Nanobodies targeting human LILRB4 and their applications

Abstract

This invention discloses a nanobody targeting LILRB4, a nucleic acid encoding the nanobody, an expression vector containing the nucleic acid, a pharmaceutical composition containing the nanobody, and its use in preparing a pharmaceutical product.

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 a nanobody targeting LILRB4, its derived protein, and its use in the preparation of drugs. 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] LILRB4 (also known as ILT3, LIR5, CD85K) is a member of leukocyte immunoglobulin-like receptors (LILRs / LIRs) and an immunomodulatory transmembrane protein found on antigen-presenting cells (APCs). LILRB4 inhibits APC activation and induces immune tolerance through T suppressor cells. It has been shown to regulate immune responses by inducing T cell unresponsiveness and differentiation of CD8+ T suppressor cells, and may play a role in establishing immune tolerance in cancer. It is primarily expressed in tolerant dendritic cells (DCs), myeloid-derived suppressor cells, and M2 macrophages, with limited expression on the surface of plasma cells, but not on hematopoietic progenitor cells or stem cells. LILRB4 is a marker molecule for monocytic acute myeloid leukemia (AML), and is highly expressed on the surface of monocytic AML cells.

[0004] Currently, two antibody drugs targeting LILRB4 are in clinical trials. NGM831, developed by NGM Biopharmaceuticals, is currently in Phase I clinical trials for indications including pancreatic cancer, breast cancer, gastric cancer, non-small cell lung cancer, cervical cancer, intracervical cancer, squamous cell carcinoma of the head and neck, urothelial carcinoma of the bladder, colorectal cancer, esophageal cancer, ovarian cancer, renal cell carcinoma, prostate cancer, melanoma, mesothelioma, and cholangiocarcinoma. IO-202, developed by Immune-Onc Therapeutics, is currently in Phase I clinical trials for indications including acute myeloid leukemia and myelomonocytic leukemia. Summary of the Invention

[0005] Existing anti-LILRB4 antibody molecules such as ScFv, Fab, or all-IgG are structurally complex and large. Although they can link active molecules to LILRB4, they affect the function of the 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.

[0006] To address the shortcomings of the existing technologies, this invention provides a series of anti-human LILRB4 nanobody sequences and preparation methods. The provided anti-LILRB4 nanobodies can bind to human LILRB4 with high affinity, and their activation effect on T cells can be clearly observed in cell models, thus demonstrating potential therapeutic value against tumors.

[0007] In a first aspect, the present invention provides an anti-LILRB4 nanobody. According to an embodiment of the present invention, the nanobody is capable of specifically binding to LILRB4, and the complementarity-determining region (CDR) of the VHH chain in the nanobody is selected from one or more of the following:

[0008] (1) CDR1 shown in SEQ ID NO: 16, CDR2 shown in SEQ ID NO: 17, and CDR3 shown in SEQ ID NO: 18;

[0009] (2) CDR1 shown in SEQ ID NO: 19, CDR2 shown in SEQ ID NO: 20, and CDR3 shown in SEQ ID NO: 21;

[0010] (3) CDR1 shown in SEQ ID NO: 22, CDR2 shown in SEQ ID NO: 23, and CDR3 shown in SEQ ID NO: 24;

[0011] (4) CDR1 shown in SEQ ID NO: 25, CDR2 shown in SEQ ID NO: 26, and CDR3 shown in SEQ ID NO: 27;

[0012] (5) CDR1 shown in SEQ ID NO: 28, CDR2 shown in SEQ ID NO: 29, and CDR3 shown in SEQ ID NO: 30;

[0013] (6) CDR1 shown in SEQ ID NO: 25, CDR2 shown in SEQ ID NO: 26, and CDR3 shown in SEQ ID NO: 31;

[0014] (7) CDR1 shown in SEQ ID NO: 32, CDR2 shown in SEQ ID NO: 33, and CDR3 shown in SEQ ID NO: 34;

[0015] (8) CDR1 shown in SEQ ID NO: 35, CDR2 shown in SEQ ID NO: 36, and CDR3 shown in SEQ ID NO: 37.

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

[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, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15.

[0018] Secondly, the present invention provides a fusion protein, according to embodiments of the present invention, comprising a functional domain capable of specifically binding to LILRB4, said functional domain being composed of an anti-LILRB4 nanobody as described in any of the preceding claims.

