CN117700555A

CN117700555A - anti-CLL 1 nanobodies and related uses thereof

Info

Publication number: CN117700555A
Application number: CN202311798887.9A
Authority: CN
Inventors: 路琪中
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2023-12-25
Filing date: 2023-12-25
Publication date: 2024-03-15

Abstract

The invention discloses an anti-CLL 1 nano antibody and related application thereof, and relates to the field of antibodies. The invention provides nanobodies with high affinity for CLL1 proteins, which are capable of specifically binding to human CLL1 positive cells, and can be used for detecting expression of CLL1 in patient cells of a related disease targeted at CLL 1; secondly, the recombinant nano antibody for resisting the CLL1 provided by the invention can effectively activate the effector function of NK cells and has stronger killing capacity on CLL1 positive cells; in addition, the nano antibody can be prepared into immune complexes or pharmaceutical compositions for preventing or treating diseases related to CLL1 targets, or detection reagents or kits for CLL1 proteins, and has wide application prospects in the fields of clinical diagnosis, prevention, treatment and the like.

Description

anti-CLL 1 nanobodies and related uses thereof

Technical Field

The invention relates to the field of antibodies, in particular to an anti-CLL 1 nanobody and related application thereof.

Background

Acute myelogenous leukemia (Acute myeloid leukemia, AML) is the most common acute adult leukemia and the second most common childhood leukemia, a disease characterized by rapid proliferation and accumulation of undifferentiated myeloid cells in the bone marrow and peripheral blood, with 5-year survival rates of less than 30% in adult AML patients. The long term survival of most AML patients is poor, chemotherapy and hematopoietic stem cell transplantation are the standard therapies for the current treatment of AML. Immunotherapy has been considered a breakthrough therapy in the area of hematological malignancies and solid tumors for the past few years. However, in AML, the use of immunotherapy in AML has generally progressed slowly due to factors such as lack of high specificity of target antigen and high heterogeneity of AML. For example, the putative antigens CD33 and CD123 on AML cells, as they are also present on the surface of normal hematopoietic stem cells (Hematopoietic stem cell, HSCs), immunotherapy targeting these antigens can lead to severe whole blood cytopenias.

In 2004, bakker et al reported for the first time that C-type lectin-like molecule 1 (CLL 1) was present in more than 90% of the myeloid cells of AML patients by phage display technology, while CLL1 was also highly expressed in leukemia hematopoietic stem cells (LSCs), but not in normal hematopoietic stem cells. Larsen et al found that the expression of CLL-1 was limited to myeloid cells compared to other stem cell antigens, indicating that CLL-1 can be used as a marker for AML diagnosis, and that, in addition, the expression of CLL-1 was stable throughout the course of the disease, with no differences in expression between diagnostic and recurrent samples from the same patient. CLL-1 can therefore be one of the important molecular markers for the detection of AML stem cell level minimal residual lesions (MRD). In addition, zhao et al developed a monoclonal antibody targeting CLL1 that was able to demonstrate strong killing activity against AML cell lines by Complement Dependent Cytotoxicity (CDC) and antibody dependent cytotoxicity (ADCC) mechanisms. Thus, the particular expression pattern of CLL1 makes it one of the potential targets for AML immunotherapy.

Currently, antibodies targeting CLL1 with high specificity and high affinity are still lacking in the market.

In view of this, the present invention has been made.

Disclosure of Invention

The object of the present invention is to provide nanobodies against CLL1 and related uses thereof.

The invention is realized in the following way:

in a first aspect, embodiments of the present invention provide an anti-CLL 1 nanobody comprising a heavy chain variable region comprising a complementarity determining region as set forth in any one of: (1) The amino acid sequence is shown as CDR1, CDR2 and CDR3 shown in SEQ ID NO 1-3 in sequence; (2) The amino acid sequence is shown as CDR1, CDR2 and CDR3 shown in SEQ ID NO 5-6 in sequence; (3) The amino acid sequences are shown as CDR1, CDR2 and CDR3 of SEQ ID NO 9-11 in sequence.

In a second aspect, embodiments of the present invention provide an antibody or antigen-binding fragment thereof comprising: the nanobody of the previous embodiment.

