CN114805568B - Nano antibody targeting human LILRB2 and application thereof - Google Patents

Abstract

The invention discloses a nanobody resisting LILRB2, a nucleic acid encoding the nanobody, an expression vector containing the nucleic acid, a pharmaceutical composition containing the nanobody, and an application of preparing a medicament.

Description

Nano antibody targeting human LILRB2 and application thereof

Technical Field

The invention belongs to the field of antibody engineering, particularly relates to a therapeutic single-domain antibody for diagnosing or treating tumors, and particularly relates to a nano antibody for resisting LILRB2, a derivative protein thereof and application of the nano antibody in preparation of a medicament.

Background

Nanobodies are the smallest antibody molecule at present, originally found in camelid blood by the belgium scientist Hamers, a class of great interest in engineered antibody products. The nano antibody has the main advantages that: one is 1/10 with volume of common antibody, because of its small volume, it has strong penetrating power in animal tissue, for example, it can reach the inside of high density tumor through human brain tissue, but common antibody can't, it can treat some tumor or brain disease through nanometer antibody; secondly, the antigen specificity is good; thirdly, the gene modification is easy, and the artificial modification is convenient to obtain antibodies for resisting different pathogens; fourthly, the stability is high, for example, the nano antibody is not naturally decomposed in vivo for a longer time than the common antibody (meaning that the drug effect time is longer), and the nano antibody can even pass through the human stomach to keep the effectiveness.

LILRB2, also known as ILT4, is expressed predominantly on cells of the myeloid lineage, including monocytes, dendritic cells, macrophages and neutrophils. Genetic studies have shown that Tumor Associated Macrophages (TAMs) in various tumor microenvironments highly express LILRB2, and that inhibition of LILRB2 (the mouse counterpart protein, Pirb) reduces the invasion of tregs and MDSCs in tumor tissues. Animal experiments show that the Pirb antibody inhibits tumor growth and has synergistic effect with the PD-1 antibody. LILRB2 is mainly expressed in bone marrow cells and has limited expression in other tissues, which makes on-target and off-tissue toxicity slightly. Human LILRB2 is an important homeostatic surface regulator in the maturation of myeloid cells, is a promising target of myeloid immune checkpoint specifically directed to the determination of myeloid cell function, and has important therapeutic value.

Disclosure of Invention

ScFv, Fab or whole IgG anti-LILRB 2 antibody molecules in the prior art have complex structure and larger molecules, and although the active molecules can be connected to LILRB2, the molecules have complex functions and methods and lower loading efficiency and influence the functions of the active molecules; the nano antibody has small molecules and is easy to operate, but the humanized degree is low, the affinity is not high, and the half-life prolonging property needs to be further improved.

Aiming at the defects of the prior art, the invention provides a series of antihuman LILRB2 nano antibody sequences and a preparation scheme.

In a first aspect, the present invention provides a nanobody against LILRB2, according to an embodiment of the present invention,

the nanobody is capable of specifically binding to LILRB2, and the complementarity determining region CDRs of the VHH chains in the nanobody are one or more selected from the group consisting of:

(1) SEQ ID NO: 14, CDR1 shown in SEQ ID NO: 15, and the CDR2 shown in SEQ ID NO: 16 CDR 3;

(2) the amino acid sequence of SEQ ID NO: 17, CDR1 shown in SEQ ID NO: 18, and the CDR2 shown in SEQ ID NO: 19 CDR3 shown in seq id no;

(3) SEQ ID NO: 20, CDR1 shown in SEQ ID NO: 21, and the CDR2 shown in SEQ ID NO: 22 CDR3 shown in seq id no;

(4) SEQ ID NO: CDR1 shown in SEQ ID NO: 21, and the CDR2 shown in SEQ ID NO: CDR3 shown in FIG. 24;

(5) the amino acid sequence of SEQ ID NO: 25, CDR1 shown in SEQ ID NO: 26, and a CDR2 shown in SEQ ID NO: 27, CDR 3.

(6) SEQ ID NO: 28, CDR1 shown in SEQ ID NO: 29, and the CDR2 shown in SEQ ID NO: 30, CDR 3.

(7) SEQ ID NO: 31, CDR1 shown in SEQ ID NO: 32, and the CDR2 shown in SEQ ID NO: 33, CDR3 shown.

(8) The amino acid sequence of SEQ ID NO: 34, CDR1 shown in SEQ ID NO: 35, and SEQ ID NO: 36, CDR3 shown in fig. 36.

(9) SEQ ID NO: 25, CDR1 shown in SEQ ID NO: 37, and SEQ ID NO: 27, CDR 3.

Further, in some embodiments of the invention, the nanobody is a humanized VHH or a camelized VHH.

Further, in some embodiments of the present invention, the nanobody has an amino acid sequence as set forth in SEQ ID NO: 3 to SEQ ID NO: 12, or an amino acid sequence substantially identical to SEQ ID NO: 3 to SEQ ID NO: 12, or a variant thereof, having at least 80% identity to the amino acid sequence set forth in any one of items 12.

"at least 80% identity" as used herein is any percentage identity greater than or equal to 80%, such as at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% identity.

In a second aspect, the present invention provides a fusion protein, according to an embodiment of the present invention, comprising a functional domain capable of specifically binding LILRB2, the functional domain consisting of a nanobody against LILRB2 as described in any one of the above.

The nano-antibody provided by the invention can be fused with any other protein or substance to achieve different purposes, such as combining with fluorescent protein, enzyme or radioactive element to achieve the purpose of easy detection, and further fusing with drug molecules for treating LILRB 2-mediated related diseases to achieve better treatment purpose. The type of protein fused with the nanobody can be reasonably selected by those skilled in the art according to actual needs or purposes, and it is also within the scope of the present invention to fuse whatever type of substance.