[0019] 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 LILRB4-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.

[0020] Thirdly, the present invention provides an antibody against LILRB4. 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-LILRB4 nanobody described in any of the preceding claims.

[0021] Furthermore, the aforementioned functional fragments are the Fab, Fab', (Fab')2, Fv, scFv, or sdFv structures of the conventional antibody.

[0022] 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 the disclosed structure of nanobodies capable of specifically binding LILRB4, those skilled in the art will readily conceive of modifying traditional antibodies using the nanobodies of this invention. For example, applying the CDR region structure of the nanobodies of this invention to traditional antibodies can yield traditional antibodies capable of specifically binding LILRB4. Such traditional antibodies also fall within the scope 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 LILRB4 binding specificity, and these also fall within the scope of this invention.

[0023] Fourthly, the present invention provides a composition for treating a disease, comprising an anti-LILRB4 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.

[0024] The pharmaceutically acceptable excipients mentioned above refer to pharmaceutical excipients used in the pharmaceutical field, such as diluents, fillers, binders, humectants, absorption enhancers, surfactants, disintegrants, adsorbent carriers, lubricants, etc. Other excipients, such as flavoring agents and sweeteners, may also be added. That is, the excipients mentioned are one or more of the following: diluents, fillers, binders, humectants, absorption enhancers, surfactants, disintegrants, adsorbent carriers, lubricants, flavoring agents, and sweeteners.

[0025] 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.

[0026] Fifthly, the present invention provides an isolated nucleic acid molecule that encodes a single-domain antibody as described in any of the preceding claims.

[0027] Based on the content disclosed in this invention, those skilled in the art can easily obtain the polynucleotide molecules encoding the above-mentioned nanoantibodies 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 single-domain antibody or fusion protein of this invention, they fall within the protection scope of this invention.

[0028] In a sixth aspect, the present invention provides a carrier containing the nucleic acid molecules described above.

[0029] In a seventh aspect, the present invention provides recombinant cells containing the vector described above.

[0030] 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.

[0031] 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.

[0032] In a ninth aspect, the present invention provides a method for activating T cells, comprising contacting the T cells with an anti-LILRB4 nanobody as described in any of the preceding claims, a fusion protein as described above, an antibody as described above, a composition as described above, a nucleic acid molecule as described above, a carrier as described above, or a recombinant cell as described above.

[0033] Optionally, the contact includes the presence of cancer cells.

[0034] LILRB4 is expressed on monocytes, macrophages, and dendritic cells, and can suppress innate immunity in a cell-autonomous manner and inhibit T cell activation through indirect mechanisms. LILRB4 is a specific marker for monocytic acute myeloid leukemia (AML), including refractory and relapsed disease. LILRB4 has been shown to support tumor cell infiltration into tissues and inhibit T cell activity through signaling pathways involving APOE, LILRB4, SHP-2, uPAR, and ARG1 in AML cells (Deng M. etah, Nature (2018) 562:605-09).

[0035] In a tenth aspect, the invention provides the use of the anti-LILRB4 nanobody as described in any of the preceding claims, 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 use in the preparation of a medicament for diseases mediated by human LILRB4 protein.

[0036] The antibodies of the present invention can be used to treat diseases that target LILRB4 and neutralize LILRB4, thereby achieving the effects of prevention, treatment and / or improvement, including but not limited to tumors.

[0037] To better understand this invention, some terms are first defined. Other definitions are listed throughout the detailed description section.

[0038] 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.

[0039] 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.

[0040] "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.

[0041] "Nanobodies" are generally defined as in WO 2008 / 020079 or WO 2009 / 138519, and therefore 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 chain or light chain variable region obtained from human or murine monoclonal antibodies, which retains antigen-binding activity but has significantly reduced affinity and solubility.

[0042] 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.

[0043] "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.

[0044] 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.

[0045] 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.

