Nanobodies targeting human lllrb2 and uses thereof

Abstract

Disclosed are an anti-LILRB2 nanobody, a nucleic acid encoding the nanobody, an expression vector comprising the nucleic acid, a pharmaceutical composition comprising the nanobody, and a use for preparing a medicament.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of antibody engineering, and particularly relates to a therapeutic single-domain antibody for diagnosing or treating tumors, and particularly relates to an anti-LILRB2 nanobody, a derivative protein thereof, and a use for preparing a medicine. BACKGROUND

[0002] Nanobody is currently the smallest antibody molecule, which was first discovered by Belgian scientist Hamers in camel blood, and is a type of engineering antibody product that has attracted much attention. The main advantages of nanobody are: first, the volume is 1 / 10 of that of ordinary antibodies, and because of the small volume, the penetration in animal tissues is strong, for example, it can pass through the brain tissue of the human body and reach the inside of high-density tumors, while ordinary antibodies cannot, which makes it possible to treat some tumors or brain diseases through nanobody; second, good antigen specificity; third, easy to genetically modify, which is convenient for artificial modification to obtain antibodies against different pathogens; fourth, high stability, for example, nanobody is not naturally decomposed in the body for a longer time than ordinary antibodies (meaning that the drug effect lasts longer), and nanobody can even pass through the human stomach while maintaining effectiveness.

[0003] LILRB2, also known as ILT4, is mainly expressed on myeloid cells, including monocytes, dendritic cells, macrophages and neutrophils. Genetic studies have shown that tumor-associated macrophages (TAM) in various tumor microenvironments highly express LILRB2, and inhibition of LILRB2 (the corresponding protein in mice is called Pirb) reduces the invasion of Treg and MDSC in tumor tissues. Animal experiments show that Pirb antibodies inhibit tumor growth and have a synergistic effect with PD-1 antibodies. LILRB2 is mainly expressed on myeloid cells and has limited expression in other tissues, which makes the on-target, off-tissue toxicity relatively mild. Human LILRB2 is an important homeostatic surface regulator during myeloid cell maturation, and is a promising myeloid immune checkpoint target specifically targeting myeloid cell function, which has important therapeutic value. SUMMARY

[0004] The anti-LILRB2 antibody molecules of ScFv, Fab or full IgG in the prior art have complex structures and large molecules, although they can connect active molecules to LILRB2, but affect the function of the active molecules, the method is complex, and the loading efficiency is also low; nanobody molecules are small and easy to operate, but the degree of humanization is low, the affinity is not high, and the performance of prolonging the half-life needs to be further improved.

[0005] In view of the deficiencies of the prior art, the present application provides a series of anti-LILRB2 nanobody sequences and preparation schemes.

[0006] In a first aspect, the present invention provides an anti-LILRB2 nanobody. According to an embodiment of the present invention, the nanobody is capable of specifically binding to LILRB2, and the complementarity-determining region (CDR) of the VHH chain in the nanobody is selected from one or more of the following:

[0007] (1) CDR1 shown in SEQ ID NO: 9, CDR2 shown in SEQ ID NO: 10, and CDR3 shown in SEQ ID NO: 11;

[0008] (2) CDR1 shown in SEQ ID NO: 12, CDR2 shown in SEQ ID NO: 13, and CDR3 shown in SEQ ID NO: 14;

[0009] (3) CDR1 shown in SEQ ID NO: 15, CDR2 shown in SEQ ID NO: 16, and CDR3 shown in SEQ ID NO: 17;

[0010] (4) CDR1 shown in SEQ ID NO: 18, CDR2 shown in SEQ ID NO: 19, and CDR3 shown in SEQ ID NO: 20;

[0011] (5) CDR1 shown in SEQ ID NO: 21, CDR2 shown in SEQ ID NO: 22, and CDR3 shown in SEQ ID NO: 23.

[0012] Furthermore, in some embodiments of the present invention, the above-mentioned nanobody is a humanized VHH or a camel-derived VHH.