In a third aspect, the present invention provides an anti-LILRB 2 antibody, according to the embodiments of the present invention, the antibody is a conventional antibody or a functional fragment thereof, and the heavy chain variable region of the antibody is composed of any one of the nanobodies against LILRB 2;

further, the functional fragment is the Fab, Fab ', (Fab') 2, Fv, scFv or sdFv structure of the conventional antibody.

Traditional antibodies consist structurally of two identical heavy chains and two identical light chains, a light chain having a light chain variable region (VL) and a light chain constant region (CL); the heavy chain has a heavy chain variable region (VH) and a heavy chain constant region (CH1, CH2, CH3 and/or CH 4). On the premise that the present invention discloses a structure of nanobody capable of specifically binding to LILRB2, those skilled in the art can easily think of using nanobody of the present invention to modify a conventional antibody, for example, applying CDR region structure of nanobody of the present invention to conventional antibody to obtain conventional antibody capable of specifically binding to LILRB2, and such conventional antibody also belongs to the protection scope of the present invention; further, based on the structure of the conventional antibody, a partial structure thereof such as a Fab, Fab ', (Fab') 2, Fv, scFv or sdFv structure, etc. also has the binding specificity of LILRB2, which also falls within the scope of the present invention.

In a fourth aspect, the present invention provides a composition for treating diseases, which includes the nanobody against LILRB2 described above, the fusion protein described above, or the antibody described above, and a pharmaceutically acceptable excipient.

The pharmaceutical compositions provided herein contain at least one (e.g., one, two, three, or four) of the antibodies or antigen-binding fragments described in the examples herein, and two or more (e.g., two, three, or four) of any of the antibodies or antigen-binding fragments described herein can be present in the pharmaceutical composition in any combination. The pharmaceutical compositions may be formulated in any manner known in the art.

The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. The pharmaceutically acceptable carrier may further comprise minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, which increase the shelf-life or effectiveness of the antibody.

The dosage form of the medicament provided by the invention is not strictly limited, and the medicament can be prepared into various dosage forms according to the existing methods in the field of medicaments, and can be applied to patients needing treatment by oral administration, nasal inhalation, rectal administration, parenteral administration, transdermal administration and the like.

In a fifth aspect, the invention provides an isolated nucleic acid molecule encoding a nanobody as claimed in any one of the above.

Based on the disclosure of the present invention, the polynucleotide molecules encoding the nanobodies and the fusion proteins are easily obtained by those skilled in the art through the conventional techniques in the art, and based on the degeneracy of the codon, the polynucleic acid molecules are varied, and there are many possibilities for their specific base sequences, and therefore, it is within the scope of the present invention that the polynucleic acid molecules may be varied, so long as they encode the nanobodies or fusion proteins of the present invention.

In a sixth aspect, the present invention provides a vector comprising a nucleic acid molecule as described above.

In a seventh aspect, the present invention provides a recombinant cell comprising a vector as described above.

The embodiments provide recombinant vectors (e.g., expression vectors) comprising the isolated polynucleotides disclosed herein (e.g., polynucleotides encoding the polypeptides disclosed herein), host cells into which the recombinant vectors are introduced (i.e., such that the host cells contain the polynucleotides and/or polynucleotide-containing vectors), and production of recombinant antibody polypeptides or fragments thereof by recombinant techniques.

As used herein, a "vector" is any construct capable of delivering one or more polynucleotides of interest to a host cell when the vector is introduced into said host cell. An "expression vector" is capable of delivering and expressing one or more polynucleotides of interest as an encoded polypeptide in a host cell into which the expression vector has been introduced. Thus, in an expression vector, a polynucleotide of interest is targeted for expression in the vector by being operably linked to regulatory elements such as a promoter, enhancer and/or polyadenylation tail, which are located at or near or on both sides of the site of integration of the polynucleotide of interest within the vector or in the genome of the host cell, such that the polynucleotide of interest will be translated in the host cell into which the expression vector is introduced.

The vector may be introduced into the host cell by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and/or transduction (e.g., with a recombinant virus). Thus, non-limiting examples of vectors include viral vectors (useful for the production of recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

The embodiments of the present invention provide host cells transformed with the vectors described above. The host cell may be a prokaryotic or eukaryotic cell. A preferred prokaryotic host cell is E.coli (Escherichia coli). Preferably, the eukaryotic cell is selected from: protist cells, animal cells, plant cells and fungal cells. More preferably, the host cell is a mammalian cell, including but not limited to CHO and COS cells. A preferred fungal cell is Saccharomyces cerevisiae.

In an eighth aspect, the present invention provides a method of preparing a nanobody as described in any one of the above, comprising: culturing the recombinant cells, and separating and purifying the culture product to obtain the nano antibody.

It should be noted that the preparation of the nanobody, fusion protein or antibody of the present invention may be achieved by chemical synthesis, genetic engineering, or other methods, and it is within the scope of the present invention to prepare the nanobody, fusion protein or antibody of the present invention by any method.

In a ninth aspect, there is provided a use of the anti-LILRB 2 nanobody of any one of the above, the fusion protein as described above, the antibody as described above, the composition as described above, the nucleic acid molecule as described above, the vector as described above, or the recombinant cell as described above, for preventing, treating and/or ameliorating a solid tumor or a hematological tumor.