[0046] The present invention achieves the following beneficial technical effects:

[0047] The anti-LILRB4 nanobody of the present invention can bind to human LILRB4 with high affinity, and its activation effect on T cells can be clearly observed in cell models, thus having potential therapeutic value for tumors. Attached Figure Description

[0048] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Furthermore, the same reference numerals denote the same parts throughout the drawings. In the drawings:

[0049] Figure 1 FACS detection of the binding activity of anti-human LILRB4 chimeric antibody on THP-1 cells

[0050] Figure 2 FACS detection of the binding activity of anti-human LILRB4 chimeric antibody on RPMI-8226 cells

[0051] Figure 3 Activation curve of T cells by anti-human LILRB4 chimeric antibody

[0052] Figure 4 FACS detection of the binding activity of anti-human LILRB4 humanized antibody on RPMI-8226 cells Detailed Implementation

[0053] Exemplary embodiments of the present disclosure will now be described in more detail with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

[0054] Example 1: Construction of a camel nanobody phage library

[0055] 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.

[0056] Specifically, camels were immunized every two weeks for a total of four times. Each injection consisted of 0.8 mg of recombinant LILRB4 extracellular protein, administered subcutaneously at multiple sites 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 inversion, 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 LILRB4 VHH antibody immunotherapy library was used for screening specific anti-human LILRB4 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%.

[0057] The constructed camel immune library was screened using a solid-phase screening method to obtain specific phage-displayed nanobodies. Through presentation, screening, and identification of the original library, ten phage-displayed nanobodies capable of simultaneously binding to the recombinant protein of human LILRB4 were obtained: D2, D5, C6, C7, H9, F1, C3, B2, E5, and B4.

[0058] Example 2: Preparation of anti-human LILRB4 nanobody and control antibody

[0059] The variable region gene of the target control antibody 193 (sequence source: WO2020056077A1, SEQ NO.231 and SEQ NO.237) was synthesized, and its light and heavy chain variable region sequences are shown in SEQ ID NO.1 and SEQ ID NO.2. The light and heavy chain sequences were cloned into eukaryotic transient expression vectors containing the light and heavy chain constant regions of human kappa / IgG1, respectively, to obtain light and heavy chain expression plasmids of the control antibody. These plasmids were transformed into *E. coli* for amplification, and a large number of plasmids containing the light and heavy chains of the control antibody were isolated. The plasmids were extracted and precipitated with ethanol. Following the instructions of the transfection reagent 293fectin (Cat:12347019, Gibco), the light and heavy chain plasmids of the control antibody 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.

[0060] Based on the sequencing results of nanobodies displayed by bacteriophages, primers were designed to clone D2, D5, C6, C7, H9, F1, C3, B2, E5, and B4 into a eukaryotic transient expression vector containing the human Fc (hFc) encoding gene via PCR, and then recombinantly expressed in HEK293 cells. Five to six days after cell transfection, the culture supernatant was collected and purified using a ProA affinity chromatography column to obtain recombinant proteins chD2, chD5, chC6, chC7, chH9, chF1, chC3, chB2, chE5, and chB4. The variable region sequence of chD2 is shown in SEQ ID NO.3, the variable region sequence of chD5 is shown in SEQ ID NO.4, the variable region sequence of chC6 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 chH9 is shown in SEQ ID NO.7, the variable region sequence of chF1 is shown in SEQ ID NO.8, the variable region sequence of chC3 is shown in SEQ ID NO.9, the variable region sequence of chB2 is shown in SEQ ID NO.10, the variable region sequence of chE5 is shown in SEQ ID NO.11, and the variable region sequence of chB4 is shown in SEQ ID NO.12. The constant region sequence is shown in SEQ ID NO.13.

[0061] SEQ.ID NO.1:193 Heavy chain variable region amino acid sequence

[0062] EVQLVESGGGLVQPGGSLRLSCAASGFSLSSSYWISWVRQAPGKGLEWIGSIDSGSVGITYYATWVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHGDNWALDLWGQGTLVTVSS

[0063] SEQ.ID NO.2:193 Light chain variable region amino acid sequence

[0064] DIQMTQSPSTLSASVGDRVTITCRASQSINSWLAWYQQKPGKAPKLLIYKASTLASGVPSRFSGSGSGTEFTLTISSLQPDDAATYYCQHGYIRGDLDNVFGGGTKVEIK

[0065] SEQ.ID NO.3:chD2 VHH amino acid sequence

[0066] QVQLQESGGGSVQAGGSLRLSCTASGYTAS SDYMG WFRQAPGKKREGVA CINTNGGETYHANSVSG RFTISRDNAKNTVYLQMNSLKPDDTAMYYCAV GRTNPDSYGGSRCLLAPEYTY WGQGTQVTVSS

[0067] The amino acid sequences of CDRs 1, 2 and 3 of the chD2 antigen complementarity-determining region are SEQ ID NO: 16, 17 and 18, respectively, and are underlined (according to the definition of Kabat CDR).

