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] This invention belongs to the field of antibody engineering, specifically relating to a therapeutic single-domain antibody for the diagnosis or treatment of tumors, and particularly to an anti-LILRB2 nanobody, its derived protein, and its use in the preparation of pharmaceuticals. Background Technology

[0002] Nanobodies are the smallest antibody molecules currently known. Initially discovered in camel blood by Belgian scientist Hamers, they represent a highly anticipated class of engineered antibody products. The main advantages of nanobodies are: first, their size is only 1 / 10 that of ordinary antibodies. Due to their small size, they have strong penetrating power in animal tissues; for example, they can pass through human brain tissue and reach the high-density interior of tumors, which ordinary antibodies cannot. This makes nanobodies suitable for treating certain tumors or brain diseases. Second, they have good antigen specificity. Third, they are easily genetically modified, facilitating the artificial creation of antibodies against different pathogens. Fourth, they have high stability; for example, nanobodies do not naturally degrade in the body for a longer time than ordinary antibodies (meaning a longer duration of efficacy). Nanobodies can even pass through the human stomach while maintaining their effectiveness.

[0003] LILRB2, also known as ILT4, is primarily expressed on myeloid cells, including monocytes, dendritic cells, macrophages, and neutrophils. Genetic studies have shown that tumor-associated macrophages (TAMs) in various tumor microenvironments highly express LILRB2, and inhibiting LILRB2 (the mouse counterpart is called Pirb) reduces the invasion of Tregs and MDSCs into tumor tissues. Animal experiments have shown that Pirb antibodies inhibit tumor growth and have a synergistic effect with PD-1 antibodies. LILRB2 is mainly expressed in bone marrow cells, with limited expression in other tissues, resulting in relatively mild on-target and off-tissue toxicity. Human LILRB2 is an important homeostatic surface regulator during myeloid cell maturation and is a promising immune checkpoint target specifically for myeloid cell function assays, possessing significant therapeutic value. Summary of the Invention

[0004] Existing anti-LILRB2 antibody molecules such as ScFv, Fab, or all-IgG are structurally complex and large. Although they can link active molecules to LILRB2, they affect the function of active molecules, are complex to operate, and have low loading efficiency. Nanobody molecules are small and easy to manipulate, but they have low humanization and low affinity, and their performance in extending half-life needs to be further improved.

[0005] To address the shortcomings of the existing technologies, this invention provides a series of anti-human LILRB2 nanobody sequences and preparation methods.

[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: 14, CDR2 shown in SEQ ID NO: 15, and CDR3 shown in SEQ ID NO: 16;

[0008] (2) CDR1 shown in SEQ ID NO: 17, CDR2 shown in SEQ ID NO: 18, and CDR3 shown in SEQ ID NO: 19;

[0009] (3) CDR1 shown in SEQ ID NO: 20, CDR2 shown in SEQ ID NO: 21, and CDR3 shown in SEQ ID NO: 22;

[0010] (4) CDR1 shown in SEQ ID NO: 23, CDR2 shown in SEQ ID NO: 21, and CDR3 shown in SEQ ID NO: 24;

[0011] (5) CDR1 shown in SEQ ID NO: 25, CDR2 shown in SEQ ID NO: 26, and CDR3 shown in SEQ ID NO: 27.

[0012] (6) CDR1 shown in SEQ ID NO: 28, CDR2 shown in SEQ ID NO: 29, and CDR3 shown in SEQ ID NO: 30.

[0013] (7) CDR1 shown in SEQ ID NO: 31, CDR2 shown in SEQ ID NO: 32, and CDR3 shown in SEQ ID NO: 33.

[0014] (8) CDR1 shown in SEQ ID NO: 34, CDR2 shown in SEQ ID NO: 35, and CDR3 shown in SEQ ID NO: 36.

[0015] (9) CDR1 shown in SEQ ID NO: 25, CDR2 shown in SEQ ID NO: 37, and CDR3 shown in SEQ ID NO: 27.

[0016] Furthermore, in some embodiments of the present invention, the above-mentioned nanobody is a humanized VHH or a camel-derived VHH.

[0017] 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 to SEQ ID NO: 12, or an amino acid sequence having at least 80% identity with the amino acid sequence shown in any one of SEQ ID NO: 3 to SEQ ID NO: 12.