Preferably, the solid tumor is lung cancer, non-small cell lung cancer (NSCLC), pancreatic cancer, pancreatic ductal carcinoma, Chronic Lymphocytic Leukemia (CLL), Acute Myelogenous Leukemia (AML), endometrial cancer, hepatocellular cancer, melanoma, ovarian cancer, breast cancer, colorectal cancer, glioma, gastric cancer, renal cancer, testicular cancer, esophageal cancer, cervical cancer, squamous lung cancer, leukemia, thyroid cancer, liver cancer, urinary tract cancer or head and neck cancer.

To better understand the present invention, certain terms are first defined. Other definitions are listed throughout the detailed description section.

In general, the antigen binding properties of an antibody can be described by 3 specific regions in the heavy chain variable region, called variable regions (CDRs), which are separated into 4 Framework Regions (FRs), the amino acid sequences of the 4 FRs being relatively conserved and not directly involved in the binding reaction. These CDRs form a loop structure, and the β -sheets formed by the FRs between them are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.

The invention includes not only intact antibodies, but also fragments of antibodies with immunological activity or fusion proteins of antibodies with other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of the antibodies.

"chimeric antibody" refers to an antibody in which the amino acid sequences of immunoglobulin molecules are derived from two or more species. Typically, the variable regions of both the light and heavy chains correspond to those of an antibody derived from one mammalian species (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capacity, while the constant regions are homologous to sequences in the antibody derived from another species (typically human) to avoid eliciting an immune response in that species.

"nanobodies" are generally as defined in WO 2008/020079 or WO 2009/138519 and in one particular aspect generally denote VHHs, humanized VHHs or camelized VH (such as camelized human VH), or generally denote VHHs that are sequence optimized (e.g. optimized for chemical stability and/or solubility, maximal overlap and maximal expression with known human framework regions). The 'nano antibody' is obtained by a genetic engineering method, and mainly has 3 types, wherein the first type is a heavy chain variable region obtained from a camelid HCAb, is a single folding unit, retains complete antigen binding activity, and is a minimum natural antibody fragment. The second type is a heavy chain variable region obtained from IgNAR of cartilaginous fish such as shark, and is denoted by VNAR. The third type is a heavy chain or light chain variable region obtained from a monoclonal antibody of human or murine origin, which retains antigen binding activity but has greatly reduced affinity and solubility.

"Fc region" or "Fc" refers to the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the hinge region, the CH2 domain, and the CH3 domain, which mediates binding of the immunoglobulin to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or to the first component of the classical complement system (e.g., C1q), including native sequence Fc regions and variant Fc regions. Typically, the human IgG heavy chain Fc region is the carboxy-terminal stretch from the amino acid residue at position Cys226 or Pro230, but the boundaries may vary. The C-terminal lysine of the Fc region (residue 447, according to the EU numbering system) may or may not be present. Fc may also refer to this region of isolation, or in the case of a protein polypeptide comprising Fc, such as "binding protein comprising an Fc region," also referred to as an "Fc fusion protein" (e.g., an antibody or immunoadhesin). The native sequence Fc region in the antibodies of the invention includes human IgG1, IgG2(IgG2A, IgG2B), IgG3 and IgG 4. In IgG, IgA, and IgD antibody isotypes, the Fc region comprises the CH2 and CH3 constant domains of each of the two heavy chains of an antibody; the IgM and IgEFc regions comprise three heavy chain constant domains (CH domains 2-4) in each polypeptide chain.

By "specific binding" is meant a non-random binding reaction between two molecules, such as between an antibody and the antigen to which it is directed. The term "immunological binding" refers to a specific binding reaction that occurs between an antibody molecule and an antigen for which the antibody is specific. The strength or affinity of an immunological binding interaction may be expressed as the equilibrium dissociation constant (KD) of the interaction, where a smaller KD value indicates a higher affinity. The immunological binding properties between two molecules can be quantified using methods well known in the art. One method involves measuring the rate of antigen binding site/antigen complex formation and dissociation. Both the "association rate constant" (Ka or Kon) and the "dissociation rate constant" (Kd or Koff) for a particular antibody-antigen interaction can be calculated by concentration and the actual rate of association and dissociation, and Kd, Ka and Kd values can be measured by any effective method. In a preferred embodiment, the dissociation constant is measured by bioluminescence interferometry. In other preferred embodiments, the dissociation constant can be measured using surface plasmon resonance techniques (e.g., Biacore) or KinExa.

"vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and an episomal mammalian vector). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operatively linked.

"nucleic acid molecule" is intended to include both DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, and may be a cDNA.

The invention achieves the following beneficial technical effects:

the anti-LILRB 2 nanobody can be combined with human LILRB2 with high affinity, and can block the combination of LILRB2 and a receptor HLA-G thereof, thereby having potential therapeutic value on tumors.

Drawings

FIG. 1 shows ELISA detection of binding of anti-LILRB 2 chimeric antibody to recombinant human LILRB2 protein;

FIG. 2 shows FACS detection of binding activity of anti-LILRB 2 chimeric antibodies on human LILRB2/293 cells;

FIG. 3 shows FACS detection of binding activity of anti-LILRB 2 chimeric antibodies on cyno LILRB2/293 cells;

FIG. 4 shows that ELISA detects the blocking activity 1 of the chimeric antibody LILRB2 on the binding of recombinant LILRB2 to HLA-G;

FIG. 5 shows ELISA detection of blocking activity of the chimeric LILRB2 antibody against HLA-G binding of recombinant LILRB2 2.

Detailed Description

The scheme of the invention will be explained with reference to the examples. It will be understood by those skilled in the art that the following examples are illustrative only and are not to be construed as limiting the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The raw materials and reagent materials used in the following examples are all commercially available products unless otherwise specified.