[0068] SEQ.ID NO.4:chD5 VHH amino acid sequence

[0069] QVQLQESGGGSVQAGGSLLRLSCATSGDTYS TLCMG WFRQAPGKEREGVA AIYRGGDSTVYADSVKG RFTISQDNAKNTVYLQMNGLKPEDTAIYYCAASSLGRCAA DIRTGPPFWAVGFRY WGQGTQVTVSS

[0070] The amino acid sequences of CDRs 1, 2 and 3 of the chD5 antigen complementarity-determining region are SEQ ID NO: 19, 20 and 21, respectively, and are underlined (according to the definition of Kabat CDR).

[0071] SEQ.ID NO.5:chC6 VHH amino acid sequence

[0072] QVQLQESGGGLVQPGGSLRLSCAASGLAFS RYYMS WVRQAPGKGLEWVS GIRSDGLSTSYADSVKG RFTISRDNAKNTLFLQMNNLKSEDTALYYCAT GVGDSGDY RGQGTQVTVSS

[0073] The amino acid sequences of CDRs 1, 2 and 3 of the chC6 antigen complementarity-determining region are SEQ ID NO: 22, 23 and 24, respectively, and are underlined (according to the definition of Kabat CDR).

[0074] SEQ.ID NO.6:chC7 VHH amino acid sequence

[0075] QVQLQESGGGSVQAGGSLLRLSCAASEYTYS RHCMA WFRQAPGKEREGVA TIYTGGGITRYADSVKG RFTISQDNAKNTVYLQMNSLKPEDTAMYYCAA DIASRACVTDPFPLKHAQFSS WGQGTQVTVSS

[0076] The amino acid sequences of CDRs 1, 2 and 3 of the chC7 antigen complementarity-determining region are SEQ ID NO: 25, 26 and 27, respectively, and are underlined (according to the definition of Kabat CDR).

[0077] SEQ.ID NO.7:chH9 VHH amino acid sequence

[0078] QVQLQESGGGSVQAGGSRLLSCAVSEYVYS RCTMA WYRQAPGKERELVS AFDSGETTWYADSVKG RFTISQDNAKNTVYLQMNSLKPEDTAMYYCNT VGVKSVKSGGGSWCFSPDY WGQGTQVTVSS

[0079] The amino acid sequences of CDRs 1, 2 and 3 of the chH9 antigen complementarity-determining region are SEQ ID NO: 28, 29 and 30, respectively, and are underlined (according to the definition of Kabat CDR).

[0080] SEQ.ID NO.8:chF1 VHH amino acid sequence

[0081] QVQLQESGGGLVQAGGSLLRLSCAASEYSYR RHCMAWFRQAPGKEREGVA TIYTGGGITRYADSVKG RFTISQDNAKNTVYLQMNSLKPEDTAMYYCAA DIASRACVTDPFPLERARFSA WGQGTQVTVSS

[0082] The amino acid sequences of CDRs 1, 2 and 3 of the chF1 antigen complementarity-determining region are SEQ ID NO: 25, 26 and 31, respectively, and are underlined (according to the definition of Kabat CDR).

[0083] SEQ.ID NO.9:chC3 VHH amino acid sequence

[0084] QVQLQESGGGLVQAGGSLRLSCVASEYIYT RCNMA WYRQAPGKERELVS AFDIGDTLYYADSVKG RFTISQDNAKNTVYLQMNSLKPEDTAMYYCNT VGVKPGGGSWCFNPDY WGQGTQVTVSS

[0085] The amino acid sequences of CDRs 1, 2 and 3 of the chC3 antigen complementarity-determining region are SEQ ID NO: 32, 33 and 34, respectively, and are underlined (according to the definition of Kabat CDR).

[0086] SEQ.ID NO.10:chB2 VHH amino acid sequence

[0087] QVQLQESGGGPVQAGGSLLRLSCAASEYSYR RHCMA WFRQAPGKEREGVA TIYTGGGITRYADSVKG RFTISQDNAKNTVYLQMNSLKPEDTAMYYCAA DIASRACVTDPFPLERARFSA WGQGTQVTVSS

[0088] The amino acid sequences of CDRs 1, 2 and 3 of the chB2 antigen complementarity-determining region are SEQ ID NO: 25, 26 and 31, respectively, and are underlined (according to the definition of Kabat CDR).

