Anti-RAGE antibodies and their applications

Abstract

The present application discloses an anti-RAGE antibody, and also discloses a conjugate, bispecific, multispecific antibody, immune cell, fusion protein, etc. comprising the anti-RAGE antibody. The anti-RAGE antibody of the present application can target RAGE antigen targets of different species such as human, mouse and monkey, and has high application value in scientific research and clinical application.

Description

Technical Field

[0001] The present application belongs to the field of bioengineering, and specifically relates to an anti-RAGE antibody, a conjugate comprising the anti-RAGE antibody, a bispecific antibody, a multispecific antibody, an immune cell, a fusion protein and applications thereof. Background Art

[0002] The receptor for advanced glycationendproducts (RAGE, or AGER) is a multiligand cell surface member of the immunoglobulin superfamily. RAGE consists of an extracellular domain, a single transmembrane domain, and a cytoplasmic tail. The extracellular domain of the receptor consists of a v-type immunoglobulin domain and two c-type immunoglobulin domains. RAGE is expressed by a variety of cell types, such as endothelial and smooth muscle cells, macrophages, and lymphocytes, in many different tissues, including the lung, heart, kidney, skeletal muscle, and brain. RAGE is involved in many important pathological responses, including Alzheimer's disease, diabetes, inflammation, and cancer. RAGE stimulates cancer growth, survival, and metastasis. In addition, RAGE expression is closely associated with gastric cancer invasion and metastasis. Moreover, there are reports that blocking RAGE can inhibit gastric cancer cell invasion. It has been reported that Ser82 enhances ligand affinity and upregulates receptor signaling through mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB). RAGE interacts with advanced glycation end products (AGEs), S100 proteins, etc. The interaction between the ligand and receptor RAGE activates important cellular pathways, including MAPK, Cdc42 / Rac and NF-κB signaling pathways.

[0003] The vast majority of anti-RAGE antibody molecules reported so far have high affinity only for RAGE from a single species, but the single antigen species limits the versatility of the antibody. Summary of the invention

[0004] In view of the above problems in the prior art, the present application provides an anti-RAGE antibody, which can target RAGE antigen targets of different species such as humans, mice and monkeys, and has high application value both in scientific research and clinical application.

[0005] The specific technical solutions of this application are as follows:

[0006] 1. An anti-RAGE antibody, wherein the antibody comprises three heavy chain complementary determining regions and three light chain complementary determining regions, the three heavy chain complementary determining regions are CDR-H1, CDR-H2 and CDR-H3, and the three light chain complementary determining regions are CDR-L1, CDR-L2 and CDR-L3, wherein:

[0007] The amino acid sequence contained in the CDR-H1 is shown in SEQ ID NO: 1, and SEQ ID NO: 1 is SSYAIS;

[0008] The amino acid sequence contained in the CDR-H2 is shown in SEQ ID NO: 2, and SEQ ID NO: 2 is RIIPILGIAN;

[0009] The amino acid sequence contained in the CDR-H3 is shown in SEQ ID NO: 3, and SEQ ID NO: 3 is QVGAIDY;

[0010] The amino acid sequence contained in the CDR-L1 is shown in SEQ ID NO: 4, and SEQ ID NO: 4 is QSISTYVS;

[0011] The amino acid sequence contained in the CDR-L2 is shown in SEQ ID NO: 5, and SEQ ID NO: 5 is IYYASILKS; and

[0012] The amino acid sequence contained in the CDR-L3 is shown in SEQ ID NO: 6, and SEQ ID NO: 6 is QSYSFPLT.

[0013] 2. An anti-RAGE antibody according to claim 1, comprising a human universal framework region.

[0014] 3. The anti-RAGE antibody according to item 1, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0015] the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO:7, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:7, wherein SEQ ID NO:7 is QVQLVQSWAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCASQVGAIDYWGQGTPVTVSS;

[0016] The amino acid sequence contained in the light chain variable region is as shown in SEQ ID NO:8, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:8, wherein SEQ ID NO:8 is NIQLTQSPSSLSASLGDRVTITCRTSQSISTYVSWYQQRSGEAPNLLIYYASILKSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSFPLTFGGGTKVEIK.

[0017] 4. The anti-RAGE antibody according to any one of items 1 to 3, which is a human antibody, a humanized antibody or a chimeric antibody.

[0018] 5. The anti-RAGE antibody according to any one of items 1 to 3, which is a full-length IgG antibody.

[0019] 6. The anti-RAGE antibody according to any one of items 1 to 3, which is a full-length IgG1 antibody.

