A kind of anti-CD16A nano antibody and its application

Abstract

The present invention discloses an anti-CD16A nanobody and its application. The heavy chain variable region of the nanobody includes CDR1, CDR2 and CDR3, wherein the amino acid sequence of CDR1 is shown in SEQ ID NO: 1, the amino acid sequence of CDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 3. The anti-CD16A nanobody of the present invention can specifically bind to the CD16A antigen, and the affinity of the nanobody is 10 ‑9 The number of M is high affinity nano-antibodies. The heterodimeric bispecific antibody constructed by the above anti-CD16A nano-antibody and anti-CD22 nano-antibody still maintains good antigen binding activity to CD16A, and also has strong killing activity against acute B lymphocytic leukemia, effectively improving the therapeutic effect, and can provide a new method for efficient and long-lasting anti-tumor.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an anti-CD16A nanobody and applications thereof. Background Art

[0002] Natural killer cells (NK cells) are important members of the innate immune system. Unlike T cells, NK cells do not express antigen-specific receptors. NK cells themselves have a broad spectrum of tumor-killing capabilities and also play an important role in enhancing antibody and T cell responses. At present, NK cell-based tumor immunotherapy is diverse and the methods used are also different. The CD16 molecule is an important marker on the surface of NK cells. It can activate the immunoreceptor tyrosine activation motif (ITAM) of IgE NK cell receptor (FcεRIγ) and CD3ζ to produce ADCC. Therefore, the CD16 target is preferentially selected in bispecific antibodies based on NK cell redirection.

[0003] B cell-acute lymphoblastic leukemia (B-ALL) is the most common type of acute lymphoblastic leukemia (ALL). It originates from B-lineage lymphoid precursor cells and accounts for about 85% of ALL. B-ALL accounts for about 80% of acute leukemia in children and about 20% in adults. Existing traditional treatments can enable 80% of children to survive long-term without disease, but only 30% to 40% of adults do. Overall, more than 50% of patients will still relapse. Therefore, in order to improve the problem of reduced efficacy during treatment, it is necessary to develop safer, more effective and more diverse new drugs.

[0004] There are three major categories of human IgG Fc receptors: FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16). The second category includes three subcategories: IIA / IIB / IIC, and the third category includes two subcategories: IIIA / IIIB. The receptor most relevant to the activation of innate immune cells with killing function (such as NK cells) is IIIA, also known as CD16A. CD16A is expressed on the surface of NK cells, monocytes and macrophages, and is also expressed in small amounts on the surface of DC cells. It is an activating receptor. Fc receptors expressed on the cell surface can directly induce or regulate cell activation, and their effects can include ADCC (antibody-dependent cell-mediated cell killing) and CDC (complement-dependent cytotoxicity). Antibody drugs targeting CD16A can be divided into two categories. One type is IgG-derived antibodies with an antibody constant region (Fc), which enhance the cell-killing effect by binding the antibody Fc segment to the CD16A receptor. Usually, the modification of the Fc region of therapeutic antibodies can enhance their binding to CD16A.

[0005] Nanobody (Nb) is a type of antibody that exists in camelids and has the ability to bind to antigens and is naturally missing the first constant region (CH1) of the light chain and heavy chain. It has a small molecular weight (15kDa), which is only 1 / 10 of the traditional monoclonal antibody. It has good stability, solubility, affinity, specificity, flexibility in coupling with different protein molecules, and easy modification. It has broad development and application value in the fields of disease treatment and detection.

[0006] Bispecific antibodies (BsAb) are artificial antibodies that can simultaneously target two antigens or different epitopes of the same antigen. Since human CD16A is mainly expressed in NK cells, most drugs targeting CD16A achieve therapeutic effects by enhancing NK cell activity. Currently, an antibody drug on the market, Margenza (Margetuximab), enhances the affinity of the antibody to CD16A and weakens the affinity to the inhibitory receptor CD32B through engineering modification of the Fc region of the Her-2-targeting antibody (MacroGenetics' proprietary Fc optimization platform), thereby enhancing the participation of the innate immune system and better killing Her2-positive tumor cells. Therefore, Margenza is also called the optimized version of Herceptin (Herceptin, trastuzumab, trastuzumab, targeting Her2).