In a third aspect, embodiments of the invention provide an isolated nucleic acid encoding a nanobody as described in the previous embodiments or encoding an antibody or antigen-binding fragment thereof as described in the previous embodiments, an expression cassette comprising the isolated nucleic acid, or a recombinant vector comprising the isolated nucleic acid.

In a fourth aspect, embodiments of the present invention provide a host cell comprising the recombinant vector of the previous embodiments.

In a fifth aspect, embodiments of the present invention provide a method for preparing an antibody, comprising: the host cells described in the previous examples were cultured.

In a sixth aspect, embodiments of the present invention provide a conjugate comprising: the nanobody of the previous embodiment or the antibody or antigen-binding fragment thereof of the previous embodiment.

In a seventh aspect, embodiments of the present invention provide an immunoconjugate or pharmaceutical composition comprising: the nanobody of the previous embodiment or the antibody or antigen-binding fragment thereof of the previous embodiment.

In an eighth aspect, embodiments of the invention provide the use of a nanobody as described in the preceding embodiments or an antibody or antigen binding fragment thereof as described in the preceding embodiments or an isolated nucleic acid as described in the preceding embodiments or a recombinant vector comprising said isolated nucleic acid or a host cell as described in the preceding embodiments or a conjugate as described in the preceding embodiments for the detection of CLL1 proteins for the diagnosis or treatment of non-diseases.

In a ninth aspect, the present embodiment provides the use of a nanobody as described in the previous embodiment or an antibody or antigen binding fragment thereof as described in the previous embodiment or an isolated nucleic acid or a recombinant vector comprising said isolated nucleic acid or a host cell as described in the previous embodiment or a conjugate as described in the previous embodiment for the preparation of a product for the prevention, diagnosis, treatment or adjuvant treatment of a disease associated with CLL1 as a target.

The invention has the following beneficial effects:

the invention provides nanobodies with high affinity for CLL1 proteins, which are capable of specifically binding to human CLL1 positive cells, and can be used for detecting expression of CLL1 in patient cells of a related disease targeted at CLL 1; secondly, the recombinant nano antibody for resisting the CLL1 provided by the invention can effectively activate the effector function of NK cells and has stronger killing capacity on CLL1 positive cells; in addition, the nano antibody can be prepared into immune complexes or pharmaceutical compositions for preventing or treating diseases related to CLL1 targets, or detection reagents or kits for CLL1 proteins, and has wide application prospects in the fields of clinical diagnosis, prevention, treatment and the like.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the detection of immunotitres by indirect ELISA in the examples of the present invention;

FIG. 2 shows the purity of Nbs-hFc recombinant nanobody detected by SDS-PAGE in the examples of the present invention;

FIG. 3 shows the detection of the reactivity of anti-CLL 1 nanobody with CLL1 protein by indirect ELISA in the examples of the present invention;

FIG. 4 is a schematic diagram showing the detection of binding of anti-CLL 1 nanobodies to CLL1-Hela cells using IFA in an example of the invention;

FIG. 5 is a flow cytometry assay for detecting binding of recombinant nanobodies to AML cell lines in an embodiment of the invention;

FIG. 6 shows the measurement of ADCC effect induced by Nbs-hFc recombinant nanobody in the examples of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

In one aspect, embodiments of the present invention provide an anti-CLL 1 nanobody comprising a heavy chain variable region comprising a complementarity determining region as set forth in any one of:

(1) The amino acid sequence is shown as CDR1, CDR2 and CDR3 shown in SEQ ID NO 1-3 in sequence;

(2) The amino acid sequence is shown as CDR1, CDR2 and CDR3 shown in SEQ ID NO 5-6 in sequence;

(3) The amino acid sequences are shown as CDR1, CDR2 and CDR3 of SEQ ID NO 9-11 in sequence.

The anti-CLL 1 nanobody with the complementarity determining region has good reactivity with an acute myelogenous leukemia cell line, and has stronger killing activity on the acute myelogenous leukemia cell line.

In some embodiments, the heavy chain variable region further comprises a framework region. Specifically, the heavy chain variable region of the nanobody has the structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

In some embodiments, the heavy chain variable region has an amino acid sequence as set forth in SEQ ID NO. 4.

In some embodiments, the heavy chain variable region has an amino acid sequence as set forth in SEQ ID NO. 8.

In some embodiments, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO. 12.