[0020] 7. The anti-RAGE antibody according to any one of items 1 to 3, which is an antigen-binding fragment selected from any one of the following: Fv, Fab, F(ab')2, Fab', Fd, dsFv, scFv, sc(Fv)2, dAb, isolated complementarity determining region, domain-specific antibody, single domain antibody, domain-deleted antibody, CDR-grafted antibody, diabody, triabody, tetrabody and minibody.

[0021] 8. The anti-RAGE antibody according to any one of items 1 to 3, which binds to a RAGE antigen derived from humans, monkeys, and mice.

[0022] 9. A conjugate comprising the anti-RAGE antibody according to any one of items 1 to 8.

[0023] 10. A conjugate according to claim 9, comprising a therapeutic agent conjugated to the anti-RAGE antibody.

[0024] 11. According to the conjugate of item 10, the therapeutic agent is selected from one or more of cytotoxic agents, hormone preparations, targeted small molecule preparations, proteasome inhibitors, immunomodulators, angiogenesis inhibitors, cell proliferation inhibitors, pro-apoptotic agents, cytokines, and activators of co-stimulatory molecules.

[0025] 12. A bispecific or multispecific antibody comprising the anti-RAGE antibody according to any one of items 1 to 8.

[0026] 13. An immune cell comprising the anti-RAGE antibody or a nucleic acid encoding the antibody according to any one of items 1 to 8.

[0027] 14. The immune cell according to item 13, which is a NK cell, a T cell or a dendritic cell.

[0028] 15. A fusion protein comprising the anti-RAGE antibody according to any one of items 1 to 8 and one or more polypeptides or proteins.

[0029] 16. A nucleic acid molecule encoding the anti-RAGE antibody according to any one of items 1 to 8.

[0030] 17. An expression vector comprising the nucleic acid molecule described in item 16.

[0031] 18. A cell comprising the nucleic acid molecule described in item 16 or the expression vector described in item 17.

[0032] 19. Use of the anti-RAGE antibody described in any one of Items 1 to 8, the conjugate described in any one of Items 9 to 11, the bispecific or multispecific antibody described in Item 12, the immune cell described in Item 13 or 14, or the fusion protein described in Item 15 in the preparation of a drug for diagnosing, treating and / or preventing a disease.

[0033] Effects of the Invention

[0034] The anti-RAGE antibody of the present application is a human, mouse and monkey cross molecule, which can target the RAGE antigen targets of different species of humans, mice and monkeys. At the same time, the anti-RAGE antibody has a high binding activity to the RAGE antigen protein and the cell surface RAGE antigen, and has a high application value both in scientific research and clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1A The Coomassie brilliant blue staining results of three His-tagged antigens; Figure 1B The following are the results of Coomassie brilliant blue staining of three Fc-tagged antigens.

[0036] Figure 2A This is the Coomassie Brilliant Blue staining result of h11E6.16 antibody; Figure 2B The figure shows the results of Coomassie brilliant blue staining with the control antibody.

[0037] Figure 3 It is the HPLC-SEC spectrum of h11E6.16 antibody.

[0038] Figure 4A Shows the binding activity of different antigens with h11E6.16 antibody (P38411); Figure 4B~Figure 4CThe binding activity of different antigens to Anti-hRAGE (P46085) is shown.

[0039] Figure 5 This is a graph showing the results of the biotin labeling effect test of the RAGE antigen.

[0040] Fig. 6A The results of the Fc-tagged RAGE antigen binding antibody activity analysis before and after Biotin labeling; Figure 6B The results of the His-tagged RAGE antigen binding antibody activity analysis before and after Biotin labeling.

[0041] Fig. 7A Flow cytometric analysis of a high-expressing RAGE cell line constructed by transiently transfecting plasmid p318 into 293F cells; Figure 7B to Figure 7D WB detection results of the high-expression RAGE cell line constructed by transiently transfecting plasmid p318 into 293F cells; Fig. 7E Confocal microscopic photograph of a high-expressing RAGE cell line constructed by transiently transfecting plasmid p318 into 293F cells.

[0042] Fig. 8A Flow cytometric analysis of a high-expressing RAGE cell line constructed by transiently transfecting plasmid p605 into HepG2 cells; Figure 8B to Figure 8D WB detection results of the high-expressing RAGE cell line constructed by transiently transferring plasmid p605 into HepG2 cells.

[0043] Fig. 9A and Fig. 9B The flow cytometric results show the specific binding ability of each candidate phage antibody group to cells expressing human RAGE antigen.

[0044] Figure 10A to Figure 10L The figure is an Elisa result diagram showing the specific binding ability of each candidate phage antibody group to RAGE antigens of different species.

[0045] Fig.11A and Fig. 11B The figure shows the flow cytometry results of each candidate antibody binding ability on p318-293F cells.