[0007] With a deeper understanding of tumor biology, it is becoming increasingly clear that personalized medical strategies are essential to improve treatment outcomes. Although a variety of treatments (such as chemotherapy, radiotherapy, and targeted therapy) are available for the treatment of acute B-lymphoblastic leukemia, many patients eventually experience disease relapse or become resistant to existing treatments. Therefore, new treatment strategies are urgently needed to improve the prognosis of these patients. Based on these factors, the development of bispecific antibodies targeting CD22 and the NK cell surface molecule CD16A, which can directly guide NK cells to the vicinity of tumor cells, thereby enhancing the immune system's ability to recognize and kill B-ALL cells, can be regarded as an innovative treatment strategy aimed at improving the survival rate and quality of life of B-ALL patients, especially those who do not respond well to traditional treatments. This research and development work can not only promote the advancement of cancer treatment, but also provide a new theoretical and practical basis for future NK cell immunotherapy.

[0008] In view of this, the present invention is proposed. Summary of the invention

[0009] The object of the present invention is to provide an anti-CD16A nanobody and application thereof, wherein the nanobody has high affinity.

[0010] The present invention screens nano antibodies with high affinity for CD16A targets. CD16A is a key factor in the intracellular signal transduction pathway. Its structure and function are relatively complex, and it is extremely difficult to screen specific nano antibodies. The present invention finally obtains specific nano antibodies through specific screening methods and large-scale screening, and at the same time identifies the stability and adaptability of the obtained nano antibodies; finally, the present invention also provides a method for preparing the nano antibody, according to which the stable production of anti-CD16A nano antibodies can be achieved.

[0011] The present invention is achieved in that:

[0012] In the first aspect, the present invention provides an anti-CD16A nanobody, whose heavy chain variable region includes CDR1, CDR2 and CDR3, the amino acid sequence of CDR1 is shown in SEQ ID NO:1, the amino acid sequence of CDR2 is shown in SEQ ID NO:2, and the amino acid sequence of CDR3 is shown in SEQ ID NO:3.

[0013] In a second aspect, the present invention provides an antibody comprising the above-mentioned anti-CD16A Nanobody or the heavy chain variable region of the anti-CD16A Nanobody.

[0014] In a third aspect, the present invention provides a bispecific antibody comprising the above-mentioned anti-CD16A nanobody and anti-CD22 nanobody.

[0015] In a fourth aspect, the present invention provides a nucleic acid encoding the above-mentioned anti-CD16A nanobody, antibody or bispecific antibody.

[0016] In a fifth aspect, the present invention provides an expression vector comprising the above-mentioned nucleic acid.

[0017] In a sixth aspect, the present invention provides a host cell, which contains the above-mentioned expression vector, or the above-mentioned nucleic acid is integrated into the genome, or the host cell expresses the above-mentioned anti-CD16A nanobody, antibody or bispecific antibody.

[0018] In a seventh aspect, the present invention provides a method for preparing nanobodies, comprising: culturing the above-mentioned host cells under conditions suitable for producing antibodies to obtain a culture containing anti-CD16A antibodies, and then isolating or recovering the anti-CD16A antibodies from the culture.

[0019] In an eighth aspect, the present invention provides an immunoconjugate or pharmaceutical composition comprising the above-mentioned anti-CD16A nanobody, antibody or bispecific antibody.

[0020] In a ninth aspect, the present invention provides the use of the above-mentioned anti-CD16A nanobody, antibody, bispecific antibody, nucleic acid, expression vector, host cell or immunoconjugate or pharmaceutical composition in the preparation of a product for preventing or treating tumors.

[0021] The present invention has the following beneficial effects:

[0022] The anti-CD16A nanobody screened from the Bactrian camel VHH immune library can specifically bind to the CD16A antigen, and the affinity of the nanobody is between 10 -9 M number, belonging to high-affinity nano antibodies. In addition, the present invention constructs a heterodimeric bispecific antibody targeting CD16A and an anti-CD22 nanoantibody, which still maintains good antigen binding activity to CD16A; at the same time, in vivo and in vitro experiments have confirmed that the heterodimeric bispecific antibody has a strong killing activity against acute B lymphocytic leukemia, which can effectively improve the treatment effect and provide a key material for the subsequent development of immunotherapy with high efficiency and lasting anti-tumor activity, which has important clinical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0024] Figure 1 The binding specificity between CD16A nanobody and CD16A antigen was analyzed by indirect ELISA in Example 2 of the present invention;

[0025] Figure 2 The binding ability of CD16A nanobody to CD16A antigen was analyzed by indirect ELISA in Example 3 of the present invention;

[0026] Figure 3 This is a diagram showing the binding effect of recombinant nanobodies and CD16A+-HeLa cells detected by flow cytometry in Example 4 of the present invention;

[0027] Figure 4 This is the construction of the bispecific antibody (CD22×CD16A) gene fragment in Example 6 of the present invention;

[0028] Figure 5 The killing efficiency of the bispecific antibody in Example 6 of the present invention on the CD22-positive cell line is detected;

[0029] Figure 6 This is an analysis of the survival of the B-ALL mouse transplant model using the bispecific antibody in Example 7 of the present invention. DETAILED DESCRIPTION

[0030] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0031] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR) Reaction" (M.μLlis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0032] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0033] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0034] In this specification, some embodiments may be disclosed in a format of being in a certain range. It should be understood that this description of "being in a certain range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the description of the range should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the range, the above rules apply.