In some embodiments, the nanobody has a KD of 10 or less ^-9 M、10 ^-10 M and 10 ^-11 The affinity of any of M binds CLL1 protein.

In another aspect, embodiments of the present invention provide an antibody or antigen-binding fragment thereof comprising: the nanobody of any of the preceding embodiments.

Nanobodies have a large number of hydrophilic residues on their surface, maintain a strict monomer structure, and bind their antigen with high specificity and high affinity only in this monomer form. The nano antibody is single gene coding due to small molecular weight, is easy to carry out genetic engineering, and can polymerize a plurality of nano antibodies through a short connecting sequence to form a multivalent or multispecific antibody structure.

In some embodiments, the antibody is selected from the group consisting of: any one of a diabody, a bispecific antibody, a multivalent antibody, a multispecific antibody, a fusion antibody, and a chimeric antibody.

Herein, a "diabody" or "multivalent antibody" is a polymer of monovalent antibodies that recognize the same epitope, with a higher antigen affinity than the corresponding monovalent antibody.

"bispecific antibodies" or "multispecific antibodies" herein are polymers of monovalent antibodies that bind to different targets or different binding regions on the same target, with greater antigen recognition capability than the corresponding monovalent antibodies. Alternatively, the multispecific antibody may be a trispecific antibody, a tetraspecific antibody, or the like.

The "chimeric antibody" herein is usually an antibody in which a variable region of a non-human antibody is fused to a constant region or a framework region of a human antibody, and an immune response induced by the non-human antibody can be reduced.

Fusion type antibodies include fusion type nanobodies, including but not limited to, enzymes, antimicrobial peptides, or imaging substances that bind to other structures (e.g., BSA, igG-Fc, etc.) by genetic engineering techniques to form novel fusion molecules, such as to extend their half-lives.

In some embodiments, the fusion antibody is fused to a constant region of an antibody and a nanobody as described in any of the examples above.

In some embodiments, the constant region of an antibody may be a heavy chain constant region of an antibody, and in particular may be selected from a heavy chain constant region of a human antibody, a murine heavy chain constant region, a rabbit heavy chain constant region, a sheep heavy chain constant region, or a monkey heavy chain constant region.

In some embodiments, the heavy chain constant region of the human antibody is selected from the heavy chain constant region of any one of hIgG1, hIgG2, hIgG3, hIgG4, or a mutation thereof. The heavy chain constant region of IgG1 is used as the constant region of the nanometer antibody against CLL1, has extremely high inhibition rate to target cells and keeps good binding activity with antigen.

The antibody constructed after the anti-CLL 1 nanobody provided by the embodiment of the invention is combined with the human Fc fragment is verified to have a longer half-life due to the binding effect with FcRn.

In some embodiments, the antigen binding fragment comprises any one selected from the group consisting of F (ab ') 2, fab', fab, fv, and scFv of an antibody, so long as they exhibit the desired antigen binding activity.

The antigen binding fragments, i.e., functional fragments of antibodies, generally have the same binding specificity as the antibody from which they were derived. It will be readily appreciated by those skilled in the art from the disclosure herein that functional fragments of the above antibodies may be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by methods of chemical reduction cleavage of disulfide bonds. The above functional fragments are readily available to those skilled in the art based on the disclosure of the structure of the intact antibodies.

The antigen binding fragments described above may also be obtained synthetically by recombinant genetic techniques also known to those skilled in the art or by automated peptide synthesizers such as those sold for example as Applied BioSystems.

In another aspect, embodiments of the invention provide an isolated nucleic acid encoding a nanobody as described in any of the previous embodiments or encoding an antibody or antigen-binding fragment thereof as described in any of the previous embodiments, an expression cassette comprising the isolated nucleic acid, or a recombinant vector comprising the isolated nucleic acid. Taking into account the degeneracy of the codons, the sequence of the genes encoding the above antibodies or antigen-binding fragments thereof may be modified in the coding region thereof without changing the amino acid sequence to obtain genes encoding the same antibodies or antigen-binding fragments thereof; the modified genes can also be artificially synthesized according to the codon preference of the host for expressing the antibody so as to improve the expression efficiency of the antibody.

The recombinant vector is an expression vector or cloning vector, preferably an expression vector, and may refer to any recombinant polynucleotide construct that can be used to directly introduce a DNA fragment of interest into a host cell by transformation, transfection or transduction for expression of the gene of interest.