[0018] 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.

[0019] 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.

[0020] The nanobodies provided by this invention can be fused with any other protein or substance to achieve different purposes. For example, they can be bound to fluorescent proteins, enzymes, or radioactive elements for easy detection, or fused with drug molecules for treating LILRB2-mediated diseases to achieve better therapeutic effects. The type of protein fused with the nanobodies can be rationally selected by those skilled in the art according to actual needs or purposes. Regardless of the type of substance fused, it is also within the scope of this invention.

[0021] Thirdly, the present invention provides an antibody against LILRB2. According to an embodiment 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 the anti-LILRB2 nanobody described in any of the preceding claims.

[0022] Furthermore, the aforementioned functional fragments are the Fab, Fab', (Fab')2, Fv, scFv, or sdFv structures of the conventional antibody.

[0023] Traditional antibodies are structurally composed of two identical heavy chains and two identical light chains. The light chains have a variable region (VL) and a constant region (CL); the heavy chains have a variable region (VH) and a constant region (CH1, CH2, CH3, and / or CH4). Given that this invention discloses a nanobody structure capable of specifically binding to LILRB2, those skilled in the art will readily conceive of modifying traditional antibodies using the nanobody of this invention. For example, applying the CDR region structure of the nanobody of this invention to a traditional antibody can yield a traditional antibody capable of specifically binding to LILRB2. Such traditional antibodies also fall within the scope of protection of this invention. Furthermore, based on the structure of traditional antibodies, some of their structures, such as Fab, Fab', (Fab')2, Fv, scFv, or sdFv structures, also possess LILRB2 binding specificity, and these also fall within the scope of protection of this invention.

[0024] Fourthly, the present invention provides a composition for treating a disease, comprising an anti-LILRB2 nanobody as described in any of the preceding claims, a fusion protein as described above, or an antibody as described above, and pharmaceutically acceptable excipients.

[0025] The pharmaceutical compositions provided by this invention contain at least one (e.g., one, two, three, or four) of the antibody or antigen-binding fragments described in the embodiments of this invention. Two or more (e.g., two, three, or four) of any antibody or antigen-binding fragments described herein may be present in the pharmaceutical composition in any combination. The pharmaceutical compositions may be formulated in any manner known in the art.

[0026] Pharmaceutical compositions may also contain pharmaceutically acceptable carriers. As used herein, "pharmaceutically acceptable carriers" include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents, etc. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate-buffered saline, dextran, glycerol, ethanol, etc., and combinations thereof. In many cases, it is preferred to include isotonic agents, such as sugars, polyols such as mannitol, sorbitol, or sodium chloride, in the composition. Pharmaceutically acceptable carriers may further contain small amounts of excipients, such as wetting agents or emulsifiers, preservatives, or buffers, which increase the shelf life or potency of the antibody.

[0027] The dosage form of the drug provided by this invention is not strictly limited and can be prepared into various dosage forms according to existing methods in the pharmaceutical field, and administered to patients in need of treatment via oral, nasal, rectal, parenteral, or transdermal administration.

[0028] Fifthly, the present invention provides an isolated nucleic acid molecule that encodes a nanobody as described in any of the preceding claims.

[0029] Based on the content disclosed in this invention, those skilled in the art can easily obtain the polynucleotide molecules encoding the above-mentioned nanobodies and fusion proteins using conventional techniques in the field. Based on the degeneracy of codons, the polynucleotide molecules are variable, and their specific base sequences have multiple possibilities. Therefore, regardless of how the polynucleotide molecules change, as long as they can encode the nanobodies or fusion proteins of this invention, they fall within the protection scope of this invention.

[0030] In a sixth aspect, the present invention provides a carrier containing the nucleic acid molecules described above.

[0031] In a seventh aspect, the present invention provides recombinant cells containing the vector described above.

[0032] Embodiments of the present invention provide a recombinant vector (e.g., an expression vector) comprising isolated polynucleotides disclosed herein (e.g., polynucleotides encoding polypeptides disclosed herein), a host cell into which the recombinant vector is introduced (i.e., such that the host cell contains polynucleotides and / or a vector containing polynucleotides), and a recombinant antibody polypeptide or fragment thereof generated by recombinant technology.