Example 1: construction of camel nano antibody immune phage library

Utilizing antigen immune camel, separating Peripheral Blood Mononuclear Cells (PBMC), extracting total RNA for reverse transcription, amplifying a variable region of a nanometer antibody heavy chain (VHH) by using a reverse transcription product as a template, connecting the VHH with a phage display carrier, and electrically transferring Escherichia coli TG1 competent cells to construct a camel immune library.

Specifically, camels were immunized twice weekly for 4 times. Each injection of 0.8 mg of human LILRB2 extracellular domain recombinant protein was administered in Freund's complete/incomplete adjuvant (Sigma, F5881, F5506) by subcutaneous multi-point injection. 1mL of blood serum was collected 2 weeks after each immunization, and the titers of whole antibody (IgG) and heavy chain antibody (HcAb) in the serum were measured by ELISA using the immunogen as the antigen. When the serum titer meets the requirement of library construction, collecting 100 mL camel peripheral blood, separating PBMC by using a separation kit (Tianjin third layer, Cat: TBD2011 CM), extracting the total RNA of the PBMC, inverting to obtain cDNA, and using the cDNA as a template for subsequent VHH fragment amplification. Searching camel source VHH antibody genes according to related literatures and databases, designing and synthesizing VHH antibody library construction primers, and amplifying antibody variable region gene sequences by PCR. The vector and amplified antibody fragment are then cleaved enzymatically using an endonuclease. The ligation product is constructed by adopting a T4 ligase ligation mode, and is transferred into TG1 strain by utilizing an electrotransfection technology. Finally construct a 1.8X 10 ⁸ A camel anti-human LILRB2 VHH antibody immune library is used for screening specific anti-human LILRB2 nano antibodies. In order to test the accuracy of the library, 50 clones were randomly selected for colony PCR, and the results showed that the insertion rate reached 90%.

The constructed camel immune bank is screened by a liquid phase panning method by using Biotinylated-LILRB2 (Acro, Cat. LI2-H82F 5) as an antigen to obtain a specific phage display nano antibody. 10 phage-displayed nanobodies that can simultaneously bind to the recombinant protein of human LILRB2 were obtained by original library presentation and screening and identification: a3, a7, B9, C7, D5, D6, E1, E5, E8 and G3.

Example 2: preparation of anti-human LILRB2 nano antibody and control antibody

Variable region gene synthesis is carried out on JTX-8064 (sequence source: CN111699196A, SEQ ID NO.53 and 54) which is the same as a target point contrast antibody, the heavy chain and light chain variable region sequences are shown as SEQ ID NO.1 and SEQ ID NO.2, and light chain and heavy chain sequences are respectively cloned into eukaryotic transient expression vectors containing human kappa/IgG1 light chain and heavy chain constant regions to obtain contrast antibody light chain and heavy chain expression plasmids. Transferring the obtained control antibody light chain and heavy chain expression plasmids into escherichia coli for amplification, separating to obtain a large number of plasmids containing the control antibody light chain and heavy chain, extracting the plasmids, performing ethanol precipitation, and transferring the light chain plasmids and the heavy chain plasmids of the control antibody into HEK293 cells for recombinant expression respectively according to the operation instruction of a transfection reagent 293fectin (Cat: 12347019, Gibco). 5-6 days after cell transfection, culture supernatant is taken and purified by a ProA affinity chromatography column to obtain a control antibody.

According to the sequencing result of the phage-displayed nanobody, primers are designed, cloned into a eukaryotic transient expression vector containing a human Fc (hFc) coding gene by a PCR method, and are recombined and expressed in HEK293 cells. After 5-6 days of cell transfection, culture supernatants were taken and purified by a ProA affinity chromatography column to obtain recombinant proteins chA3, chA7, chB9, chC7, chD5, chD6, chE1, chE5, chE8 and chG 3. The variable region sequence of chA3 is shown in SEQ ID NO.3, the variable region sequence of chA7 is shown in SEQ ID NO.4, the variable region sequence of chB9 is shown in SEQ ID NO.5, the variable region sequence of chC7 is shown in SEQ ID NO.6, the variable region sequence of chD5 is shown in SEQ ID NO.7, the variable region sequence of chD6 is shown in SEQ ID NO.8, the variable region sequence of chE1 is shown in SEQ ID NO.9, the variable region sequence of chE5 is shown in SEQ ID NO.10, the variable region sequence of chE8 is shown in SEQ ID NO.11, and the variable region sequence of chG3 is shown in SEQ ID NO.12, wherein the underline shows the corresponding CDRs (according to the definition of Kabat CDRs) and the constant region sequence is shown in SEQ ID NO. 13.

SEQ ID NO.1 JTX-8064 heavy chain variable region amino acid sequence

qitlkesgptlvkptqtltltctfsgfslntyamgvswirqppgkalewlasiwwngnkynnpslksrltvtkdtsknqvvltmtnmdpvdtatyycahsriirftdyvmdawgqgtlvtvss

SEQ ID NO.2 JTX-8064 light chain variable region amino acid sequence

diqmtqspsslstsvgdrvtitcrasediyndlawyqqkpgkapklliynanslhtgvasrfsgsgsgtdftftisslqpedvatyfcqqyydypltfgqgtkleik

Amino acid sequence of SEQ ID NO.3 chA3 VHH

Qvqlqesggglvqaggsltlscavsgisfgtramawfrqapgkeregvasiqadtyisyadsvkgrftiskdsgkntlnlqmnnlkpedtavyycavgdlwgshlqphgynywgqgtqvtvss

The amino acid sequences of CDRs 1, 2 and 3 of the chA3 antigen complementarity determining region are SEQ ID NOs: 14. 15 and 16.