In another aspect, embodiments of the present invention provide a host cell comprising a recombinant vector according to any of the preceding embodiments.

Specifically, host cells include 293 cells, 293T cells, 293FT cells, CHO cells, per6 cells. 293 series cells, per6 cells and CHO cells are common mammalian cells used for the production of antibodies or recombinant proteins and are well known to those of ordinary skill in the art.

On the basis of the present invention, which discloses the amino acid sequence of an antibody or a functional fragment thereof, it is easy for a person skilled in the art to prepare the antibody or the functional fragment thereof by genetic engineering techniques or other techniques (chemical synthesis, recombinant expression), for example, by separating and purifying the antibody or the functional fragment thereof from a culture product of recombinant cells capable of recombinantly expressing the antibody or the functional fragment thereof according to any one of the above, and on the basis of this, it is within the scope of the present invention to prepare the antibody or the functional fragment thereof by any technique.

In another aspect, embodiments of the present invention provide a method for preparing an antibody, comprising: culturing the host cell of any of the preceding embodiments. Specifically, the culture conditions for the host cells are not particularly limited in the present invention, and culture conditions capable of allowing the host cells to express and produce the antibody can be obtained based on conventional technical knowledge.

In another aspect, embodiments of the present invention provide a conjugate comprising: the nanobody of any of the embodiments above or the antibody or antigen-binding fragment thereof of any of the embodiments above.

In some embodiments, the conjugate further comprises: a coupling moiety coupled to the nanobody or the antibody or antigen binding fragment thereof.

In some embodiments, the coupling moiety comprises: any one of a protein tag for purification, a label for detection or tracking, and a solid support.

In some embodiments, the label is selected from at least one of a fluorescent dye, an enzyme, a radioisotope, a chemiluminescent reagent, and a nanoparticle-based label.

In another aspect, embodiments of the present invention provide an immunoconjugate or pharmaceutical composition comprising: the nanobody of any of the embodiments above or the antibody or antigen-binding fragment thereof of any of the embodiments above.

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

In some embodiments, the therapeutic agent comprises: at least one of a chemotherapeutic agent, a radionuclide, a photosensitizer, a photothermal agent, an immune checkpoint inhibitor, a toxin, a factor, a kinase inhibitor, an antibody to an inhibitory second signaling molecule, a PD-L1 inhibitor, a PD-1 mab agent, and a PD-L1 mab agent.

In some embodiments, the biomarkers associated with immune checkpoint inhibitor treatment include PD-L1, MSI/bMSI, TMB/bTMB, TNB and EGFR mutations, ALK fusions, TP53 mutations, KRAS mutations.

In some embodiments, the chemotherapeutic agent is selected from any one or more of taxanes, vinca alkaloids, anthracyclines, epipodophyllotoxins, tyrosine kinase inhibitors, fraapine, irinotecan and its metabolite SN-38, topotecan, teniposide, etoposide, imatinib, gefitinib, darnusertib, doxorubicin, daunorubicin, mitoxantrone, methotrexate, camptothecine, and saquinavir.

In some embodiments, the photosensitizer is selected from: (a) 5-aminolevulinic acid (ALA) or a derivative thereof; (b) a photosensitive compound containing a tetrapyrrole ring; (c) a traditional Chinese medicine photosensitizer; or (d) ALA or a derivative thereof in combination with the compound of (b) or (c), respectively.

In some embodiments, the photothermal agent is selected from the group consisting of IR-780, IR-783, IR-805, IR-808, IR-825, IR-1045, IR-1048, IR-1061, and IR-26.

In some embodiments, the inhibitory second signal molecule may be PD-1; CTLA-4; PD-1 and CTLA-4.

In a preferred embodiment of the invention, the PD-1/PD-L1 mab is selected from at least one of the following groups: nivolumab (Nivolumab), pembrolizumab (Pembrolizumab), picolizumab (pimelizumab), BMS-936559, atuzumab (Atezolizumab), AMP-224, AMP224, AUNP12, BGB108, MCLA134, MEDI0680, PDROOl, REGN2810, SHR1210, TSR042, BMS-936558, BGB-a317, BCD-100, and JS001.

In other embodiments, the therapeutic agent further comprises a cytotoxic agent.