[0033] 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.

[0034] 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.

[0035] 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*.

[0036] 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.

[0037] 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 scope of protection of the present invention.

[0038] 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.

[0039] Preferably, the solid tumor is lung cancer, non-small cell lung cancer (NSCLC), pancreatic cancer, pancreatic ductal carcinoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), endometrial cancer, hepatocellular carcinoma, melanoma, ovarian cancer, breast cancer, colorectal cancer, glioma, gastric cancer, kidney cancer, testicular cancer, esophageal cancer, cervical cancer, squamous cell lung cancer, leukemia, thyroid cancer, liver cancer, urinary tract cancer, or head and neck cancer.

[0040] To better understand this invention, some terms are first defined. Other definitions are listed throughout the detailed description section.

[0041] Generally, the antigen-binding properties of an antibody can be described by three specific regions located in the variable region of the heavy chain, called the variable region (CDR). This segment is divided into four frame regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a ring structure, and are spatially close to each other through the β-sheets formed by the FRs between them. The CDRs on the heavy chain and the corresponding CDRs on the 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.

[0042] This invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies.

[0043] "Chimeric antibodies" are antibodies in which the amino acid sequence of the immunoglobulin molecule is derived from two or more species. Typically, the variable regions of both the light and heavy chains correspond to the variable regions of the antibody derived from one mammalian species (e.g., mouse, rat, rabbit) that have the desired specificity, affinity, and ability, while the constant regions are homologous to sequences in the antibody derived from another species (usually human) to avoid triggering an immune response in that species.

[0044] "Nanobodies" are generally defined as in WO 2008 / 020079 or WO 2009 / 138519, and in a specific context generally refer to VHH, humanized VHH, or camel-derived VH (such as camel-derived human VH), or generally refer to sequence-optimized VHH (e.g., optimized for chemical stability and / or solubility, maximum overlap with known human framework regions, and maximum expression). Nanobodies are obtained through genetic engineering methods and mainly fall into three categories: the first category is the heavy chain variable region obtained from camel HCAbs, which is a single folded unit that retains complete antigen-binding activity and is the smallest natural antibody fragment. The second category is the heavy chain variable region obtained from IgNARs of cartilaginous fish such as sharks, denoted as VNAR. The third category is the heavy or light chain variable region obtained from human or murine monoclonal antibodies, which retains antigen-binding activity but has significantly reduced affinity and solubility.

[0045] The term "Fc region" or "Fc" refers to the C-terminal region of an immunoglobulin heavy chain, containing at least a portion of a hinge region, a CH2 domain, and a CH3 domain, which mediates the binding of immunoglobulins to host tissues or factors, including binding to Fc receptors 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 a segment from an amino acid residue at its Cys226 or Pro230 position to its carboxyl terminus, but its boundaries may vary. The C-terminal lysine residue (residue 447, according to the EU numbering system) of the Fc region may or may not be present. Fc can also refer to this isolated region, or in the case of a protein polypeptide containing Fc, such as "a binding protein containing an Fc region," also referred to as an "Fc fusion protein" (e.g., an antibody or an immunoadhesive). The native sequence Fc regions in the antibodies of this invention include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. In IgG, IgA, and IgD antibody isotypes, the Fc region contains the CH2 and CH3 constant domains of each of the two heavy chains of the antibody; the IgM and IgEFc regions contain the three heavy chain constant domains (CH domains 2-4) of each polypeptide chain.

[0046] "Specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its targeted antigen. The term "immunobinding" refers to a specific binding reaction occurring between an antibody molecule and an antigen (for which the antibody is specific). The strength or affinity of an immunobinding interaction can be expressed by the equilibrium dissociation constant (KD) of the interaction, where a smaller KD value indicates higher affinity. The immunobinding properties between two molecules can be quantified using methods known in the art. One method involves measuring the rate of formation and dissociation of the antigen binding site / antigen complex. Both the "binding rate constant" (Ka or Kon) and the "dissociation rate constant" (Kd or Koff) of a particular antibody-antigen interaction can be calculated from the concentration and the actual rates of association and dissociation, and the KD, Ka, and Kd values ​​can be measured using any effective method. In a preferred embodiment, the dissociation constant is measured using bioluminescent interferometry. In other preferred embodiments, the dissociation constant can be measured using surface plasmon resonance techniques (e.g., Biacore) or KinExa.