[0013] Furthermore, in some embodiments of the present invention, the above-mentioned nanobody has an amino acid sequence as shown in any one of SEQ ID NO: 3, 4, 5, 6, 7, or an amino acid sequence having at least 80% identity with the amino acid sequence shown in any one of SEQ ID NO: 3, 4, 5, 6, 7.

[0014] The “at least 80% identity” as described in this invention refers to any percentage of identity of ≥80%, such as at least 80%, preferably at least 85%, more preferably at least 90%, further preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identity.

[0015] Secondly, the present invention provides a fusion protein, according to an embodiment of the present invention, comprising a functional domain capable of specifically binding to LILRB2, said functional domain being composed of an anti-LILRB2 nanobody as described in any of the preceding claims.

[0016] The nanobody provided by the present application can be fused with any other protein or substance to achieve different purposes, for example, combined with fluorescent protein, enzyme or radioactive element to achieve the purpose of easy detection, and combined with a drug molecule for treating a disease related to LILRB2 to achieve the purpose of better treatment. The type of protein fused with the nanobody can be reasonably selected by a person skilled in the art according to actual needs or purposes, and no matter what type of substance is fused, it is within the scope of the present application.

[0017] In a third aspect, the present application provides an anti-LILRB2 antibody, according to an embodiment of the present application, the antibody is a traditional antibody or a functional fragment thereof, the heavy chain variable region of the antibody is composed of any one of the aforementioned anti-LILRB2 nanobodies.

[0018] Further, the functional fragment is a Fab, Fab', (Fab')2, Fv, scFv or sdFv structure of the traditional antibody.

[0019] The traditional antibody is structurally composed of two identical heavy chains and two identical light chains, the light chain has 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 CH4). Under the premise that the present application discloses the structure of the nanobody capable of specifically binding to LILRB2, a person skilled in the art can easily conceive that the nanobody of the present application is used to modify the traditional antibody, for example, the CDR region structure of the nanobody of the present application is applied to the traditional antibody to obtain a traditional antibody capable of specifically binding to LILRB2, and such a traditional antibody also belongs to the protection scope of the present application; further, based on the structure of the traditional antibody, part of the structure such as Fab, Fab', (Fab')2, Fv, scFv or sdFv structure also has LILRB2 binding specificity, which also belongs to the protection scope of the present application.

[0020] In a fourth aspect, the present application provides a composition for treating a disease, which comprises the anti-LILRB2 nanobody according to any one of the above, the fusion protein according to the above or the antibody according to the above, and a pharmaceutically acceptable excipient.

[0021] The pharmaceutical composition provided by the present application contains at least one (for example, one, two, three or four) of the antibodies or antigen binding fragments described in the embodiments of the present application, and two or more (for example, two, three or four) of any of the antibodies or antigen binding fragments described herein can exist in any combination in the pharmaceutical composition. The pharmaceutical composition can be formulated in any manner known in the art.

[0022] The pharmaceutical compositions can also contain pharmaceutically acceptable carriers. 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 that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as 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. Pharmaceutically acceptable carriers can further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody.

[0023] The dosage form of the medicament provided by the present application is not strictly limited, and can be prepared into various dosage forms according to the existing methods in the field of pharmaceutical agents, and is administered to patients in need of treatment by oral, nasal inhalation, rectal, parenteral or transdermal administration and the like.

[0024] In a fifth aspect, the present application provides an isolated nucleic acid molecule encoding the Nanobody according to any one of the above.

[0025] Based on the disclosure of the present application, the polynucleotide molecule encoding the Nanobody and the fusion protein described above can be easily obtained by those skilled in the art through the conventional techniques in the art, and based on the degeneracy of codons, the polynucleotide molecule is variable, and there are multiple possibilities for the specific base sequence, and based on this, as long as the polynucleotide molecule can encode the Nanobody or the fusion protein of the present application, it belongs to the protection scope of the present application regardless of the changes in the polynucleotide molecule.