[0046] Figure 12A to Figure 12L The results are for analysis of the binding activity of the eukaryotic expressed antibody supernatant with the antigens Human-RAGE-Fc (P38406), Cyno-RAGE-Fc (P38408), and Mouse-RAGE-Fc (P38410).

[0047] Fig.13A This is a diagram showing the SDS-PAGE electrophoresis results of the antiRAGE-Fc antibody of the present application; Fig. 13BThe figure is a flow cytometric graph showing the specific binding of the antiRAGE-Fc antibody of the present application to p605 transiently transfected HepG2 cells. DETAILED DESCRIPTION

[0048] The specific embodiments of the present application will be described in more detail below with reference to the accompanying drawings. Although the specific embodiments of the present application are shown in the accompanying drawings, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present application and to enable the scope of the present application to be fully communicated to those skilled in the art.

[0049] It should be noted that the words "include" or "comprising" mentioned throughout the specification and claims are open-ended terms and should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment of the present application, but the description is for the purpose of illustrating the general principles of the present application and is not intended to limit the scope of the present application. The scope of protection of the present application shall be determined by the definition of the attached claims.

[0050] definition

[0051] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as understood by one of ordinary skill in the art.

[0052] As used herein, the term "Receptor for advancedglycation endproducts (RAGE, or AGER)" is a multi-ligand cell surface member of the immunoglobulin superfamily. RAGE consists of an extracellular domain, a single transmembrane domain, and a cytoplasmic tail. The extracellular domain of the receptor consists of one v-type immunoglobulin domain and two c-type immunoglobulin domains. RAGE is expressed by a variety of cell types, such as endothelial and smooth muscle cells, macrophages, and lymphocytes, in many different tissues, including the lung, heart, kidney, skeletal muscle, and brain.

[0053] As used herein, the term "antibody" is a class of immunoglobulins that can specifically bind to an antigen. Such molecules typically include two heavy chains (H) and two light chains (L) that are interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The variable regions of the heavy and light chains of antibodies contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as C1q (the first component in the classical pathway of complement activation).

[0054] The heavy chain of an antibody can be divided into three functional regions: the Fd region, the hinge region, and the Fc region (crystallizable fragment). The Fd region contains the VH and CH1 domains and combines with the light chain to form a Fab (antigen binding fragment). The Fc fragment is responsible for immunoglobulin effector functions, including, for example, complement binding and binding to the cognate Fc receptors of effector cells. The hinge region found in the IgG, IgA, and IgD immunoglobulin classes acts as a flexible spacer, allowing the Fab portion to move freely in space relative to the Fc region. The hinge domain is structurally diverse, with different sequences and lengths between immunoglobulin classes and subclasses.

[0055] The "light chain variable region" (VL) and the "heavy chain variable region" (VH) both comprise the following framework regions (FR) and CDR regions from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The CDR1, CDR2, and CDR3 of the light chain variable region are also referred to herein as CDR-L1, CDR-L2, and CDR-L3, respectively; the CDR1, CDR2, and CDR3 of the heavy chain variable region are also referred to herein as CDR-H1, CDR-H2, and CDR-H3, respectively.

[0056] CDRs can be determined according to the Kabat definition, the Chothia definition, the accumulation of both the Kabat definition and the Chothia definition, the AbM definition, the contact definition, the IMGT unique numbering definition and / or the conformational definition, or any CDR determination method known in the art. In the present application, the Kabat definition is used to determine the CDR sequences.

[0057] Based on the amino acid sequence of the constant region of the heavy chain of an antibody, immunoglobulin molecules can be divided into five classes (isotypes): IgA, IgD, IgE, IgG, and IgM, and can be further divided into different subtypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc. Based on the amino acid sequence of the light chain, the light chain of an antibody can be divided into lambda (λ) chain and kappa (κ) chain.

[0058] In this article, the term "antibody" should be understood in its broadest sense, including but not limited to monoclonal antibodies, polyclonal antibodies, antigen-binding fragments, etc. Antibodies may contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations in glycosylation sites. Antibodies also include post-translationally modified antibodies, fusion proteins containing antigenic determinants of antibodies, and immunoglobulin molecules containing any other modifications to the antigen recognition site, as long as these antibodies exhibit the desired biological activity.