[0047] A "vector" is a nucleic acid molecule capable of transporting another nucleic acid it is linked to. One type of vector is a "plasmid," which is a circular double-stranded DNA loop in which an additional DNA segment can be linked. Another type of vector is a viral vector, in which an additional DNA segment can be linked to the viral genome. Some vectors can replicate autonomously in the host cells they are introduced into (e.g., bacterial vectors with bacterial origins of replication and free mammalian vectors). Other vectors (e.g., non-attached mammalian vectors) can integrate into the host cell's genome after introduction and thereby replicate along with the host genome. Furthermore, some vectors can guide the expression of genes to which they are effectively linked.

[0048] The term "nucleic acid molecule" is intended to include both DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, and can be cDNA.

[0049] The present invention achieves the following beneficial technical effects:

[0050] The anti-LILRB2 nanobody of the present invention can bind to human LILRB2 with high affinity and block the binding of LILRB2 to its receptor HLA-G, thereby having potential therapeutic value against tumors. Attached Figure Description

[0051] Figure 1 The experiment demonstrated the binding of the anti-LILRB2 chimeric antibody to the recombinant human LILRB2 protein as detected by ELISA.

[0052] Figure 2The binding activity of the anti-LILRB2 chimeric antibody on human LILRB2 / 293 cells was demonstrated by FACS detection.

[0053] Figure 3 The binding activity of the anti-LILRB2 chimeric antibody on cyno LILRB2 / 293 cells was demonstrated by FACS detection.

[0054] Figure 4 The ELISA assay showed the blocking activity of the LILRB2 chimeric antibody against recombinant LILRB2 binding to HLA-G.

[0055] Figure 5 The results show the blocking activity of the LILRB2 chimeric antibody against the binding of recombinant LILRB2 to HLA-G as detected by ELISA. Detailed Implementation

[0056] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are exemplary and only used to explain the present invention, and should not be construed as limiting the present invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the raw materials, reagents, and other materials used in the following embodiments are commercially available products.

[0057] Example 1: Construction of a camel nanobody phage library

[0058] Camels were immunized with antigens, peripheral blood mononuclear cells (PBMCs) were isolated and total RNA was extracted for reverse transcription. The reverse transcription product was used as a template to amplify the variable domain of the heavy-chain antibody (VHH) and ligated into a phage display vector. The VHH was then electroporated into E. coli TG1 competent cells to construct a camel immune library.

[0059] Specifically, camels were immunized every two weeks for a total of four times. Each injection consisted of 0.8 mg of recombinant human LILRB2 extracellular protein, administered subcutaneously via multiple sites, along with Freund's complete / incomplete adjuvant (Sigma, F5881, F5506). Two weeks after each immunization, 1 mL of blood was collected to separate serum. Using the immunogen as the assay antigen, the titers of total antibodies (IgG) and heavy chain antibodies (HcAb) in the serum were measured by ELISA. Once the serum titers met the requirements for library construction, 100 mL of camel peripheral blood was collected, and PBMCs were isolated using a separation kit (Tianjin Haoyang, Cat: TBD2011CM). Total RNA was extracted from the PBMCs, and cDNA was obtained by reverse transfection, serving as a template for subsequent amplification of the VHH fragment. Based on relevant literature and databases, camel-derived VHH antibody genes were retrieved, and primers for VHH antibody library construction were designed and synthesized. The variable region gene sequence of the antibody was amplified by PCR. Subsequently, the vector and the amplified antibody fragment were digested with restriction enzymes. The ligation product was constructed using T4 ligase, and then transferred into TG1 bacteria using electroporation. A 1.8 × 10⁻⁶ cell line was ultimately constructed. 8 A camel anti-human LILRB2 VHH antibody immunotherapy library was used for screening specific anti-human LILRB2 nanobodies. To test the accuracy of the library, 50 clones were randomly selected for colony PCR, and the results showed an insertion rate of 90%.