[0035] The present invention provides a CD16A nanobody, whose heavy chain variable region includes CDR1, CDR2 and CDR3, the amino acid sequence of CDR1 is shown in SEQ ID NO: 1, the amino acid sequence of CDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 3.

[0036] The nanobodies of the present invention generally include four framework regions (FRs) and three complementary determining regions (CDRs), which are respectively referred to as FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The above FRs and CDRs constitute the heavy chain variable region, and its structure is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0037] In some embodiments, the Nanobody is at least one of a monovalent Nanobody, a multivalent Nanobody, a multispecific antibody, and a fusion Nanobody.

[0038] Monovalent nanoantibodies: Antigen-specific nanoantibodies obtained by screening from a nanoantibody library using specific antigens. Because of the large number of hydrophilic residues on their surface, they can maintain a strict monomer structure and can bind to their antigens with high specificity and high affinity only in this monomer form.

[0039] Multivalent Nanobodies: Multivalent antibodies are polymers of monovalent antibodies that recognize the same epitope and have higher antigen affinity than the corresponding monovalent Nanobodies.

[0040] Multispecific antibodies are polymers of monovalent antibodies that recognize different epitopes. They can bind to different targets or different epitopes of the same target and have higher antigen recognition ability than monovalent antibodies.

[0041] Fusion nanoantibodies: Antibodies include but are not limited to new fusion molecules formed by combining with other structures (such as BSA, IgG-Fc, etc.) through genetic engineering technology, such as enzymes that can prolong their half-life, antimicrobial peptides or imaging substances.

[0042] In some embodiments, when the Nanobody is a monovalent Nanobody, its amino acid sequence is as shown in SEQ ID NO:4.

[0043] The present invention also provides an antibody comprising the above-mentioned anti-CD16A nanobody or the heavy chain variable region of the anti-CD16A nanobody.

[0044] In some embodiments, the antibody can be any of a full-length antibody, a heavy chain antibody, a chimeric antibody, a multispecific antibody (e.g., a bispecific antibody, a trispecific antibody, a tetraspecific antibody, etc.), a murine antibody, a humanized antibody, or an antigen-binding fragment.

[0045] The antigen-binding fragment includes any one selected from F(ab')2, Fab', Fab, Fv and scFv of an antibody, as long as they exhibit the desired antigen-binding activity.

[0046] Chimeric antibodies are antibodies formed by fusing the variable region of a non-human antibody with the constant region or framework region of a human antibody, which can reduce the immune response induced by non-human antibodies.

[0047] The above antigen-binding fragments, i.e., functional fragments of antibodies, generally have the same binding specificity as the antibodies from which they are derived. It is easy for a person skilled in the art to understand based on the contents described in the present invention that the functional fragments of the above antibodies can be obtained by, for example, enzymatic digestion (including pepsin or papain) and / or by chemical reduction to split disulfide bonds. Based on the structure of the complete antibody disclosed in the present invention, a person skilled in the art can easily obtain the above functional fragments.

[0048] The above antigen-binding fragments can also be synthesized by recombinant genetic techniques also known to those skilled in the art or by, for example, an automatic peptide synthesizer, such as those sold by Applied BioSystems and the like.

[0049] The present invention also provides a bispecific antibody, which comprises an anti-CD16A nanobody and an anti-CD22 nanobody.

[0050] In some embodiments, the above-mentioned bispecific antibody sequentially connects the anti-CD22 nanobody and the anti-CD16A nanobody via a connecting peptide.

[0051] In some embodiments, the above-mentioned connecting peptide is (G4S)n, wherein n is a non-zero integer.

[0052] In some embodiments, n is an integer between 1 and 20; specifically, n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0053] In some embodiments, n is 3 or 4.

[0054] In some embodiments, when the anti-CD22 nanobody is a monovalent nanobody, the amino acid sequence of its heavy chain variable region is as shown in SEQ ID NO: 5 (granted patent number: CN202310419011.2).