[0026] In a sixth aspect, the present application provides a vector containing the nucleic acid molecule described above.

[0027] In a seventh aspect, the present application provides a recombinant cell containing the vector described above.

[0028] The embodiments of the present application provide a recombinant vector (e.g., an expression vector) comprising an isolated polynucleotide disclosed herein (e.g., a polynucleotide encoding a polypeptide disclosed herein), a host cell into which the recombinant vector has been introduced (i.e., such that the host cell contains the polynucleotide and / or a vector containing the polynucleotide), and the production of a recombinant antibody polypeptide or fragment thereof by recombinant techniques.

[0029] As used herein, a “vector” is any construct capable of delivering one or more target polynucleotides to a host cell when introduced into the host cell. An “expression vector” is capable of delivering and expressing one or more target polynucleotides as encoded polypeptides in a host cell into which the expression vector has been introduced. Thus, in an expression vector, the target polynucleotide is expressed within the vector by operatively linking to regulatory elements such as promoters, enhancers, and / or polyadenylate tails, said regulatory elements being located within the vector or at, near, or flanking, the integration site of the target polynucleotide in the genome of the host cell, such that the target polynucleotide will be translated in the host cell into which the expression vector has been introduced.

[0030] Vectors can be introduced into host cells using methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., with recombinant viruses). Therefore, non-limiting embodiments of vectors include viral vectors (which can be used to generate recombinant viruses), naked DNA or RNA, plasmids, granules, phage vectors, and DNA or RNA expression vectors associated with cationic condensers.

[0031] This invention provides host cells transformed using the vector described above. The host cell can be a prokaryotic or eukaryotic cell. A preferred prokaryotic host cell is *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*.

[0032] Eighthly, the present invention provides a method for preparing nanobodies as described in any of the preceding claims, comprising: culturing recombinant cells as described above, and isolating and purifying the nanobodies from the culture product.

[0033] It should be noted that the nanobodies, fusion proteins and antibodies of the present invention can be prepared by chemical synthesis, genetic engineering technology or other methods. Regardless of the method used to prepare the aforementioned nanobodies, fusion proteins or antibodies of the present invention, they are all within the protection scope of the present invention.

[0034] In a ninth aspect, the invention provides the use of the anti-LILRB2 nanobody, 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 the prevention, treatment, and / or improvement of solid tumors or hematologic malignancies.

[0035] Preferably, the solid tumor is lung cancer, non-small cell lung cancer, pancreatic cancer, pancreatic ductal carcinoma, chronic lymphocytic leukemia, acute myeloid leukemia, endometrial cancer, hepatocellular carcinoma, melanoma, ovarian cancer, breast cancer, colorectal cancer, glioma, gastric cancer, renal cancer, testicular cancer, esophageal cancer, cervical cancer, squamous cell lung cancer, leukemia, thyroid cancer, liver cancer, upper urinary tract cancer, or head and neck cancer.

[0036] For better understanding of the present application, some terms are defined first. Other definitions are listed throughout the DETAILED DESCRIPTION section.

[0037] Generally, the antigen-binding properties of an antibody can be described by three specific regions in the variable region of the heavy chain, referred to as complementarity determining regions (CDRs), which are interspaced by four framework regions (FRs), the amino acid sequences of which are relatively conserved and do not directly participate in the binding reaction. These CDRs form loop structures, which are brought into close proximity in space by the beta sheets formed by the intervening FRs. The CDRs on the heavy chain and the corresponding CDRs on the light chain form the antigen binding site of the antibody. Which amino acids constitute the FR or CDR regions can be determined by comparing the amino acid sequences of antibodies of the same class.

[0038] The present application includes not only intact antibodies, but also fragments of antibodies having immunological activity or fusion proteins of antibodies with other sequences. Therefore, the present application also includes fragments, derivatives, and analogs of the antibodies.