[0060] Using Biotinylated-LILRB2 (Acro, Cat.LI2-H82F5) as the antigen, a constructed camel immune library was screened using liquid-phase panning to obtain specific phage-displayed nanobodies. Through presentation, screening, and identification of the original library, ten phage-displayed nanobodies capable of simultaneously binding to recombinant human LILRB2 protein were obtained: A3, A7, B9, C7, D5, D6, E1, E5, E8, and G3.

[0061] Example 2: Preparation of anti-human LILRB2 nanobody and control antibody

[0062] The variable region gene of the target control antibody JTX-8064 (sequence source: CN111699196A, SEQ ID NO. 53 and 54) was synthesized. The heavy and light chain variable region sequences are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively. The light and heavy chain sequences were cloned into eukaryotic transient expression vectors containing the human kappa / IgG1 light and heavy chain constant regions to obtain control antibody light and heavy chain expression plasmids. The obtained control antibody light and heavy chain expression plasmids were transformed into *E. coli* for amplification, and a large number of plasmids containing the control antibody light and heavy chains were isolated. The plasmids were extracted and precipitated with ethanol. Following the instructions of the transfection reagent 293fectin (Cat: 12347019, Gibco), the control antibody light and heavy chain plasmids were transformed into HEK293 cells for recombinant expression. Five to six days after cell transfection, the culture supernatant was collected and purified using a ProA affinity chromatography column to obtain the control antibody.

[0063] Based on the sequencing results of the nanobodies displayed by bacteriophages, primers were designed, and the vectors were cloned into a eukaryotic transient expression vector containing the human Fc (hFc) encoding gene by PCR. The vectors were then recombinantly expressed in HEK293 cells. Five to six days after cell transfection, the culture supernatant was collected and purified using ProA affinity chromatography to obtain recombinant proteins chA3, chA7, chB9, chC7, chD5, chD6, chE1, chE5, chE8, and chG3. The variable region sequence chA3 is shown in SEQ ID NO.3, the variable region sequence chA7 is shown in SEQ ID NO.4, the variable region sequence chB9 is shown in SEQ ID NO.5, the variable region sequence chC7 is shown in SEQ ID NO.6, the variable region sequence chD5 is shown in SEQ ID NO.7, the variable region sequence chD6 is shown in SEQ ID NO.8, the variable region sequence chE1 is shown in SEQ ID NO.9, the variable region sequence chE5 is shown in SEQ ID NO.10, the variable region sequence chE8 is shown in SEQ ID NO.11, and the variable region sequence chG3 is shown in SEQ ID NO.12. The underlined parts indicate the corresponding CDRs (according to the definition of Kabat CDRs). The constant region sequence is shown in SEQ ID NO.13.

[0064] SEQ.ID NO.1: JTX-8064 Heavy Chain Variable Region Amino Acid Sequence

[0065] QITLKESGPTLVKPTQTLTLTCTFSGFSLNTYAMGVSWIRQPPGKALEWLASIWWNGNKYNNPSLKSRLTVTKDTSKNQVVLTMTNMDPVDTATYYCAHSRIIRFTDYVMDAWGQGTLVTVSS

[0066] SEQ.ID NO.2: JTX-8064 light chain variable region amino acid sequence

[0067] DIQMTQSPSSSLSTSVGDRVTITCRASEDIYNDLAWYQQKPGKAPKLLIYNANSLHTGVASRFSGSGSGTDFTFTISSLQPEDVATYFCQQYYDYPLTFGQGTKLEIK

[0068] SEQ.ID NO.3:chA3 VHH amino acid sequence

[0069] QVQLQESGGGLVQAGGSLTLSCAVSGISFG TRAMA WFRQAPGKEREGVA SIQADTYISYADSVKG RFTISKDSGKNTLNLQMNNLKPEDTAVYYCAV GDLWGSHLQPHGYNY WGQGTQVTVSS

[0070] The amino acid sequences of CDRs 1, 2 and 3 of the chA3 antigen complementarity-determining region are SEQ ID NO: 14, 15 and 16, respectively.