[0055] The present invention also provides a nucleic acid encoding the above-mentioned anti-CD16A nanobody, antibody or bispecific antibody.

[0056] Taking into account the degeneracy of codons, the gene sequence encoding the above-mentioned antibody can be modified in its coding region without changing the amino acid sequence to obtain a gene encoding the same antibody; the gene can also be artificially synthesized and modified according to the codon preference of the host expressing the antibody to improve the expression efficiency of the antibody.

[0057] The present invention also provides an expression vector comprising the above nucleic acid.

[0058] The expression vector in the present invention refers to any recombinant polynucleotide construct, which can directly introduce the target DNA fragment into the host cell by transformation, transfection or transduction to express the target gene.

[0059] The vector of the present invention can be selected from various vectors known in the art. For example, a commercially available vector is selected, and then the nucleotide sequence encoding the antibody of the present invention is operably linked to an expression regulatory sequence to form an expression vector.

[0060] The present invention also provides a host cell, which contains the above expression vector, or the above nucleic acid is integrated into the genome, or the host cell expresses the above anti-CD16A nanobody, antibody or bispecific antibody.

[0061] In some embodiments, the host cell is selected from at least one of a prokaryotic host cell, a eukaryotic host cell, and a bacteriophage.

[0062] In some embodiments, the eukaryotic host cell is an animal cell, a plant cell, or a fungus, wherein the animal cell includes a mammalian cell.

[0063] In some embodiments, mammalian cells include 293 cells, 293T cells, 293FT cells, CHO cells and Per6 cells, which are commonly used mammalian cells for producing antibodies or recombinant proteins and are well known to ordinary technicians in the field.

[0064] The present invention also provides a method for preparing a nanobody, comprising: culturing the above host cell under conditions suitable for producing the antibody, thereby obtaining a culture containing the anti-CD16A antibody, and then separating or recovering the anti-CD16A antibody from the culture. Specifically, the present invention does not specifically limit the culturing conditions of the host cell, and based on conventional technical knowledge, the culturing conditions that enable the host cell to express and produce the antibody can be obtained.

[0065] The present invention also provides an immunoconjugate or pharmaceutical composition containing the above-mentioned anti-CD16A nanobody, antibody or bispecific antibody.

[0066] In some embodiments, the immunoconjugate further comprises a therapeutic agent.

[0067] In some embodiments, the therapeutic agent includes at least one of a chemotherapeutic drug, a radionuclide, a photosensitizer, a photothermal agent, an immune checkpoint inhibitor, a toxin, a factor, a kinase inhibitor, an antibody to an inhibitory second signal molecule, a PD-L1 inhibitor, and a PD-1 / PD-L1 monoclonal antibody drug.

[0068] Relevant biomarkers for immune checkpoint inhibitor treatment include PD-L1, MSI / bMSI, TMB / bTMB, TNB, EGFR mutation, ALK fusion, TP53 mutation, and KRAS mutation.

[0069] In some embodiments, the chemotherapy drug is selected from any one or more of taxanes, vinca alkaloids, anthracyclines, epipodophyllotoxins, tyrosine kinase inhibitors, flavopiridol, irinotecan and its metabolite SN-38, topotecan, teniposide, etoposide, imatinib, gefitinib, danucetinib, doxorubicin, daunorubicin, mitoxantrone, methotrexate, camptothecin and saquinavir.

[0070] The photosensitizer is selected from: (1) 5-aminolevulinic acid (ALA) or its derivatives; (2) photosensitizer compounds containing a tetrapyrrole ring; (3) traditional Chinese medicine photosensitizers; or (4) a combination of ALA or its derivatives and the compounds in (2) or (3).

[0071] The photothermal agent is selected from IR-780, IR-783, IR-805, IR-808, IR-825, IR-1045, IR-1048, IR-1061 and IR-26.

[0072] The inhibitory second signal molecule can be PD-1; CTLA-4; PD-1 and CTLA-4.

[0073] In some embodiments, the PD-1 / PD-L1 monoclonal antibody drug is selected from at least one of the following groups: Nivolumab, Pembrolizumab, Pidilizumab, Lambrolizumab, BMS-936559, Atezolizumab, AMP-224, AMP224, AUNP12, BGB108, MCLA134, MEDI0680, PDROOl, REGN2810, SHR1210, STIAl lOX, STIAl l lO, TSR042, BMS-936558, BGB-A317, BCD-100 and JS001.

