim 9.

11. The disease treatment agent according to claim 10, characterized in that: The diseases mentioned are cancer, autoimmune diseases, or viral infections.

12. A method for detecting the presence of NKG2A protein in a sample, characterized in that: The sample is brought into contact with the NKG2A antibody or its antigen-binding portion according to any one of claims 1-3 under conditions that allow the formation of a complex between the antibody or its antigen-binding portion and the NKG2A protein, and the formation of the complex is detected.

13. Use of the NKG2A antibody or its antigen-binding portion according to any one of claims 1-3 or the pharmaceutical composition according to claim 9 in the preparation of a medicament for treating a disease.

14. Use of the NKG2A antibody or its antigen-binding portion according to any one of claims 1-3, or the pharmaceutical composition according to claim 9, in enhancing the immune activity of a patient, treating the patient's cancer, or treating the patient's autoimmune disease.

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