Amino acid sequence of SEQ ID NO.4 chA7 VHH

Qvqlqesgggsvqpggrlrlscetsgytssrnwmgwfrqapgkeregvasiytdngsayyadsvkgrftisvdnakntvylqmnslkpedtamyycagrirppagtrwpgplvesayntwgrgtqvtvss

The amino acid sequences of CDRs 1, 2 and 3 of the chA7 antigen complementarity determining region are SEQ ID NOs: 17. 18 and 19.

SEQ ID NO.5: chB9 VHH amino acid sequence

Qvqlqesgggsvqaggslrlscaasgytdsrycmgwfrqvpgkerekvatiyngdgntyyddsvkgrftisqgnakntlflemnnlkpedtamyycaaikaygsnwcpaveysdwgqgtqvtvss

chB9 the amino acid sequences of CDRs 1, 2 and 3 of the antigen complementarity determining regions are respectively SEQ ID NO: 20. 21 and 22.

SEQ ID NO.6: chC7 VHH amino acid sequence

Qvqlqesggglvqaggslrlscaaseytdsrycmawfrqvpgkerekvaiiyngdgntyyddsvkgrftisqsvakntlylqmnnlkpedtgmyycaaikaygsnwcraveysdwgqgtqvtvss

chC7 the amino acid sequences of CDRs 1, 2 and 3 of the antigen complementarity determining regions are respectively SEQ ID NO: 23. 21 and 24

SEQ ID NO.7: chD5 VHH amino acid sequence

Qvqlqesggglvqaggslrlscvasgyprssvcmgwyrqapgkeregvaaiftgggtpyygdsvkgrftisqdnavntvslqmndlkpedtamyycaaeltycsggpwtdpvgywgqgtqvtvss

chD5 the amino acid sequences of CDRs 1, 2 and 3 of the antigen complementarity determining regions are respectively SEQ ID NO: 25. 26 and 27.

SEQ ID NO.8: chD6 VHH amino acid sequence

Qvqlqesggglvqaggslrlscaasgftgsgycmgwfrqvpgkerekvatiyngypgdgdpsyddsvk grftisqdtakntvylqmndlkpedtamyycaainaygsnwcqiveyahwgvgtqvtvss

chD6 the amino acid sequences of CDRs 1, 2 and 3 of the antigen complementarity determining region are respectively SEQ ID NO: 28. 29 and 30.

SEQ ID NO.9: chE1 VHH amino acid sequence

Qvqlqesggglvqpggslrlscvgsgftfrhyamnwarqapgkgiewvasigtpglpeayadsvkgrftisrddakntlylqmdnlktedtgvyycarssdcgggtcrppgqgtqvtvss

chE1 the amino acid sequences of CDRs 1, 2 and 3 of the antigen complementarity determining regions are respectively SEQ ID NO: 31. 32 and 33.

SEQ ID NO.10: chE5 VHH amino acid sequence

Qvqlqesgggsvqaggslrlscvasgyprssvcmgwyrqapgkeregvaaiftgggtpyygdsvkgrftisqdnavntvslqmndlkpedtamyycaveltycsggpwtdpvgywgqgtqvtvss

chE5 the amino acid sequences of CDRs 1, 2 and 3 of the antigen complementarity determining regions are respectively SEQ ID NO: 25. 26 and 27.

SEQ ID NO.11: chE8 VHH amino acid sequence

Qvqlqesggglvqpggslnlscaasgftfsnsfmtwvrqapgkrpewvrewvagisgdgqytsyadfa kgrftisrdnakstmylqlnnlktedtaiyyceksstergqgtqvtvss

chE8 the amino acid sequences of CDRs 1, 2 and 3 of the antigen complementarity determining regions are respectively SEQ ID NO: 34. 35 and 36.

Amino acid sequence of SEQ ID NO.12 chG3 VHH

Qvqlqesgggsvqaggslrlscaasgyprssvcmgwyrqapgkeregvaaiftgggrpyyadsvkgrftisqdnavntvslqmnnlkpedtamyycaaeltycsggpwtdpvgywgqgtqvtvss

The amino acid sequences of CDRs 1, 2 and 3 of the chG3 antigen complementarity determining region are SEQ ID NOs: 25. 37 and 27.

hFc constant region amino acid sequence of SEQ ID NO.13

asepkssdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspg

Example 3: chimeric antibody affinity detection

The antibody affinity was determined by capturing the Fc fragment of the antibody with an Ocet QKe system instrument from Fortebio using an anti-human antibody Fc fragment capture Antibody (AHC) biological probe. For the measurement, the chA3, chA7, chB9, chC7, chD5, chD6, chE1, chE5, chE8 and chG3 chimeric antibody and the control antibody JTX-8064 were diluted to 4ug/ml with PBS buffer, and passed over the surface of an AHC probe (Cat: 18-0015, PALL) for 120 s. The LILRB2 recombinant protein (purchased from ACRO, Cat # LI2-H5220) is 60 nm; as the mobile phase, the binding time was 300s, and the dissociation time was 300 s. After the experiment, blank control response values were deducted, and the software was run for 1: 1 Langmuir binding pattern was fitted and kinetic constants for antigen-antibody binding were calculated.

The kinetic parameters are shown in table 1 below, and the results show that the 10 chimeric antibodies all bind to LILRB2 recombinant protein with binding activity comparable to that of the control antibody.