[0074] In some embodiments, the above therapeutic agents further include a cytotoxic agent.

[0075] In some embodiments, the pharmaceutical composition includes at least one of a pharmaceutical excipient, a pharmaceutically acceptable carrier, and a diluent.

[0076] The above-mentioned pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active ingredient (antibody or antigen-binding portion thereof, conjugate / conjugate or bispecific molecule) can be coated in a substance to protect the active ingredient from the action of acids and other natural conditions that may inactivate the active ingredient.

[0077] Pharmaceutical compositions must generally be sterile and stable under production and storage conditions. Pharmaceutical compositions can be formulated into solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or dispersion medium comprising, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and a suitable mixture thereof.

[0078] In some embodiments, pharmaceutically acceptable carriers include, but are not limited to, fillers, lubricants, disintegrants, binders, glidants, and the like.

[0079] In some embodiments, pharmaceutically acceptable carriers include, but are not limited to, polyvinyl pyrrolidone and its derivatives, polyvinyl alcohol and its derivatives, methyl cellulose and its derivatives, ethyl cellulose and its derivatives, hydroxypropyl cellulose and its derivatives, starch and its derivatives, polyethylene glycol and its derivatives, lactose, sucrose, mannitol, trehalose, sorbitol, dextrin, microcrystalline cellulose, acrylic resin, calcium hydrogen phosphate, calcium stearate, sodium stearyl fumarate, silicon dioxide, titanium dioxide, talc, indigo or a combination thereof.

[0080] The present invention also provides the use of the above-mentioned anti-CD16A nanobody, antibody, bispecific antibody, nucleic acid, expression vector, host cell or immunoconjugate or pharmaceutical composition in the preparation of products for preventing or treating tumors.

[0081] In some embodiments, the product includes at least one of: an immune cell, a reagent, a kit, a drug, and a pharmaceutical composition.

[0082] In some embodiments, the product is used to prevent or treat at least one of acute B-cell leukemia, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, hairy cell leukemia, systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis.

[0083] In the present invention, the term "treatment" includes curing a disease, inhibiting or delaying the development or progression of a disease, and alleviating and / or stabilizing the disease state.

[0084] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0085] Example 1

[0086] This example is the preparation of CD16A recombinant protein, which is as follows:

[0087] The purchased plasmid encoding human pCMV3-untagged-CD16A was used as a template for PCR amplification. The extracellular domain (ECD) fragment was obtained by agarose gel electrophoresis. The extracellular domain of CD16A was cloned into the pcDNA3.1 expression vector with Fc or His tag at the C-terminus using the pcDNA3.1 vector as the backbone. The Fc tag includes human Fc (hFc) and mouse Fc (mFc). Then, 293FT was transiently transfected and FreeStyle TM The cells were cultured in shake flasks in serum-free medium (Life Technologies) for 5-7 days, and the supernatant was collected and purified by Protein A / G or nickel column affinity chromatography and molecular sieve chromatography column to purify the recombinant CD16A protein carrying the Fc or His tag.

[0088] Example 2

[0089] This example is the construction, panning and ELISA preliminary screening of phage nanobody library, including the following steps:

[0090] (1) Bactrian camel immunization

[0091] Take 2 mg of the expressed and purified CD16A extracellular domain recombinant protein, add 2 mL of Freund's complete adjuvant, and fully emulsify it using an emulsifier; immunize the Bactrian camel subcutaneously at multiple points in the neck, and then immunize once every two weeks (2 mg protein) with Freund's incomplete adjuvant for a total of 4 immunizations. After the last immunization, collect peripheral blood for titer detection. One week after the impact immunization, collect Bactrian camel peripheral blood to isolate lymphocytes.

[0092] (2) Construction of Nanobody Library

[0093] When the camel reaches a certain immune titer, it will be subjected to the last impact immunization. After 7 days, 200 mL of peripheral blood will be collected in a blood collection bag for lymphocyte separation. Take out the above-isolated lymphocytes and extract RNA according to the steps of Promega's RNA extraction kit. Immediately after RNA extraction from lymphocytes, reverse transcribe cDNA using the TaKaRa reverse transcription kit, and then amplify the VHH gene using nested PCR; insert the amplified VHH gene into the pMECS phage display vector and electrotransform TG1 competent cells. Take the culture solution after electroporation and dilute it in multiples (10-fold dilution) using LB / Amp-GLU medium, and then take 10 -4 , 10 -5 , 10 -6 , 10 -7 100 μL of the dilution solution was spread on LB / Amp-GLU plates and incubated at 37°C. After 8 h of incubation, the number of colonies at different dilutions was counted to calculate the capacity of the antibody library, which was 6.56×10 9 At the same time, 50 colonies with similar morphology and size were randomly selected and cultured for 4-6 hours, and PCR was performed on the bacterial solution to identify the positive rate of the library, that is, the insertion rate of the library reached 97%.