[0071] SEQ.ID NO.4:chA7 VHH amino acid sequence

[0072] QVQLQESGGGSVQPGGRLRLSCETSGYTSS RNWMG WFRQAPGKEREGVA SIYTDNGSAYYADSVKG RFTISVDNAKNTVYLQMNSLKPEDTAMYYCAG RIRPPAGTRWPGPLVESAYNT WGRGTQVTVSS

[0073] The amino acid sequences of CDRs 1, 2 and 3 of the chA7 antigen complementarity-determining region are SEQ ID NO: 17, 18 and 19, respectively.

[0074] SEQ.ID NO.5:chB9 VHH amino acid sequence

[0075] QVQLQESGGGSVQAGGSLLRLSCAASGYTDS RYCMGWFRQVPGKEREKVA TIYNGDGNTYYDDSVKG RFTISQGNAKNTLFLEMNNLKPEDTAMYYCAA IKAYGSNWCPAVEYSD WGQGTQVTVSS

[0076] The amino acid sequences of CDRs 1, 2 and 3 of the chB9 antigen complementarity-determining region are SEQ ID NO: 20, 21 and 22, respectively.

[0077] SEQ.ID NO.6:chC7 VHH amino acid sequence

[0078] QVQLQESGGGLVQAGGSLLRLSCAASEYTDS RYCMA WFRQVPGKEREKVA IIYNGDGNTYYDDSVKG RFTISQSVAKNTLYLQMNNLKPEDTGMYYCAA IKAYGSNWCRAVEYSD WGQGTQVTVSS

[0079] The amino acid sequences of CDRs 1, 2, and 3 in the chC7 antigen complementarity-determining region are SEQ ID NO: 23, 21, and 24, respectively.

[0080] SEQ.ID NO.7:chD5 VHH amino acid sequence

[0081] QVQLQESGGGLVQAGGSLRLSCVASGYPRS SVCMG WYRQAPGKEREGVA AIFTGGGTPYYGDSVKG RFTISQDNAVNTVSLQMNDLKPEDTAMYYCAA ELTYCSGGPWTDPVGY WGQGTQVTVSS

[0082] The amino acid sequences of CDRs 1, 2 and 3 of the chD5 antigen complementarity-determining region are SEQ ID NO: 25, 26 and 27, respectively.

[0083] SEQ.ID NO.8:chD6 VHH amino acid sequence

[0084] QVQLQESGGGLVQAGGSLLRLSCAASGFTGS GYCMG WFRQVPGKEREKVA TIYNGYPGDGDPSYDDSVKG RFTISQDTAKNTVYLQMNDLKPEDTAMYYCAA INAYGSNWCQIVEYAH WGVGTQVTVSS

[0085] The amino acid sequences of CDRs 1, 2 and 3 of the chD6 antigen complementarity-determining region are SEQ ID NO: 28, 29 and 30, respectively.

[0086] SEQ.ID NO.9:chE1 VHH amino acid sequence

[0087] QVQLQESGGGLVQPGGSLRLSCVGSGFTFR HYAMN WARQAPGKGIEWVA SIGTPGLPEAYADSVKG RFTISRDDAKNTLYLQMDNLKTEDTGVYYCAR SSDCGGGTCRP PGQGTQVTVSS

[0088] The amino acid sequences of CDRs 1, 2 and 3 of the chE1 antigen complementarity-determining region are SEQ ID NO: 31, 32 and 33, respectively.

[0089] SEQ.ID NO.10:chE5 VHH amino acid sequence

[0090] QVQLQESGGGSVQAGGSLRLSCVASGYPRS SVCMG WYRQAPGKEREGVA AIFTGGGTPYYGDSVKG RFTISQDNAVNTVSLQMNDLKPEDTAMYYCAV ELTYCSGGPWTDPVGY WGQGTQVTVSS

[0091] The amino acid sequences of CDRs 1, 2 and 3 of the chE5 antigen complementarity-determining region are SEQ ID NO: 25, 26 and 27, respectively.

[0092] SEQ.ID NO.11:chE8 VHH amino acid sequence

[0093] QVQLQESGGGLVQPGGSLNLSCAASGFTFS NSFMT WVRQAPGKRPEWVREWV AGISGDGQYTSYADFAK G RFTISRDNAKSTMYLQLNNLKTEDTAIYYCEK SSTE RGQGTQVTVSS

[0094] The amino acid sequences of CDRs 1, 2 and 3 of the chE8 antigen complementarity-determining region are SEQ ID NO: 34, 35 and 36, respectively.