[0039] A "chimeric antibody" refers to an antibody in which the amino acid sequences of the immunoglobulin molecule are derived from two or more species. Typically, the variable regions of both the light and heavy chains correspond to the variable regions of an antibody of a desired specificity, affinity, and capability derived from one mammalian species (e.g., mouse, rat, rabbit, etc.), while the constant regions are homologous to sequences in an antibody derived from another species (typically human) to avoid eliciting an immune response in that species.

[0040] A "nanobody" is generally as defined in WO 2008 / 020079 or WO 2009 / 138519 and in one particular aspect generally denotes a VHH, a humanized VHH or a camelized VH (such as a camelized human VH), or generally denotes a sequence optimized VHH (e.g. optimized for chemical stability and / or solubility, maximal overlap with known human framework regions and maximal expression). "Nanobodies" are obtained by genetic engineering methods, and there are mainly three types, the first type is the heavy chain variable region obtained from camelid HCAb, which is a single folding unit, retains the complete antigen binding activity, and is the smallest natural antibody fragment. The second type is the heavy chain variable region obtained from cartilaginous fish IgNAR such as sharks, denoted as VNAR. The third type is the heavy chain or light chain variable region obtained from human or murine monoclonal antibodies, which retains the antigen binding activity, but the affinity and solubility are greatly reduced.

[0041] "Fc region" or "Fc" refers to a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a hinge region, a CH2 domain and a CH3 domain, which mediates the binding of an 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 (e.g., Clq) of the classical complement system, including native-sequence Fc regions and variant Fc regions. Generally, a human IgG heavy chain Fc region is the segment from an amino acid residue at its Cys226 or Pro230 position, or to the carboxy-terminus, but can vary in its boundaries. The C-terminal lysine (residue 447, according to EU numbering system) of the Fc region can or can not be present. Fc can also refer to this region isolated, or in the context of a protein polypeptide comprising Fc, e.g., a "binding protein comprising an Fc region," also referred to as an "Fc fusion protein" (e.g., an antibody or immunoadhesin). Native-sequence Fc regions in the antibodies of the application include human IgGl, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. The Fc region in the IgG, IgA, and IgD antibody isotypes comprises the CH2 and CH3 constant domains of each of the heavy chains of the antibody; the IgM and IgE Fc regions comprise the three heavy chain constant domains (CH domains 2-4) in each polypeptide chain.

[0042] "Specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen against which it is directed. The term "immunological binding" refers to the specific binding reaction that occurs between an antibody molecule and an antigen to which the antibody is specific. The strength or affinity of an immunological binding interaction can be expressed in terms of the equilibrium dissociation constant (KD) of the interaction, where a smaller value for KD 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 formation and dissociation of antigen binding site / antigen complexes. Both the "association rate constant" (Kaor Kon) and the "dissociation rate constant" (Kdor Koff) for a particular antibody-antigen interaction can be calculated from the concentration and the actual rates of association and dissociation, and KD, Kaand Kdvalues can be measured using any effective method. In preferred embodiments, the dissociation constant is measured using bioluminometric interferometry. In other preferred embodiments, the dissociation constant is measured using surface plasmon resonance technology (e.g., Biacore) or KinExa.

[0043] A "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been 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 episomal mammalian vectors). 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. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Host cells can be prokaryotic or eukaryotic, and include but are not limited to bacterial cells, yeast cells, fungal cells, plant cells and animal cells.

[0044] A "nucleic acid molecule" is intended to include DNA and RNA molecules. A nucleic acid molecule can be single-stranded or double-stranded, and can be cDNA.