[0094] (3) Screening of CD16A Nanobodies

[0095] First, the preparation and concentration of auxiliary phages and the rescue of phage libraries were performed. The panning steps of the nanoantibody phage library are as follows: ① Antigen coating: After diluting the CD16A-mFc recombinant protein with PBS, 20 μg per well (the antigen coating amount for the subsequent two rounds of panning was 10 μg / well and 5 μg / well, respectively) was coated in a 96-well ELISA plate and coated overnight at 4°C; ② Washing: After overnight coating, the liquid in the well was discarded and each well was washed 5 times with 200 μL PBST; ③ Blocking: Add 200 μL 5% skimmed milk powder to each well and place it at 37°C for 1 hour; ④ Washing: Discard the liquid in the well and wash each well 3 times with 200 μL PBST; ⑤ Incubation of recombinant phage: Dilute the recombinant phage to 5×10 with 5% skimmed milk powder. 11 pfu / mL, add 100 μL to each well and incubate at room temperature for 2 h; ⑥ Washing: discard the liquid in the wells and wash each well with 200 μL PBST for 15 times. Add 100 μL of freshly prepared 0.1 M triethylamine to each well, let it stand at room temperature for 10 min, aspirate the eluate into a 1.5 mL centrifuge tube and quickly add an equal volume of 1 M Tris-HCl (pH = 7.4) for neutralization; ⑦ Determination of recombinant phage titer: collect the neutralized phage solution and determine the phage titer; infect 2 mL of TG1 in the logarithmic growth phase with the remaining phage solution, and let it stand at 37°C for 30 min; add 8 mL of 2×YT / Amp GLU culture medium, and culture at 37°C 220 rpm until the logarithmic growth phase; ⑧ Rescue: add 8 mL of 2×YT ampicillin resistance culture medium, add 4% glucose, and culture at 37°C 220 rpm; ⑨ Phage concentration; ⑩ Repeat the above steps ①-⑨ for the second and third rounds of screening.

[0096] (4) Detection of specific recombinant phage enrichment

[0097] Antigen coating: After diluting the two antigens with PBS, 400 ng per well was coated in a 96-well ELISA plate and coated overnight at 4°C. Washing: After overnight coating, the liquid in the wells was discarded and each well was washed three times with 200 μL PBST. Blocking: 200 μL 5% skim milk powder was added to each well and placed at 37°C for blocking for 1 hour. Washing: The liquid in the wells was discarded and each well was washed three times with 200 μL PBST. Incubation of recombinant phages: Dilute the phage concentrate (1:10), add 100 μL to each well, and incubate at 37°C for 1 hour. Washing: The liquid in the wells was discarded and each well was washed three times with 200 μL PBST. Secondary antibody: HRP-labeled mouse anti-M13 secondary antibody was diluted 1:2000, 100 μL / well, and incubated at 37°C for 1 hour. Washing: The liquid in the wells was discarded and each well was washed three times with 200 μL PBST. Color development: Add 100 μL of TMB color development solution to each well and place at room temperature away from light for 10-15 minutes. Stop and read: After color development, add 50 μL of 2M H2SO4 to each well to stop the reaction; read the absorbance at 450 nm. Analyze the data.

[0098] (5) Sequencing analysis of specific nanobodies

[0099] Through the ELISA test results, clones with a value greater than 3 times the negative value were determined to be positive, and the bacterial solution was sent for sequencing and comparison analysis, and finally a CD16A nanobody sequence was obtained, named Nb60 (1#). The sequence of the nanobody is shown in Table 1 below.

[0100] Table 1 Amino acid sequences of Nanobodies

[0101]

[0102]

[0103] (6) Specificity analysis of anti-CD16a protein nanoantibodies

[0104] The recombinant supernatant of the screened nanoantibodies was used as the primary antibody and incubated with CD16A recombinant protein and other irrelevant antigens (CD16B, NKP30, CD22) and detected using HRP anti-M13 antibody. The results are as follows Figure 1 As shown, the obtained CD16A nanobody can bind to CD16A recombinant protein and does not react with other unrelated antigens, indicating that the above nanobody has good specific binding activity.