[0095] SEQ.ID NO.12:chG3 VHH amino acid sequence

[0096] QVQLQESGGGSVQAGGSLRLSCAASGYPRS SVCMG WYRQAPGKEREGVA AIFTGGGRPYYADSVKGRFTISQDNAVNTVSLQMNNLKPEDTAMYYCAA ELTYCSGGPWTDPVGY WGQGTQVTVSS

[0097] The amino acid sequences of CDRs 1, 2 and 3 of the chG3 antigen complementarity-determining region are SEQ ID NO: 25, 37 and 27, respectively.

[0098] SEQ.ID NO.13: hFc constant region amino acid sequence

[0099] ASEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0100] Example 3: Detection of chimeric antibody affinity

[0101] Antibody affinity was determined using the Fortebio Octet QKe system with an AHC (antibody capture antibody) bioprobe to capture the Fc fragment of the anti-human antibody. For the assay, chimeric antibodies chA3, chA7, chB9, chC7, chD5, chD6, chE1, chE5, chE8, and chG3, along with the control antibody JTX-8064, were diluted to 4 μg / ml with PBS buffer and flowed through the AHC probe (Cat: 18-0015, PALL) for 120 s. Recombinant LILRB2 protein (purchased from ACRO, Cat#LI2-H5220) at 60 nm was used as the mobile phase, with binding and dissociation times of 300 s. After the experiment, the blank control response was subtracted, and the antigen-antibody binding kinetic constants were calculated using a 1:1 Langmuir binding model fitting.

[0102] The kinetic parameters are shown in Table 1 below. The results show that all 10 chimeric antibodies bound to the LILRB2 recombinant protein, and their binding activity was comparable to that of the control antibody.

[0103] Table 1. Affinity assay results between chimeric antibody and LILRB2 recombinant protein

[0104] sample 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

[0105] Example 4: ELISA detection of the binding activity of anti-LILRB2 chimeric antibody

[0106] Human LILRB2 recombinant protein (KN expression) was coated overnight at 4°C at a concentration of 1 μg / mL. After washing the plate three times with PBS, 5% BSA PBS was added, and the plate was blocked at 37°C for 60 min. The plate was then washed three times with PBST. 10 μg / mL of chimeric antibodies against chA3, chA7, chB9, chC7, chD5, chD6, chE1, chE5, chE8, and chG3, as well as the control antibody JTX-8064, were added, and the plate was incubated at 37°C for 60 min. The plate was washed four times with PBST. Finally, 1:5000 dilution of HRP-anti-human Fc (Cat: 109-035-098, Jackson) was added. ImmunoResearch), incubate at 37℃ for 45 min, wash the plate 4 times with PBST; add TMB substrate for color development, incubate at 37℃ for 10 min, and then add 2M HCl to terminate the reaction; use 630nm as the reference wavelength, read and record the absorbance A450nm-630nm of the plate at a wavelength of 450nm.

[0107] Experimental results showed that chimeric antibodies against chA3, chA7, chB9, chC7, chD5, chD6, chE1, chE5, chE8, and chG3, as well as the control antibody JTX-8064, could specifically bind to recombinant human LILRB2 protein. Figure 1 ).

[0108] Example 5: FACS detection of the binding activity of anti-LILRB2 chimeric antibody to recombinant human LILRB2-expressing 293 cells (humanLILRB2 / 293).

[0109] Using 293 cells transiently transfected with human LILRB2 (human LILRB2 / 293), the binding of chimeric antibodies to human LILRB2 was detected. 2E5 cells were conjugated with different concentrations of anti-LILRB2 antibody (chA3, chA7, chB9, chC7, chD5, chD6, chE1, chE5, chE8, and chG3 chimeric antibodies and the control antibody JTX-8064), serially diluted 4-fold starting at 132 nm. Cells were incubated at 4°C in the dark for 60 min, washed thoroughly with PBS, and then incubated at 4°C in the dark for 30 min with FITC-labeled goat anti-human antibody (Sigma, F9512) diluted 1:200. After washing thoroughly with PBS, the cells were resuspended in 200 μL of PBS and analyzed by flow cytometry.