[0045] The present application has the following beneficial technical effects:

[0046] The anti-LILRB2 nanobodies of the present application can bind human LILRB2 with high affinity, and have potential therapeutic value for tumors. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 ELISA was used to detect the binding of anti-LILRB2 chimeric antibodies to recombinant human LILRB2 protein;

[0048] Figure 2 FACS was used to detect the binding activity of anti-LILRB2 chimeric antibodies on human LILRB2 / 293 cells;

[0049] Figure 3 FACS was used to detect the binding activity of anti-LILRB2 chimeric antibodies on cyno LILRB2 / 293 cells. DETAILED DESCRIPTION

[0050] The scheme of the present application will be explained below in conjunction with the examples. Those skilled in the art will understand that the examples described below are exemplary and are only used to explain the present application, and cannot be understood as a limitation of the present application. The experimental methods in the following examples are conventional methods, unless otherwise specified. The raw materials, reagents and materials used in the following examples are commercially available, unless otherwise specified.

[0051] Example 1: Construction of camelid nanobody immunophage library

[0052] The camel is immunized with the antigen, peripheral blood mononuclear cells (PBMC) are isolated and total RNA is extracted for reverse transcription, the variable domain of the heavy-chain of heavy chain antibody (VHH) is amplified with the reverse transcription product as a template and connected into a phage display vector, and the electrotransformation is performed into E. coli TG1 competent cells to construct a camel immune library.

[0053] Specifically, the camel is immunized twice a week for 4 times. Each time, 0.8 mg of human LILRB2 extracellular region recombinant protein is injected, and Freund's complete / incomplete adjuvant (Sigma, F5881, F5506) is used, and the method of subcutaneous multiple injection is adopted. 1 mL of blood is collected 2 weeks after each immunization to separate serum, and the immunogen is used as the determination antigen, and the titers of total antibody (IgG) and heavy chain antibody (HcAb) in the serum are determined by ELISA method, respectively. When the serum titer reaches the requirement of library construction, 100 mL of camel peripheral blood is collected and PBMC is separated by a separation kit (Tianjin Haoyang, Cat: TBD2011CM), and the total RNA of PBMC is extracted to obtain cDNA, which is used as a template for subsequent amplification of VHH fragments. According to the camel VHH antibody gene searched in the relevant literature and database, the VHH antibody library construction primer is designed and synthesized, and the antibody variable region gene sequence is amplified by PCR. Then the vector and the amplified antibody fragment are cut by endonuclease. The connection product is constructed by using T4 ligase, and the connection product is transferred into TG1 bacteria by electroporation technology. Finally, a 1.8×10 8 The camel anti-human LILRB2 VHH antibody immune library is used for specific anti-human LILRB2 nanobody screening. In order to detect the accuracy of the library, 50 clones are randomly selected for colony PCR, and the results show that the insertion rate reaches 90%.

[0054] The camel immune library constructed is screened by solid phase screening method to obtain specific phage display nanobodies. Through the original library presentation and screening and identification, 5 strains of phage display nanobodies capable of binding to human LILRB2 recombinant protein are obtained: A2, C3, C9, D9 and E2.

[0055] Example 2: Preparation of anti-human LILRB2 nanobody and control antibody

[0056] MK-4830 (sequence source: CN110719917A, SEQ ID NO. 2 and 7) is a control antibody for the same target, the variable region sequences of its heavy and light chains are shown in SEQ ID NO. 1 and SEQ ID NO. 2. The light and heavy chain sequences are cloned into eukaryotic transient expression vectors containing human lambda / IgG4 light and heavy chain constant regions, respectively, to obtain control antibody MK-4830 light and heavy chain expression plasmids, which are transformed into E. coli for amplification. A large amount of plasmids containing control antibody light and heavy chains are obtained by separation, extraction, and ethanol precipitation of the plasmids. According to the operating instructions of the transfection reagent 293fectin (Cat: 12347019, Gibco), the light and heavy chain plasmids of the control antibody are separately transformed into HEK293 cells for recombinant expression. Five to six days after cell transfection, the culture supernatant is taken and purified using a ProA affinity chromatography column to obtain the control antibody.