[0105] Example 3

[0106] This example is an experiment on the expression, purification and reactivity identification of CD16A-specific nanobodies with antigens, as follows:

[0107] A recombinant nanobody expression platform was constructed based on the pcDNA3.1 eukaryotic expression vector, and the CD16A-specific nanobody sequences were cloned into the pcDNA3.1 expression vector. The constructed expression vector was expressed and purified using the HEK293T eukaryotic protein expression system. SDS-PAGE results showed that after affinity chromatography purification, a high-purity recombinant nanobody was obtained, and the band size was around 55kDa.

[0108] To identify the reactivity of recombinant nanoantibodies with antigens, 200 ng / well of CD16A recombinant protein was coated on an ELISA plate in advance, the plate was blocked after overnight at 4°C, and different amounts of recombinant nanoantibodies (dilution: 10 -6 ~10μg / mL), add secondary antibody for washing, color development, terminate the reaction, and measure the optical density (OD450) at 450nm using a microplate reader, and determine the binding capacity using four-parameter nonlinear regression curve fitting.

[0109] Figure 2 The results showed that the CD16A nanobody could bind to the CD16A recombinant protein with high specificity.

[0110] Example 4

[0111] This example is a flow cytometric detection of recombinant nanobodies and CD16A + -Hela cell binding experiment, as follows:

[0112] First, Hela cells were infected with a lentivirus containing the full-length CD16A gene, and high-purity CD16A cells stably expressing the CD16A gene were obtained by flow cytometry. + -Hela cells, and then the recombinant nanobody prepared in Example 3 was combined with CD16A + -Hela cells were incubated at 37°C for 40 min, washed 3 times with PBS, and then incubated with APC@goat anti-human secondary antibody. After washing 3 times with PBS, the cells were detected by flow cytometry.

[0113] The results are as follows Figure 3 As shown, the results showed that the recombinant nanobody Nb60 could bind well to the above cells.

[0114] Example 5

[0115] This example is a CD16A nanobody affinity determination experiment, which is as follows:

[0116] The CD16A nanobody was used to verify the affinity of the recombinant nanobody and determine its binding kinetic constant (Kd). TM8K instrument, the anti-mouse IgG antibody was fixed on the surface of CM5 chip using the coupling buffer in the amino coupling kit, and CD16A (ECD)-mFc was captured on the CM5 chip in a 2-fold serial dilution; then the purified CD16A nanobody was allowed to flow over the surface of the chip, and the machine read Ka (1 / Ms), kd (1 / s), and KD (M), that is, the affinity of the recombinant nanobody was measured. The affinity measurement results showed that the CD16A nanobody could specifically bind to the CD16A (ECD)-mFc protein, with an affinity of 10 -9 M order of magnitude, belonging to high-affinity antibodies. The specific data of the test are shown in Table 2.

[0117] Table 2 Summary of affinity data of nanobodies

[0118] Antibody Ka(1 / Ms) kd(1 / s) KD(M) Nb60 1.16E-03 7.33E+05 1.58E-09

[0119] Example 6

[0120] This example is the preparation of anti-CD22 and CD16A bispecific antibodies, as follows:

[0121] The nanobody of the present invention and the anti-CD22 molecule (named Nb513 (2#) in this example, whose sequence is shown in SEQ ID NO: 5) were constructed in series into a eukaryotic expression vector, transfected into HEK293T cells for expression, and purified using a Ni column. Figure 4 The purified bispecific antibodies were incubated with NK cells and NALM-6-luciferase and Raji-luciferase for 24 hours, and the killing activity was evaluated by detecting the fluorescence intensity.

[0122] The results are as follows Figure 5 As shown in the figure, compared with adding only NK cells, the killing ability of the bispecific antibody and NK cells group on tumor cells was significantly enhanced, indicating that the bispecific antibody can promote the killing ability of NK cells.

[0123] Example 7

[0124] This example is an in vivo test of the anti-tumor activity of a bispecific antibody in a B-ALL mouse transplant model, as follows:

[0125] NALM-6 and Raji cells purchased from ATCC were prepared into cell lines stably expressing luciferase, NALM-6--mCherry.ffLuc and Raji--mCherry.ffLuc. 2×10 6The cells were inoculated into 6-8 week old NCG mice through the tail vein. Ten days after inoculation, the tumor growth was detected in a live imaging instrument and the mice were randomly divided into 8 groups, with 5 mice in each group. On the 10th day after inoculation of tumor cells, a control group was set up with an equal volume of PBS as the drug administration. The bispecific antibody was administered through the tail vein at 30 μg / mouse. The control group was injected with 200 μL of normal saline per time, 3 times a week, and the NK group was injected with 1×10 NK cells through the tail vein. 7 1×10 NK cells / time, 3 times a week; CD22×CD16A group was injected with 1×10 NK cells through tail vein 7 Each mouse was treated 3 times a week for 2 consecutive weeks. The survival status of each mouse in the experimental group (double antibody + NK), control group (NK) and blank group (PBS) was observed and counted for 80 days. The Kaplan-Meier method was used to draw the mouse survival curve and the log-rank (Mantel-Cox) test was used to compare the differences in the survival period of mice in each group.