[0110] The results show ( Figure 2The chimeric antibodies JTX-8064 containing chA3, chA7, chB9, chC7, chD5, chD6, chE1, chE5, chE8 and chG3, as well as the control antibody, have considerable binding ability.

[0111] Example 6: FACS detection of the binding activity of anti-LILRB2 chimeric antibody to recombinant cynomolgus monkey 293 cells (cynoLILRB2 / 293) expressing LILRB2.

[0112] Using cynomolgus monkey LILRB2 / 293 cells transiently transfected with LILRB2, the binding of chimeric antibodies to LILRB2 was detected. 2E5 cells were conjugated with different concentrations of anti-LILRB2 antibodies (chA3, chA7, chB9, chC7, chD5, chD6, chE1, chE5, chE8, and chG3) and the control antibody JTX-8064, serially diluted 4-fold starting at 132 nM. Cells were incubated at 4°C in the dark for 60 min, washed thoroughly with PBS, and then incubated at 4°C in the dark for 30 min with FITC-labeled goat anti-human antibody (Sigma, F9512) diluted 1:200. After washing thoroughly with PBS, the cells were resuspended in 200 μL of PBS and analyzed by flow cytometry.

[0113] The results show ( Figure 3 chE1 exhibits strong binding activity to LILRB2 in cynomolgus monkeys, comparable to the control antibody JTX-8064.

[0114] Example 7: ELISA detection of the blocking activity of anti-LILRB2 chimeric antibody against recombinant LILRB2 binding to HLA-G.

[0115] Recombinant human HLA-G extracellular region protein (Kactus, Cat.HLG-HE41F) was diluted to 1 μg / mL with PBS, 100 μL / well, and coated onto an ELISA plate. The plate was incubated overnight at 4°C. Blocking was performed with 5% BSA at 37°C for 120 min, followed by washing three times with PBST. 100 μL of the following test samples (starting at 66 nM, with 12 3-fold serial dilutions) containing chimeric antibodies (chA3, chA7, chB9, chC7, chD5, chD6, chE1, chE5, chE8, and chG3) and control antibody JTX-8064 were added, along with 100 μL of 0.4 μg / mL LILRB2-mFc (NCBI No.: AAH36827, amino acids 22-461). After mixing, the mixture was incubated at 37°C for 30 min. 100 μL of the incubated sample was then added to the HLA-G coated wells and incubated at 37°C for 60 min. Wash the plate four times with PBST; add 1:5000 diluted HRP-anti-mouse IgG (Cat: 115-035-071, Jackson Immuno Research) and react for 45 min, then wash four times with PBST; finally add TMB substrate for color development, incubate at 37℃ for 15 min, stop the reaction with 2M HCl, and read and record the absorbance of the plate at a wavelength of 450 nm. The results showed ( Figure 4 , Figure 5 (Table 2) Except for chA7 and chE1 which have no blocking activity, chE8 has partial blocking activity, and the remaining chimeric antibodies all have clear blocking activity, and their blocking activity is comparable to that of the control antibody.

[0116] Table 2. ELISA detection of the blocking activity (IC50) of LILRB2 chimeric antibody against recombinant LILRB2 binding to HLA-G.

[0117] JTX-8064 chE1 chE5 chE8 chG3 chC7 IC50(nM) 0.860 Non-blocking 0.604 1.193 0.593 0.834 JTX-8064 chA3 chA7 chB9 chD5 chD6 IC50(nM) 1.001 0.928 Non-blocking 1.039 0.537 0.952

[0118] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

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: CDR1 shown in SEQ ID NO: 23, CDR2 shown in SEQ ID NO: 21, and CDR3 shown in SEQ ID NO:

24.

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 nanobody has the amino acid sequence shown in SEQ ID NO:

6.

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. The 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, wherein the medicament is used to treat and / or improve solid tumors or hematologic malignancies, wherein the solid tumor is lung cancer, pancreatic cancer, endometrial cancer, ovarian cancer, breast cancer, colorectal cancer, kidney cancer, esophageal cancer, or liver cancer; and the hematologic malignancies are chronic lymphocytic leukemia or acute myeloid leukemia.

10. The use according to claim 9, wherein the lung cancer is non-small cell lung cancer.

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