[0057] According to the sequencing results of the phage-displayed nanobodies, primers are designed and cloned into eukaryotic transient expression vectors containing human Fc (hFc) coding genes by PCR, and recombinantly expressed in HEK293 cells. Five to six days after cell transfection, the culture supernatant is taken and purified using a ProA affinity chromatography column to obtain chA2, chC3, chC9, chD9, and chE2 recombinant proteins. The variable region sequence of chA2 is shown in SEQ ID NO. 3, the variable region sequence of chC3 is shown in SEQ ID NO. 4, the variable region sequence of chC9 is shown in SEQ ID NO. 5, the variable region sequence of chD9 is shown in SEQ ID NO. 6, and the variable region sequence of chE2 is shown in SEQ ID NO. 7. The corresponding CDRs are shown in underlined (according to the definition of Kabat CDR), and the constant region sequence is shown in SEQ ID NO. 8.

[0058] SEQ.ID NO. 1: MK-4830 heavy chain variable region amino acid sequence

[0059] EVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHAGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLPTRWVTTRYFDLWGRGTLVTVSS

[0060] SEQ.ID NO. 2: MK-4830 light chain variable region amino acid sequence

[0061] ESVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGDSNRPSGVPDRFSVSKSGASASLAITGLQAEDEADYYCQSFDNSLSAYVFGGGTQLTVLGQPK

[0062] SEQ. ID NO. 3: chA2 VHH amino acid sequence

[0063] QVQLQESGGGSVQAGESLRLSCRASGSTSS YYYLG WFRQAPGKEREAVA AADNPKNLFAEHYADSVKG RFTISQDNTKNTLYLQMNSLKPEDTAVYFCAA RLKGYDWRDAPHYTY WGQGTQVTVSS

[0064] The amino acid sequences of the complementarity determining regions CDRs 1, 2, and 3 of the chA2 antigen are SEQ ID NOs: 9, 10, and 11, respectively.

[0065] SEQ. ID NO. 4: chC3 VHH amino acid sequence

[0066] QVQLQESGGGSVQAGGSLRLSCAVSTYTYS TYSLG WFRQAAGKEREGVA VISTATGISWYADSVKG RFTISQDSTKNTLFLEMNDLKPEDTAVYYCAA GFHEADAPTLGVAGSTYPLSSFGY WGQGTQVTVSS

[0067] The amino acid sequences of the complementarity determining regions CDRs 1, 2, and 3 of the chC3 antigen are SEQ ID NOs: 12, 13, and 14, respectively.

[0068] SEQ. ID NO. 5: chC9 VHH amino acid sequence

[0069] QVQLQESGGGSVQAGGSLNLSCTASHYTHS ANYMG WFRQVSGKEREGVA RIFIGSGSTIYADSVKG RFTISQDNAKRTTYLQMTSLEPEDTAVYYCAG CVASPWMCDLDPKNYGY WRQGTQVTVSS

[0070] The amino acid sequences of the complementarity determining regions CDRs 1, 2, and 3 of the chC9 antigen are SEQ ID NOs: 15, 16, and 17, respectively.

[0071] SEQ. ID NO. 6: chD9 VHH amino acid sequence

[0072] QVQLQESGGGSVQAGGSRRLSCAASGNTEN AGCMA WFRQAPGKGRQGVA RIHGISGATYYTDSVKG RFTISQDKAKNTLYLQMNNLEPEDTAMYYCAA TRLLYCSGAIVQGEYNY WGQGTQVTVSS

[0073] The amino acid sequences of chD9 antigen complementarity determining regions CDRs 1, 2, and 3 are SEQ ID NOs: 18, 19, and 20, respectively.

[0074] SEQ.ID NO.7: chE2 VHH amino acid sequence

[0075] QVQLQESGGGSVQAGGSLRLSCTVSGYTGS FYLMA WFRQTPGKGPEGVA VIYPGDGSTDYDSSVKG RFTISRDNAENTIYLQMNNLKPADTATYYCAA DVRPYGRRWDQGSEFDI WGQGTQVTVSS

[0076] The amino acid sequences of chE2 antigen complementarity determining regions CDRs 1, 2, and 3 are SEQ ID NOs: 21, 22, and 23, respectively.