[0089] SEQ.ID NO.11:chE5 VHH amino acid sequence

[0090] QVQLQESGGGLVQAGGSLLRLSCAASEYTYS RHCMA WFRQAPGKEREGVA TIYTGGGITRYADSVKG RFTISQDNAKNTVYLQMNSLKPEDTAMYYCAA DIASRACVTDPFPLKHAQFSS 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, and are underlined (according to the definition of Kabat CDR).

[0092] SEQ.ID NO.12:chB4 VHH amino acid sequence

[0093] QVQLQESGGGPVQAGGSLLRLSCAASRYDIE TKCIT WLRQAPGKERERVA SISPGDGSTYYADSVKG RFTISQEYAKNTVDLQMNSLKSEDTAMYYCAA ARAPWGRCAQWTAGIDFDY WGQGTQVTVSS

[0094] The amino acid sequences of CDRs 1, 2 and 3 of the chB4 antigen complementarity-determining region are SEQ ID NO: 35, 36 and 37, respectively, and are underlined (according to the definition of Kabat CDR).

[0095] SEQ.ID NO.13: hFc constant region amino acid sequence

[0096] ASEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0097] Example 3: Detection of chimeric antibody affinity

[0098] Antibody affinity was determined using the Octet QKe system from Fortebio, employing an anti-human antibody Fc fragment capture antibody (AHC) bioprobe to capture the antibody Fc fragment. For the assay, chimeric antibodies chD2, chD5, chC6, chC7, chH9, chF1, chC3, chB2, chE5, and chB4, along with control antibody 193, were diluted to 4 μg / ml with PBS buffer and flowed through the surface of an AHC probe (Cat: 18-0015, PALL) for 120 s. Human LILRB4 recombinant protein (purchased from Yiqiao, Cat. 16742-H08H) or cynomolgus monkey LILRB4 recombinant protein (purchased from ACRO, Cat. 3CDK-C5227) at 60 nm was used as the mobile phase, with a binding time of 300 s and a dissociation time of 300 s. After the experiment was completed, the blank control response value was subtracted, and the kinetic constant of antigen-antibody binding was calculated by fitting the 1:1 Langmuir binding mode using software.

[0099] The kinetic parameters are shown in Table 1 below. The results indicate that all 10 chimeric antibodies bound to the human LILRB4 recombinant protein, with chD2, chD5, and chC6 showing affinity superior to or equivalent to the control antibody 193. All 10 chimeric antibodies bound to the cynomolgus monkey LILRB4 recombinant protein; except for chC6, the other nine antibodies showed affinity superior to or equivalent to the control antibody 193.

[0100] Table 1. Affinity assay results between chimeric antibody and recombinant LILRB4 protein

[0101]

[0102]

[0103] Example 4: FACS detection of binding activity of anti-human LILRB4 chimeric antibody on THP-1 cells

[0104] 2E5 THP-1 cells were conjugated with different concentrations of anti-LILRB4 chimeric antibody, chD2, chD5, chC6, chC7, chH9 chimeric antibodies, and control antibody 193, in 11 serial dilutions starting at 198 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.

[0105] The results show ( Figure 1), chD2, chD5, chC6, chC7, and chH9 can bind to human LILRB4-positive THP-1 cells in a dose-dependent manner. The EC50 values ​​are shown in Table 2. Their binding ability on THP-1 cells is superior to that of the control antibody.

[0106] Table 2. EC50 values ​​of anti-human LILRB4 chimeric antibody binding on THP-1 cells as detected by FACS

[0107] 193 chD2 chD5 chC6 chC7 chH9 EC50 (nM) 1.43 0.27 0.31 0.22 0.39 0.44

[0108] Example 5: FACS detection of binding activity of anti-LILRB4 chimeric antibody on RPMI-8226 cells

[0109] 2E5 RPMI-8226 cells were conjugated with different concentrations of anti-LILRB4 chimeric antibody, chF1, chC3, chB2, chE5, and chB4 chimeric antibodies, and control antibody 193, in 8 serially diluted 3-fold starting from 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 2 ), chF1, chC3, chB2, chE5 and chB4 can bind to human LILRB4 positive cells RPMI-8226 cells in a dose-dependent manner, and the binding activity is not weaker than that of the control 193.