TABLE 1 affinity assay of chimeric antibodies to LILRB2 recombinant protein

Sample(s)	KD (M)	kon(1/Ms)	kdis(1/s)
				JTX-8064	4.93E-09	4.28E+05	2.11E-03
chA3	1.43E-10	3.47E+05	4.95E-05
				chA7	6.03E-09	4.38E+05	2.64E-03
chB9	<1.0E-12	4.24E+05	<1.0E-07
				chC7	<1.0E-12	3.88E+05	<1.0E-07
chD5	<1.0E-12	3.98E+05	<1.0E-07
				chD6	4.11E-10	387000	0.000159
chE1	3.66E-09	1.62E+05	5.93E-04
				chE5	1.65E-10	3.41E+05	5.61E-05
chE8	6.36E-09	3.96E+05	2.52E-03
				chG3	4.40E-10	2.95E+05	1.30E-04

Example 4: ELISA for detecting the binding activity of anti-LILRB 2 chimeric antibody

Human LILRB2 recombinant protein (KN expression) was coated overnight at 4 ℃ at a concentration of 1 ug/mL; after washing the plate for 3 times with PBS, adding 5% BSA PBS, blocking for 60min at 37 ℃, and washing the plate for 3 times with PBST; adding 10ug/ml of chA3, chA7, chB9, chC7, chD5, chD6, chE1, chE5, chE8 and chG3 chimeric antibody and control antibody JTX-8064, incubating at 37 deg.C for 60min, and washing the plate with PBST for 4 times; HRP-anti-human Fc (Cat: 109-; adding a TMB substrate for color development, incubating at 37 ℃ for 10 min, and adding 2M HCl to stop the reaction; the absorbance A450nm-630nm of the well plate at a wavelength of 450nm was read and recorded using 630nm as the reference wavelength.

The experimental results show that the chA3, chA7, chB9, chC7, chD5, chD6, chE1, chE5, chE8 and chG3 chimeric antibody and the control antibody JTX-8064 can be specifically combined with the human LILRB2 recombinant protein (figure 1).

Example 5: FACS detection of binding Activity of anti-LILRB 2 chimeric antibody and 293 cell (human LILRB 2/293) recombinantly expressing human LILRB2

The binding condition of the chimeric antibody and human LILRB2 is detected by using 293 cells (human LILRB 2/293) transiently transformed with human LILRB2, 2E5 cells are taken to be respectively bound with anti-LILRB 2 antibodies with different concentrations, and the chimeric antibody of chA3, chA7, chB9, chC7, chD5, chD6, chE1, chE5, chE8, chG3 and a control antibody JTX-8064 are diluted by 3 gradients in 4-fold gradient from 132 nm. Incubating at 4 ℃ in the dark for 60min, washing with PBS, adding FITC-labeled goat anti-human antibody (sigma, F9512) diluted at a ratio of 1:200, incubating at 4 ℃ in the dark for 30min, washing with PBS, resuspending in 200ul of PBS, and detecting with flow cytometry.

The results show (fig. 2) that the chA3, chA7, chB9, chC7, chD5, chD6, chE1, chE5, chE8 and chG3 chimeric antibodies and the control antibody JTX-8064 have comparable binding capacity.

Example 6: FACS detection of binding activity of anti-LILRB 2 chimeric antibody and 293 cell (cyno LILRB 2/293) of recombinant expression cynomolgus monkey LILRB2

The binding condition of the chimeric antibody and cynomolgus monkey LILRB2 is detected by using 293 cells (cyno LILRB 2/293) of transient cynomolgus monkey LILRB2, 2E5 cells are taken to be respectively bound with anti-LILRB 2 antibodies with different concentrations, and the chimeric antibodies of chA3, chA7, chB9, chC7, chD5, chD6, chE1, chE5, chE8 and chG3 and a control antibody JTX-8064 are diluted by 4-fold gradient from 132 nM. Incubating at 4 ℃ in the dark for 60min, washing with PBS, adding FITC-labeled goat anti-human antibody (sigma, F9512) diluted at a ratio of 1:200, incubating at 4 ℃ in the dark for 30min, washing with PBS, suspending in 200ul of PBS, and detecting with flow cytometry.

The results show (fig. 3), chE1 has stronger binding activity with cynomolgus monkey LILRB2, comparable to control antibody JTX-8064.

Example 7: ELISA detection of blocking Activity of anti-LILRB 2 chimeric antibody on binding of recombinant LILRB2 to HLA-G

Diluting human HLA-G extracellular region recombinant protein (Kactus, Cat. HLG-HE 41F) with PBS to 1ug/mL, 100 ul/well, coating enzyme linked plate, and coating overnight at 4 deg.C; 5% BSA blocking solution, sealing in a constant temperature incubator at 37 ℃ for 120min, and washing the plate for 3 times by PBST; taking 100uL of chA3, chA7, chB9, chC7, chD5, chD6, chE1, chE5, chE8 and chG3 chimeric antibody and a contrast antibody JTX-8064 to-be-detected sample (66 nM is started, and 3 times of gradient dilution is carried out for 12 gradients), adding 100uL of 0.4ug/ml LILRB2-mFc (NCBI number: AAH36827 and 22-461 amino acid), mixing uniformly, reacting at 37 ℃ for 30min, taking 100uL, and adding into HLA-G coated wells; washing the plate for 4 times in a constant temperature incubator at 37 ℃ for 60min by PBST; adding HRP-anti-mouse IgG (Cat: 115-; and finally adding a TMB substrate for color development, reacting for 15min in a constant-temperature incubator at 37 ℃, stopping the reaction by using 2M HCl, and reading and recording the absorbance of the pore plate with the wavelength of 450 nm. The results show (fig. 4, fig. 5, table 2) that chE8 has partial blocking activity except for chA7 and chE1 without blocking activity, and the remaining chimeric antibodies all have clear blocking activity, and the blocking activity is comparable to the control antibody.

TABLE 2 ELISA detection of blocking Activity of LILRB2 chimeric antibody against recombinant LILRB2 binding to HLA-G IC50

JTX-8064

chE1

chE5

chE8

chG3

chC7

IC50（nM）

0.860

Does not block

0.604

1.193

0.593

0.834

JTX-8064

chA3

chA7

chB9

chD5

chD6

IC50（nM）

1.001

0.928

Does not block

1.039

0.537

0.952

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.