The term "pharmaceutical composition" as used herein means a combination of at least one drug and optionally a pharmaceutically acceptable carrier or adjuvant, which are combined together to achieve a particular purpose. In certain embodiments, the pharmaceutical compositions comprise combinations that are separated in time and/or space, so long as they are capable of co-acting to achieve the objects of the present invention.

In some embodiments, the pharmaceutical composition further comprises: at least one of a pharmaceutically acceptable excipient, carrier and diluent.

Such carriers are pharmaceutically acceptable carriers including, but not limited to, fillers, lubricants, disintegrants, binders, glidants, and the like.

The pharmaceutically acceptable carrier includes, but is not limited to, one or a combination of polyvinylpyrrolidone and its derivatives, polyvinyl alcohol and its derivatives, methylcellulose and its derivatives, ethylcellulose 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, dibasic calcium phosphate, calcium stearate, sodium stearyl fumarate, silicon dioxide, titanium dioxide, talc, and color indigo.

In another aspect, the embodiments of the present invention provide the use of a nanobody as described in any of the preceding embodiments or an antibody or antigen-binding fragment thereof as described in any of the preceding embodiments or an isolated nucleic acid or a recombinant vector comprising said isolated nucleic acid or a host cell as described in any of the preceding embodiments or a conjugate as described in any of the preceding embodiments in the detection of CLL1 protein for the purpose of diagnosis or treatment of a non-disease.

There are many cases of detection for the purpose of diagnosis or treatment of non-disease, for example, when the sample to be detected is selected from a manually made sample, a negative sample and an environmental sample, the detection is for the purpose of diagnosis or treatment of non-disease.

Furthermore, embodiments of the present invention provide the use of a nanobody as described in any of the preceding embodiments or an antibody or antigen-binding fragment thereof as described in any of the preceding embodiments or an isolated nucleic acid as described in any of the preceding embodiments or a recombinant vector comprising said isolated nucleic acid or a host cell as described in any of the preceding embodiments or a conjugate as described in any of the preceding embodiments for the preparation of a product for the prevention, diagnosis, treatment or adjuvant treatment of a tumor or a related disease.

"treating" in the present invention includes preventing or alleviating a condition, reducing the rate at which a condition is raised or developed, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or terminating symptoms associated with a condition, producing a complete or partial reversal of a condition, curing a condition, or a combination thereof.

For cancer, "treatment" may refer to inhibiting or slowing the growth, proliferation, or metastasis of a tumor or malignant cell, or some combination of the foregoing. For tumors, "treatment" includes clearing all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or slowing tumor progression, or some combination thereof.

In some embodiments, the product comprises: at least one of immune cells, reagents, kits, medicaments and pharmaceutical compositions.

In some embodiments, the tumor or related disease is: related diseases (CLL 1 expression positive diseases) targeting CLL 1.

In some embodiments, the related disease comprises: any one or more of myelodysplastic syndrome, acute myelogenous leukemia, and chronic myelogenous leukemia.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

EXAMPLE 1 preparation of anti-CLL 1 protein-specific nanobodies

Firstly, CLL1-His protein (Cat: 11896-H07H) (1 mg/time) purchased from the company of the biological technology of the state of the sense perk is uniformly mixed with an equal volume of aluminum adjuvant, then the mixture is continuously immunized for 3 times, the antibody titer is detected by indirect ELISA (enzyme-linked immunosorbent assay) on collected peripheral blood, and the result shows that the antibody titer of anti-CLL 1 in the peripheral blood of the camel is 1:256,000 (figure 1), so that the titer standard of a subsequent constructed library is reached. 200mL of peripheral blood is aseptically collected through jugular vein after one week of impact immunization, PBMCs are obtained by utilizing Ficoll-Paque Plus lymphocyte separation liquid through centrifugation, total lymphocyte RNA is extracted by utilizing an RNA extraction kit, VHH genes are amplified through RT-PCR, the amplified VHH genes (400 bp) are cloned into a phage display carrier pMECS through enzyme digestion connection, and the amplified VHH genes are transformed into escherichia coli TG1 competent cells through electrotransformation, so that the result shows that the reservoir capacity is successfully constructed to be 5.68x10 ⁹ Is a VHH phage antibody library of (C).