[0077] SEQ.ID NO.8: hFc constant region amino acid sequence

[0078] ASEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0079] Example 3: Chimeric antibody affinity testing

[0080] The antibody affinity was determined by the method of capturing the Fc segment of the antibody with the Fortebio company's Octet QKe system instrument using the capture antibody (AHC) bioprobe of the Fc segment of the anti-human antibody. When determining, chA2, chC3, chC9, chD9, chE2 chimeric antibodies and control antibody MK-4830 were diluted to 4 ug / ml with PBS buffer, flowed through the surface of the AHC probe (Cat: 18-0015, PALL) for 120 s. The LILRB2 recombinant protein (purchased from ACRO, Cat#LI2-H5220) was 60 nm; as the flow phase, the binding time was 300 s, and the dissociation time was 300 s. After the experiment was completed, the blank control response value was deducted, and the software was used for 1:1 Langmuir binding mode fitting to calculate the kinetic constant of antigen-antibody binding.

[0081] The kinetic parameters are shown in Table 1 below, and the results show that the five chimeric antibodies are all combined with the LILRB2 recombinant protein, and the binding activity is comparable to the control antibody.

[0082] Table 1. Affinity determination results of chimeric antibodies and LILRB2 recombinant protein

[0083] Sample KD (M) kon (1 / Ms) kdis (1 / s) MK-4830 7.84E-09 1.71E+05 1.34E-03 chA2 6.55E-10 1.37E+05 8.95E-05 chC3 9.57E-09 3.43E+05 3.28E-03 chC9 2.86E-09 2.02E+05 5.77E-04 chD9 8.30E-09 1.54E+05 1.28E-03 chE2 3.97E-10 2.73E+05 1.08E-04

[0084] Example 4: ELISA detection of the binding activity of anti-LILRB2 chimeric antibodies

[0085] The human LILRB2-mFc recombinant protein (NCBI number: AAH36827, 22-461 amino acids) was coated overnight at 4°C, and the coating concentration was 1 ug / mL; after washing the plate with PBS for 3 times, 5% BSA PBS was added, and the plate was blocked at 37°C for 60 min, and the plate was washed with PBST for 3 times; different dilution multiples of chA2, chC3, chC9, chD9, chE2 chimeric antibodies and control antibody MK-4830 (starting from 10 ug / ml, 3 times gradient dilution of 4 gradients) were added, and the plate was incubated at 37°C for 60 min, and the plate was washed with PBST for 4 times; 1:5000 diluted HRP-anti-human Fc (Cat: 109-035-098, Jackson Immuno Research) was added, and the plate was incubated at 37°C for 45 min, and the plate was washed with PBST for 4 times; TMB substrate was added for color development, and after incubation at 37°C for 10 min, 2M HCl was added to terminate the reaction; the absorbance A450nm-630nm of the well plate at wavelength 450nm was read and recorded with 630nm as the reference wavelength.

[0086] The experimental results show that chA2, chC3, chC9, chD9, chE2 chimeric antibodies and control antibody MK-4830 can be specifically combined with human LILRB2 recombinant proteinFigure 1 ).

[0087] Example 5: FACS detection of the binding activity of anti-LILRB2 chimeric antibodies to human LILRB2 / 293 cells

[0088] The binding of chimeric antibodies to cyno LILRB2 was detected using transiently transfected cyno LILRB2 / 293 cells. 2E5 cells were incubated with different concentrations of anti-LILRB2 antibodies, chA2, chC3, chC9, chD9, chE2 and control antibody MK-4830, which were diluted at 4 gradients from 10 ug / ml, 3 times gradient. After incubation at 4°C for 60 min in the dark, the cells were washed with PBS and then incubated with 1:200 diluted FITC-labeled goat anti-human antibody (Sigma, F9512) at 4°C for 30 min in the dark. After washing with PBS, the cells were resuspended in 200 ul PBS and detected by flow cytometry.