Sequence listing

<110> Beijing Kenuo sincerity science and technology Co Ltd

<120> nano antibody targeting human LILRB2 and application thereof

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Gln Ile Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln

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Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Asn Thr Tyr

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Ala Met Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu

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Trp Leu Ala Ser Ile Trp Trp Asn Gly Asn Lys Tyr Asn Asn Pro Ser

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Leu Lys Ser Arg Leu Thr Val Thr Lys Asp Thr Ser Lys Asn Gln Val

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Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

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Cys Ala His Ser Arg Ile Ile Arg Phe Thr Asp Tyr Val Met Asp Ala

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Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Thr Ser Val Gly

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Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asp Ile Tyr Asn Asp

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Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

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Tyr Asn Ala Asn Ser Leu His Thr Gly Val Ala Ser Arg Phe Ser Gly

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Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

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Glu Asp Val Ala Thr Tyr Phe Cys Gln Gln Tyr Tyr Asp Tyr Pro Leu

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Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

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Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

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Ser Leu Thr Leu Ser Cys Ala Val Ser Gly Ile Ser Phe Gly Thr Arg

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Ala Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

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Ala Ser Ile Gln Ala Asp Thr Tyr Ile Ser Tyr Ala Asp Ser Val Lys

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Gly Arg Phe Thr Ile Ser Lys Asp Ser Gly Lys Asn Thr Leu Asn Leu

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Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala

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Val Gly Asp Leu Trp Gly Ser His Leu Gln Pro His Gly Tyr Asn Tyr

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Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

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Arg Leu Arg Leu Ser Cys Glu Thr Ser Gly Tyr Thr Ser Ser Arg Asn

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Trp Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

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Ala Ser Ile Tyr Thr Asp Asn Gly Ser Ala Tyr Tyr Ala Asp Ser Val

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Lys Gly Arg Phe Thr Ile Ser Val Asp Asn Ala Lys Asn Thr Val Tyr

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Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys

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Ala Gly Arg Ile Arg Pro Pro Ala Gly Thr Arg Trp Pro Gly Pro Leu

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Val Glu Ser Ala Tyr Asn Thr Trp Gly Arg Gly Thr Gln Val Thr Val

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Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Asp Ser Arg Tyr

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Cys Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Lys Val

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Ala Thr Ile Tyr Asn Gly Asp Gly Asn Thr Tyr Tyr Asp Asp Ser Val

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Lys Gly Arg Phe Thr Ile Ser Gln Gly Asn Ala Lys Asn Thr Leu Phe

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Leu Glu Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys

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Ala Ala Ile Lys Ala Tyr Gly Ser Asn Trp Cys Pro Ala Val Glu Tyr

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Ser Asp Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

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Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Glu Tyr Thr Asp Ser Arg Tyr

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Cys Met Ala Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Lys Val

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Ala Ile Ile Tyr Asn Gly Asp Gly Asn Thr Tyr Tyr Asp Asp Ser Val

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Lys Gly Arg Phe Thr Ile Ser Gln Ser Val Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Gly Met Tyr Tyr Cys

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Ala Ala Ile Lys Ala Tyr Gly Ser Asn Trp Cys Arg Ala Val Glu Tyr

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Ser Asp Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

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Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

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Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Tyr Pro Arg Ser Ser Val

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Cys Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

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Ala Ala Ile Phe Thr Gly Gly Gly Thr Pro Tyr Tyr Gly Asp Ser Val

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Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Val Asn Thr Val Ser

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Leu Gln Met Asn Asp Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys

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Ala Ala Glu Leu Thr Tyr Cys Ser Gly Gly Pro Trp Thr Asp Pro Val

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Gly Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

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Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Gly Ser Gly Tyr

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Cys Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Lys Val

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Ala Thr Ile Tyr Asn Gly Tyr Pro Gly Asp Gly Asp Pro Ser Tyr Asp

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Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Thr Ala Lys Asn

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Thr Val Tyr Leu Gln Met Asn Asp Leu Lys Pro Glu Asp Thr Ala Met

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Tyr Tyr Cys Ala Ala Ile Asn Ala Tyr Gly Ser Asn Trp Cys Gln Ile

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Val Glu Tyr Ala His Trp Gly Val Gly Thr Gln Val Thr Val Ser Ser

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Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

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Ser Leu Arg Leu Ser Cys Val Gly Ser Gly Phe Thr Phe Arg His Tyr

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Ala Met Asn Trp Ala Arg Gln Ala Pro Gly Lys Gly Ile Glu Trp Val

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Ala Ser Ile Gly Thr Pro Gly Leu Pro Glu Ala Tyr Ala Asp Ser Val

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Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asn Thr Leu Tyr

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Leu Gln Met Asp Asn Leu Lys Thr Glu Asp Thr Gly Val Tyr Tyr Cys

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Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

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Cys Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

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Ala Ala Ile Phe Thr Gly Gly Gly Thr Pro Tyr Tyr Gly Asp Ser Val

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Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Val Asn Thr Val Ser

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Leu Gln Met Asn Asp Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys

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Ala Val Glu Leu Thr Tyr Cys Ser Gly Gly Pro Trp Thr Asp Pro Val

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Gly Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

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Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

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Ser Leu Asn Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ser

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Phe Met Thr Trp Val Arg Gln Ala Pro Gly Lys Arg Pro Glu Trp Val

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Arg Glu Trp Val Ala Gly Ile Ser Gly Asp Gly Gln Tyr Thr Ser Tyr

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Ala Asp Phe Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys

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Ser Thr Met Tyr Leu Gln Leu Asn Asn Leu Lys Thr Glu Asp Thr Ala

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Ile Tyr Tyr Cys Glu Lys Ser Ser Thr Glu Arg Gly Gln Gly Thr Gln

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Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Pro Arg Ser Ser Val

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Cys Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

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Ala Ala Ile Phe Thr Gly Gly Gly Arg Pro Tyr Tyr Ala Asp Ser Val

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Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Val Asn Thr Val Ser

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Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys

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Ala Ala Glu Leu Thr Tyr Cys Ser Gly Gly Pro Trp Thr Asp Pro Val

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Gly Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

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Ala Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys

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Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

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Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

35 40 45

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

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Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

65 70 75 80

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

85 90 95

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

100 105 110

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

115 120 125

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

130 135 140

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

145 150 155 160

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

165 170 175

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

180 185 190

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

195 200 205

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

210 215 220

Gln Lys Ser Leu Ser Leu Ser Pro Gly

225 230

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Thr Arg Ala Met Ala

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Ser Ile Gln Ala Asp Thr Tyr Ile Ser Tyr Ala Asp Ser Val Lys Gly

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Gly Asp Leu Trp Gly Ser His Leu Gln Pro His Gly Tyr Asn Tyr

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Arg Asn Trp Met Gly

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Ser Ile Tyr Thr Asp Asn Gly Ser Ala Tyr Tyr Ala Asp Ser Val Lys

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Gly

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Arg Ile Arg Pro Pro Ala Gly Thr Arg Trp Pro Gly Pro Leu Val Glu

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Arg Tyr Cys Met Gly

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Thr Ile Tyr Asn Gly Asp Gly Asn Thr Tyr Tyr Asp Asp Ser Val Lys

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Gly

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Ile Lys Ala Tyr Gly Ser Asn Trp Cys Pro Ala Val Glu Tyr Ser Asp

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Arg Tyr Cys Met Ala

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Ile Lys Ala Tyr Gly Ser Asn Trp Cys Arg Ala Val Glu Tyr Ser Asp

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Ser Val Cys Met Gly

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Ala Ile Phe Thr Gly Gly Gly Thr Pro Tyr Tyr Gly Asp Ser Val Lys

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Gly

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Glu Leu Thr Tyr Cys Ser Gly Gly Pro Trp Thr Asp Pro Val Gly Tyr

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Gly Tyr Cys Met Gly

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Thr Ile Tyr Asn Gly Tyr Pro Gly Asp Gly Asp Pro Ser Tyr Asp Asp

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Ser Val Lys Gly

20

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Ile Asn Ala Tyr Gly Ser Asn Trp Cys Gln Ile Val Glu Tyr Ala His

1 5 10 15

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His Tyr Ala Met Asn

1 5

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Ser Ile Gly Thr Pro Gly Leu Pro Glu Ala Tyr Ala Asp Ser Val Lys

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Gly

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Ser Ser Asp Cys Gly Gly Gly Thr Cys Arg Pro

1 5 10

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Asn Ser Phe Met Thr

1 5

<210> 35

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Ala Gly Ile Ser Gly Asp Gly Gln Tyr Thr Ser Tyr Ala Asp Phe Ala

1 5 10 15

Lys Gly

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Ser Ser Thr Glu

1

<210> 37

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Ala Ile Phe Thr Gly Gly Gly Arg Pro Tyr Tyr Ala Asp Ser Val Lys

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Gly

Claims (12)

1. A nanobody against LILRB2, wherein the nanobody is capable of specifically binding to LILRB2, and the CDRs of the VHH chain in the nanobody comprise:

SEQ ID NO: 14, CDR1 shown in SEQ ID NO: 15, and the CDR2 shown in SEQ ID NO: 16, CDR3 shown in fig. 16.

2. The nanobody according to claim 1, wherein the nanobody is a humanized VHH or a camelized VHH.

3. Nanobody according to claim 1 or 2, characterized in that it has the amino acid sequence shown in SEQ ID NO: 3.

4. A fusion protein comprising a functional domain capable of specifically binding LILRB2, said functional domain consisting of nanobody against LILRB2 of any one of claims 1 to 3.

5. A pharmaceutical composition, comprising the nanobody against LILRB2 of any one of claims 1 to 3 or the fusion protein of claim 4, and a pharmaceutically acceptable excipient.

6. An isolated nucleic acid molecule encoding the nanobody of any of claims 1 to 3 against LILRB2 or encoding the fusion protein of claim 4.

7. An expression vector comprising the nucleic acid molecule of claim 6.

8. A recombinant cell comprising the expression vector of claim 7.

9. A method for preparing nanobody according to any one of claims 1 to 3, characterized in that it comprises: culturing the recombinant cell of claim 8, and separating and purifying the culture product to obtain the nanobody.

10. Use of the nanobody against LILRB2 of any one of claims 1 to 3, the fusion protein of claim 4, the composition of claim 5, the nucleic acid molecule of claim 6, the vector of claim 7, or the recombinant cell of claim 8 in the preparation of a medicament for the prevention, treatment and/or amelioration of a solid or hematological tumor.

11. The use of claim 10, wherein the solid tumor is lung cancer, pancreatic cancer, endometrial cancer, melanoma, ovarian cancer, breast cancer, colorectal cancer, glioma, gastric cancer, renal cancer, testicular cancer, esophageal cancer, cervical cancer, thyroid cancer, or liver cancer; the hemangioma is chronic lymphocytic leukemia and acute myelogenous leukemia.

12. The use of claim 10, wherein the solid tumor is non-small cell lung cancer, pancreatic ductal carcinoma, hepatocellular carcinoma, urothelial cancer, or head and neck cancer.

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