Coating a CLL1-His recombinant protein on an ELISA plate, continuously screening 3 rounds by using a phage display technology with the constructed phage library as an antibody source to obtain a nanometer antibody (see table 1) of anti-CLL 1, amplifying a VHH gene by using the nanometer antibody as a template, constructing the nanometer antibody into a eukaryotic expression vector pcDNA3.1-MCS-hFc-His by using a homologous recombination mode, and then using a transfection reagent PEI (POlyscicles, cat: 24765-1) into HEK293T cells in logarithmic growth phase, 8h after transfection, were replaced with FreeStyle ^TM 293 expression medium, continuously culturing for 5 days, collecting supernatant, purifying by using NTA-Ni column and obtaining high-purity recombinant nano antibody (figure 2).

TABLE 1 sequence information of anti-CLL 1 nanobodies

Example 2 indirect ELISA detection of binding of recombinant nanobody Nbs-hFc to CLL1 protein

The CLL1-His recombinant protein was coated in a 96-well ELISA plate at 200ng per well, blocked with 3% nonfat milk powder at 37℃for 1h, washed 3 times with PBST, and 100. Mu.L of different concentrations (10 ² ～10 ^-5 mu.g/mL) Nbs-hFc recombinant nanobody prepared in example 1 and incubated at 37℃for 1h, after PBST washing 3 times, 100. Mu.L of HRP@coat anti-human antibody (1:4000) was added to each well and incubated at 37℃for 1h, after PBST washing 3 times, TMB was developed for 5min and then incubated with 2M H ₂ SO ₄ Stop reaction, read OD ₄₅₀ The nm absorbance values are shown in figure 3, and the prepared recombinant nanometer antibodies Nb34-hFc, nb37-hFc and Nb38-hFc of the anti-CLL 1 have good binding activity with the CLL1-His protein, and the control protein hFc is not bound.

Example 3 affinity detection of nanobodies with CLL1 protein

The binding affinity of the recombinant nanobodies Nb34-hFc, nb37-hFc, nb38-hFc and anti-CLL 1 positive control antibody M26-scFv-hFc (patent number: CN 113248621A) provided in example 1 with the antigen CLL1-His coated on the CM5 chip was measured by using a Biacore 8k instrument, and the results are shown in Table 2, wherein the affinity of the nanobodies with the CLL1 protein was 10 ^-10 ～10 ^-9 M, significantly better than positive antibody M26.

TABLE 2 affinity and kinetic analysis of anti-CLL 1 nanobody binding to CLL1 protein

EXAMPLE 4IFA detection of binding of recombinant nanobodies to CLL1-Hela cells

The Hela cells were infected with lentivirus containing CLL1 full-length gene (gene number nm_ 138337.6), hela cells stably expressing CLL1 gene were obtained in high purity by flow sorting and named CLL1-Hela cells, and then the recombinant nanobodies Nb34-hFc, nb37-hFc, nb38-hFc and positive control antibodies M26-scFv-hFc (2.5 μg/mL) against CLL1 prepared in example 1 were incubated with CLL1-Hela cells at 37 ℃ for 40min, PBS washed 3 times, and then incubated with 594@goat anti-human secondary antibodies, PBS washed 3 times, and imaged with a fluorescence microscope, as shown in fig. 4, and the recombinant nanobodies Nb34-hFc, nb37-hFc and Nb38-hFc were all able to bind well to CLL1-Hela cells.

Example 5 flow detection of binding of recombinant nanobodies to AML cell lines

The recombinant nanobodies Nb34-hFc, nb37-hFc and Nb38-hFc prepared in example 1 and the positive control antibody M26-scFv-hFc (2.5. Mu.g/mL) from Guangzhou Bai and anti-CLL 1 were incubated with AML cell lines THP-1 and U937 at 37℃for 40min, washed 3 times with PBS and then incubated with APC@goat anti-human antibodies, and washed 3 times with PBS and analyzed by a flow cytometer, and the results show that the recombinant nanobodies Nb34-hFc, nb37-hFc and Nb38-hFc all have good reactivity with the positive control antibodies M26 and THP-1 and U937 cells.