[0111] Example 6: Evaluation of the activation effect of anti-human LILRB4 chimeric antibody on T cells using a LILRB4 cell model

[0112] Log-grown APOE / TCR Activator / CHO cells were harvested, trypsinized, centrifuged at 1000 rpm for 5 minutes to remove the supernatant, and resuspended in fresh F12K medium containing 10% FBS. The cell density of the resuspended cells was adjusted to 4 × 10⁶ cells / year. 5 / ml. The resuspended cells were seeded into 96-well cell culture plates with clear white walls, 100 μL / well of cell suspension, and incubated overnight at 37°C with 5% CO2. The next day, the F12K+10% FBS medium in the 96-well plates seeded with APOE / TCR Activator / CHO cells was aspirated dry, and the plates were rinsed once with 150 μL / well of DPBS. The DPBS was then removed. The chD2, chD5, chH9 chimeric antibodies and control antibody 193 were serially diluted in RPMI 1640 medium containing 0.5% BSA (starting at 256 nM, 11 4-fold serial dilutions). 2* concentration samples (50 μL / well) of the serially diluted samples were added to the cell-seeded 96-well plates. Control wells without antibody were also included. LILRB4 Effector Reporter cells in the logarithmic growth phase were harvested, centrifuged, and the supernatant was discarded. The cells were washed once with DPBS, centrifuged again to remove the DPBS, and then resuspended in fresh RPMI 1640 medium containing 0.5% BSA. The cell density of the resuspended cells was adjusted to 8 × 10⁶ cells / mL. 5 / ml, then add 50ul of cells to each well of the 96-well plate containing the antibody, and incubate at 37°C for 5 to 6 hours. Remove the 96-well plate from the incubator and add 100ul / well of Bright-Glo TM After incubating the luciferase assay reagent for 3 minutes, the values ​​were read using a microplate reader. Based on the readings for each concentration gradient well, the Prism Graphpad software was used to fit the gradient curve of cell activation by the sample, and the half-maximal effective binding concentration (EC50) of the sample was calculated.

[0113] Activation gradient curves such as Figure 3 As shown in Table 3, the corresponding EC50 values ​​are similar to those of the control antibody 193. The activation effects of chD2, chD5, and chH9 on T cells are similar.

[0114] Table 3. EC50 values ​​of anti-human LILRB4 chimeric antibody on T cell activation

[0115] [[ID= ​ ​ 193 ​ 0.153 0.043 0.064 0.094

[0116] Example 7: Humanization and Recombinant Expression Analysis of Anti-human LILRB4 Nanobody

[0117] First, a comprehensive analysis of the camel-derived antibody VHH sequence was performed to identify the antigen complementarity determinant (CDR) region for antibody-antigen binding and the framework region supporting the conserved three-dimensional conformation of the antibody. Subsequently, based on homology alignment results, the template of the most similar human antibody was selected as the base template. Combined with full-sequence BLAST results, CDR transplantation was performed, achieving humanization of the chD2 variable region (VH) in the Framework region. The amino acid sequence of the humanized D2 molecule hzD2-1 is shown in Sequence 14; the amino acid sequence of the humanized D2 molecule hzD2-2 is shown in Sequence 15. The humanized hzD2-1 and hzD2-2 variable region sequences were fully synthesized and cloned into a eukaryotic expression vector containing the human Fc (hFc) encoding gene. After obtaining the expression plasmid with the correct sequence, it was transfected 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 humanized recombinant antibody protein.

[0118] SEQ.ID NO.14: hzD2-1 amino acid sequence

[0119] EVQLVESGGGLVQPGGSLRLSCAASGFTVS ​ WFRQAPGKEREGVA ​ RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR ​ WGQGTLVTVSS

[0120] The amino acid sequences of hzD2-1 antigen complementarity-determining regions CDRs 1, 2 and 3 are SEQ ID NO: 16, 17 and 18, respectively, and are underlined (according to the definition of Kabat CDR).