[0126] The results are as follows Figure 6 As shown, the survival period of the bispecific antibody group was significantly longer than that of the negative control antibody and PBS groups, indicating that the bispecific antibody effectively controlled the proliferation and growth of B-ALL cells and significantly prolonged the survival period of mice.

[0127] The above experiments collectively illustrate that the anti-CD16A nanoantibodies screened by the present invention can specifically bind to the CD16A target with high affinity, and can be widely used in the prevention and treatment of tumors or autoimmune diseases, providing a new option for the prevention or treatment of tumors or autoimmune diseases.

[0128] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An anti-CD16A nanobody, characterized in that: The heavy chain variable region of the nanobody includes CDR1, CDR2 and CDR3, the amino acid sequence of the CDR1 is shown in SEQ ID NO: 1, the amino acid sequence of the CDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of the CDR3 is shown in SEQ ID NO:

3.

2. The Nanobody according to claim 1, characterized in that The Nanobody also includes a framework region.

3. The Nanobody according to claim 2, characterized in that When the Nanobody is a monovalent Nanobody, the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO:

4.

4. A bispecific antibody, characterized in that: Comprising the anti-CD16A nanobody and the anti-CD22 nanobody according to any one of claims 1 to 3; The bispecific antibody sequentially connects the anti-CD22 nanobody and the anti-CD16A nanobody via a connecting peptide; when the anti-CD22 nanobody is a monovalent nanobody, the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO:

5.

5. The bispecific antibody according to claim 4, characterized in that The connecting peptide is (G4S)n, wherein n is a non-zero integer.

6. The bispecific antibody according to claim 5, characterized in that n is an integer between 1 and 20.

7. The bispecific antibody according to claim 5, characterized in that n is 3 or 4.

8. A nucleic acid, characterized in that The nucleic acid encodes the anti-CD16A nanobody according to any one of claims 1 to 3 or the bispecific antibody according to any one of claims 4 to 7.

9. An expression vector, characterized in that: Comprising the nucleic acid of claim 8.

10. The expression vector according to claim 9, characterized in that The vectors include plasmids and viruses.

11. The expression vector according to claim 10, characterized in that The virus is selected from any one of adenovirus, adeno-associated virus, retrovirus, lentivirus or oncolytic virus.

12. A host cell, characterized in that The host cell contains the expression vector according to any one of claims 9 to 11, or the nucleic acid according to claim 8 is integrated into the genome, or the host cell expresses the anti-CD16A nanobody according to any one of claims 1 to 4 or the bispecific antibody according to any one of claims 4 to 7.

13. The host cell according to claim 12, characterized in that The host cell is selected from at least one of a prokaryotic host cell, a eukaryotic host cell and a bacteriophage.

14. The host cell according to claim 13, characterized in that The eukaryotic host cell is an animal cell, a plant cell or a fungus.

15. The host cell according to claim 14, characterized in that The animal cells include mammalian cells.

16. The host cell according to claim 15, characterized in that The mammalian cells include 293 cells, 293T cells, 293FT cells, CHO cells and Per6 cells.

17. A method for preparing a nanobody, characterized in that: The method comprises: culturing the host cell according to any one of claims 12 to 16 under conditions suitable for producing antibodies, thereby obtaining a culture containing the anti-CD16A antibody, and then isolating or recovering the anti-CD16A antibody from the culture.

18. Use of the anti-CD16A nanobody according to any one of claims 1 to 3, the bispecific antibody according to any one of claims 4 to 7, the nucleic acid according to claim 8, the expression vector according to any one of claims 9 to 11, or the host cell according to any one of claims 12 to 16 in the preparation of a product for preventing or treating tumors, characterized in that: Such tumors include B-cell acute lymphoblastic leukemia.

19. The use according to claim 18, characterized in that The product includes at least one of: immune cells, reagents, kits, drugs and pharmaceutical compositions.

Images