[0089] The results showed that Figure 2 ), chA2, chC3, chC9, chD9, chE2 chimeric antibodies and control antibody MK-4830 had comparable binding ability.

[0090] Example 6: FACS detection of the binding activity of anti-LILRB2 chimeric antibodies to cyno LILRB2 / 293 cells

[0091] The binding of chimeric antibodies to cyno LILRB2 was detected using transiently transfected cyno LILRB2 / 293 cells. 2E5 cells were incubated with different concentrations of anti-LILRB2 antibodies, chA2, chC3, chC9, chD9, chE2 and control antibody MK-4830, which were diluted at 4 gradients from 10 ug / ml, 3 times gradient. After incubation at 4°C for 60 min in the dark, the cells were washed with PBS and then incubated with 1:200 diluted FITC-labeled goat anti-human antibody (Sigma, F9512) at 4°C for 30 min in the dark. After washing with PBS, the cells were resuspended in 200 ul PBS and detected by flow cytometry.

[0092] The results showed that Figure 3 YYYLG AADNPKNLFAEHYADSVKG RLKGYDWRDAPHYTY TYSLG VISTATGISWYADSVKG GFHEADAPTLGVAGSTYPLSSFGY ANYMG RIFIGSGSTIYADSVKG CVASPWMCDLDPKNYGY AGCMA RIHGISGATYYTDSVKG TRLLYCSGAIVQGEYNY FYLMA VIYPGDGSTDYDSSVKG DVRPYGRRWDQGSEFDI Sample KD (M) kon (1 / Ms) kdis (1 / s) MK-4830 chA2 chC3 chC9 chD9 chE2 Figure 1 Figure 2 Figure 3 YYYLG ), chC9 had strong binding activity to cyno LILRB2, and other antibodies had weak binding to cyno LILRB2, suggesting that ChC9 and the other several molecules might have different binding epitopes.

[0093] The above merely describes preferred specific embodiments of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can easily think of changes or replacements within the technical scope disclosed by the present application, which should be covered within the protection scope of the present application.

Claims

1. A nanobody against LILRB2, characterized in that, The nanobody can specifically bind to LILRB2, and the complementarity-determining region (CDR) of the VHH chain in the nanobody is selected from the following group: (1) CDR1 shown in SEQ ID NO: 12, CDR2 shown in SEQ ID NO: 13, and CDR3 shown in SEQ ID NO: 14; (2) CDR1 shown in SEQ ID NO: 21, CDR2 shown in SEQ ID NO: 22, and CDR3 shown in SEQ ID NO:

23.

2. The nanobody according to claim 1, characterized in that, The nanobody is a humanized VHH or a camel-derived VHH.

3. The nanobody according to claim 1 or 2, characterized in that, The amino acid sequences of the nanobody are shown in SEQ ID NO: 4 and 7.

4. A pharmaceutical composition, characterized in that, It includes the anti-LILRB2 nanobody as described in any one of claims 1-3, and pharmaceutically acceptable excipients.

5. An isolated nucleic acid molecule, characterized in that, It encodes the anti-LILRB2 nanobody as described in any one of claims 1-3.

6. An expression carrier, characterized in that, The expression vector contains the nucleic acid molecule as described in claim 5.

7. A recombinant cell, characterized in that, The recombinant cell contains the expression vector of claim 6; the recombinant cell is a prokaryotic or eukaryotic cell; wherein the eukaryotic cell is selected from: protist cells, animal cells and fungal cells.

8. A method for preparing the nanobody according to any one of claims 1-3, characterized in that, It includes: The recombinant cells of claim 7 are cultured, and the nanobody is isolated and purified from the culture product.

9. Use of the anti-LILRB2 nanobody according to any one of claims 1-3, the composition according to claim 4, the nucleic acid molecule according to claim 5, the carrier according to claim 6, or the recombinant cell according to claim 7 in the preparation of a medicament for the treatment of colorectal cancer, pancreatic cancer, endometrial cancer, lung cancer, breast cancer, ovarian cancer, or gastric cancer.

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