[0121] SEQ.ID NO.15: hzD2-2 amino acid sequence

[0122] EVQLVESGGGLVQPGGSLRLSCAASGYTAS ​ WFRQAPGKEREGVA ​ RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR ​ WGQGTLVTVSS

[0123] The amino acid sequences of hzD2-2 antigen complementarity-determining regions CDRs 1, 2 and 3 are SEQ ID NO: 16, 17 and 18, respectively, and are underlined (according to the definition of Kabat CDR).

[0124] Example 8: Detection of affinity for humanized LILRB4 antibody

[0125] Antibody affinity was determined using the Fortebio Octet QKe system with an AHC (Antibody Capture Cell) bioprobe to capture the Fc fragment of the anti-human antibody. During the assay, the chimeric antibody and its corresponding humanized antibody, along with control antibody 128, were diluted to 4 μg / ml with PBS buffer and flowed through the surface of the AHC probe (Cat: 18-0015, PALL) for 120 s. Recombinant LILRB4 protein (purchased from Yiqiao, Cat. 16742-H08H) at 100 nm was used as the mobile phase, with a binding time of 300 s and a dissociation time of 300 s. After the experiment, the blank control response value was subtracted, and the kinetic constants of antigen-antibody binding were calculated using a 1:1 Langmuir binding model fitting.

[0126] The kinetic parameters are shown in Table 4 below. The results indicate that the affinity of the humanized antibody and the chimeric antibody remains essentially the same.

[0127] Table 4. Affinity assay results between humanized antibodies and recombinant human LILRB4 protein

[0128]

[0129]

[0130] Example 9: FACS detection of binding activity of anti-LILRB4 humanized antibody on RPMI-8226 cells

[0131] 2E5 RPMI-8226 cells were conjugated with different concentrations of anti-LILRB4 humanized antibody. hzD2-1 and hzD2-2 humanized antibodies were serially diluted 3-fold starting at 22 nM for 10 increments. Cells were incubated at 4°C in the dark for 60 min. After thorough washing with PBS, FITC-labeled goat anti-human antibody (Sigma, F9512) diluted 1:200 was added, and the cells were incubated at 4°C in the dark for 30 min. After thorough washing with PBS, the cells were resuspended in 200 μL of PBS and analyzed by flow cytometry.

[0132] The results show ( ​ hzD2-1 and hzD2-2 can bind to human LILRB4-positive RPMI-8226 cells, and their binding activity is consistent with that of the control antibody 193. The EC50 values ​​are shown in Table 5.

[0133] Table 5. EC50 values ​​of anti-human LILRB4 humanized antibody on RPMI-8226 cells as detected by FACS

[0134] ​ ​ 193 ​ 0.221 0.215 0.227

[0135] 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 LILRB4, characterized in that, The nanobody can specifically bind to LILRB4, and the amino acid sequence of the heavy chain variable region of the nanobody is shown in either SEQ ID NO: 14 or 15.

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

3. A fusion protein, characterized in that, It includes a functional domain capable of specifically binding to LILRB4, said functional domain being composed of the anti-LILRB4 nanobody as described in any one of claims 1-2.

4. A pharmaceutical composition, characterized in that, It includes the anti-LILRB4 nanobody as described in any one of claims 1-2, or the fusion protein as described in claim 3, and pharmaceutically acceptable excipients.

5. An isolated nucleic acid molecule, characterized in that, It encodes the anti-LILRB4 nanobody as described in any one of claims 1-2, or the fusion protein as described in claim 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 cells contain the expression vector as described in claim 6.

8. A method for preparing the nanobody according to any one of claims 1-2, 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. A method for activating T cells for non-diagnostic or therapeutic purposes, comprising contacting the T cells with the anti-LILRB4 nanobody of any one of claims 1-2, the fusion protein of claim 3, the composition of claim 4, or the recombinant cells of claim 7, said contact being in the presence of cancer cells.

10. The use of the anti-LILRB4 nanobody according to any one of claims 1-2, the fusion protein according to claim 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, said medicament for the treatment and / or improvement of pancreatic cancer, breast cancer, gastric cancer, non-small cell lung cancer, cervical cancer, intracervical carcinoma, head and neck squamous cell carcinoma, bladder urothelial carcinoma, colorectal cancer, esophageal cancer, ovarian cancer, renal cell carcinoma, prostate cancer, melanoma, mesothelioma, cholangiocarcinoma, acute myeloid leukemia, myelomonocytic leukemia, and multiple myeloma.

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