Example 6 anti-CLL 1 recombinant nanobody-mediated detection of anti-tumor Activity

Firstly, preparing an AML cell line THP-1-luciferase and U937-luciferase which stably express luciferase, mixing NK cells derived from peripheral blood with the target cells according to the ratio of the effective target ratio of 8:1, respectively adding Nbs-hFc recombinant nanobodies of 40, 20, 10, 5 and 2.5 mug/mL and irrelevant control recombinant nanobodies Nb1-hFc of anti-PSMA (the patent application number: CN 202211408304.2) of Guizhou people's Hospital, wherein 3 repeated holes are arranged for each antibody, adding potassium salt of fluorescein (10 mug/mL) after co-culturing for 4 hours, measuring fluorescence values in a Tecan full-band enzyme-labeling instrument after blowing and mixing, and calculating the specific killing activity of the NK cells according to the average value of 3 repeated holes for each antibody.

The calculation formula is as follows: lysis (%) = [1- (experimental well reading/control well reading) ] ×100%

Wherein, the experimental hole is a coculture hole of the anti-CLL 1 recombinant nano antibody, NK cells and AML target cells; control wells were anti-PSMA antibody, NK cells and AML target cell co-culture wells.

As shown in FIG. 6, the anti-CLL 1 recombinant nanobody provided by the invention can mediate good ADCC activity, and the higher the concentration of the anti-CLL 1 recombinant nanobody is, the stronger the specific killing activity of NK cells is shown, so that the anti-CLL 1 recombinant nanobody prepared by the invention can obviously induce ADCC action of NK cells, and further has stronger killing capacity on AML cells positive to CLL1 expression.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A nanobody against CLL1, comprising a heavy chain variable region comprising a complementarity determining region set forth in any one of:

2. The nanobody of claim 1, wherein the heavy chain variable region further comprises a framework region;

alternatively, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 4;

optionally, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 8;

alternatively, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 12.

3. An antibody or antigen-binding fragment thereof, comprising: the nanobody of claim 1 or 2;

alternatively, the antibody is selected from the group consisting of: any one of a diabody, a bispecific antibody, a multivalent antibody, a multispecific antibody, a fusion antibody, and a chimeric antibody.

4. An isolated nucleic acid, an expression cassette comprising the isolated nucleic acid, or a recombinant vector comprising the isolated nucleic acid, wherein the isolated nucleic acid encodes the nanobody of claim 1 or 2 or encodes the antibody or antigen-binding fragment thereof of claim 3.

5. A host cell comprising the recombinant vector of claim 4.

6. A method of producing an antibody comprising: culturing the host cell of claim 5.

7. A conjugate, comprising: the nanobody of claim 1 or 2 or the antibody or antigen-binding fragment thereof of claim 3;

optionally, the conjugate further comprises: a coupling moiety coupled to the nanobody or the antibody or antigen binding fragment thereof;

optionally, the coupling moiety comprises: any one of a protein tag for purification, a label for detection or tracking, and a solid support.

8. An immunoconjugate or pharmaceutical composition, characterized in that it comprises: the nanobody of claim 1 or 2 or the antibody or antigen-binding fragment thereof of claim 3;

optionally, the immunoconjugate further comprises a therapeutic agent;

optionally, the therapeutic agent comprises: at least one of a chemotherapeutic agent, a radionuclide, a photosensitizer, a photothermal agent, an immune checkpoint inhibitor, a toxin, a factor, a kinase inhibitor, an antibody to an inhibitory second signaling molecule, a PD-L1 inhibitor, a PD-1 mab agent, and a PD-L1 mab agent;

optionally, the pharmaceutical composition further comprises: at least one of a pharmaceutically acceptable excipient, carrier and diluent.

9. Use of a nanobody according to claim 1 or 2 or an antibody or antigen-binding fragment thereof according to claim 3 or an isolated nucleic acid according to claim 4 or a recombinant vector comprising said isolated nucleic acid or a host cell according to claim 5 or a conjugate according to claim 7 for CLL1 protein detection for the purpose of diagnosis or treatment of a non-disease.

10. Use of a nanobody according to claim 1 or 2 or an antibody or antigen-binding fragment thereof according to claim 3 or an isolated nucleic acid according to claim 4 or a recombinant vector comprising said isolated nucleic acid or a host cell according to claim 5 or a conjugate according to claim 7 for the preparation of a product for the prevention, diagnosis, treatment or co-treatment of a disease associated with CLL1 as target;

optionally, the related diseases include: any one or more of myelodysplastic syndrome, acute myelogenous leukemia, and chronic myelogenous leukemia.