[0059] As used herein, the term "antigen-binding fragment" can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Examples of antigen-antibody fragments described herein include, but are not limited to: (1) an Fv fragment having the VL and VH domains of a single antibody arm; (2) a Fab fragment having VL, CL, VH and CH1 domains; (3) a Fab' fragment, i.e., a Fab fragment having one or more cysteine ​​residues at the C-terminus of the CH1 domain; (4) a F(ab')2 fragment, i.e., a bivalent fragment comprising two Fab' fragments bridged by a disulfide bond at the hinge region; (5) a Fd fragment having VH and CH1 domains; (6) a dsFv fragment, i.e., a disulfide-stabilized Fv antibody; (7) a scFv fragment consisting of a heavy chain variable region connected to a light chain variable region; (8) a sc(Fv)2 fragment, a single-chain antibody consisting of four variable regions, two light chain variable regions (VL) and two heavy chain variable regions (VH), connected by a linker; (9) a dAb fragment consisting of a VH domain; (10) Isolated complementary determining regions; (12) antibodies with specific binding domains; (13) single domain antibodies that are antibody fragments that contain only a single variable domain of the entire antibody (such as nanobodies, which are single domain antibodies with only a heavy chain variable region); (14) domain-deleted antibodies; (15) CDR-grafted antibodies, which refer to antibodies that contain heavy chain variable region and light chain variable region sequences from one species, but in which the sequences of one or more CDR regions of VH and / or VL are replaced with CDR sequences of another species, such as antibodies with mouse heavy chain variable regions and light chain variable regions, in which one or more mouse CDRs (e.g., CDR3) have been replaced with human CDR sequences; (16) diabodies, which are dimerized scFvs formed when the VH domain of a first scFv is assembled with the VL domain of a second scFv, and the VL domain of the first scFv is assembled with the VH domain of a second scFv, and the two antigen-binding domains of the diabody can be directed against the same or different epitopes; (17) Triabodies are trimeric scFvs formed in a manner similar to diabodies, but in which three antigen-binding domains are produced in a single complex; the three antigen-binding domains can be directed against the same or different epitopes; (18) tetrabodies are tetrameric scFvs formed in a manner similar to diabodies, but in which four antigen-binding domains are produced in a single complex; the four antigen-binding domains can be directed against the same or different epitopes; (19) minibodies are scFvs fused to a CH3 domain (see Olafsen et al., Protein Eng Des Sel., Vol. 17: 315-23, 2004).

[0060] Herein, the term "bispecific antibody" refers to an artificial antibody that has fragments derived from two different monoclonal antibodies and is capable of binding to two different epitopes. The two epitopes may be present on the same antigen, or they may be present on two different antigens.

[0061] As used herein, the term "multispecific antibody" refers to an antibody that specifically binds to at least two different antigens or at least two different epitopes of the same antigen. A multispecific antibody can bind to, for example, two, three, four, five or more different antigens, or can bind to two, three, four, five or more different epitopes of the same antigen.

[0062] As used herein, the term "monoclonal antibody (mAb)" or "mAb" or "Mab" refers to a homogeneous antibody population, i.e., the individual antibodies constituting the population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in small amounts. Monoclonal antibodies are highly specific, directed against a single antigen or epitope. "Monoclonal" indicates that the antibody is a feature obtained from a substantially homogeneous antibody population and should not be construed as limiting the structure, source, or preparation of the antibody. In some embodiments, monoclonal antibodies are prepared by hybridoma methods, phage display methods, yeast display methods, recombinant DNA methods, single cell screening, or single cell sequencing methods.

[0063] In this article, the term "human antibody" is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and / or has been prepared using any technology for preparing human antibodies. This definition of human antibodies specifically excludes humanized antibodies comprising non-human antigen binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries (Hoogenboom and Winter, 1991, J.Mol.Biol.227:381; Marks et al., 1991, J.Mol.Biol.222:581) and yeast display libraries (Chao et al., 2006, Nature Protocols 1:755-68). Methods for preparing human monoclonal antibodies are also described in Cole et al., Monoclonal Antibodies and Cancer Therapy 77 (1985); Boerner et al., 1991, J. Immunol. 147 (1): 86-95; and van Dijk and van de Winkel, 2001, Curr. Opin. Pharmacol. 5: 368-74. Human antibodies can be prepared by administering an antigen to a transgenic animal (e.g., a mouse) that has been modified to produce such antibodies in response to antigenic challenge but whose endogenous loci have been disabled (see, e.g., Jakobovits, 1995, Curr. Opin. Biotechnol. 6 (5): 561-66; Brüggemann and Taussing, 1997, Curr. Opin. Biotechnol. 8 (4): 455-58, and the methods involving the XENOMOUSE TM For human antibodies generated by human B cell hybridoma technology, see also, for example, Li et al., 2006, Proc. Natl. Acad. Sci. USA 103:3557-62.

[0064] As used herein, the term "chimeric antibody" refers to an antibody that is a combination of antibody fragments from different species. Specifically, for example, a monoclonal antibody from one species (e.g., mouse) has its Fc constant region replaced with an Fc constant region from another species (e.g., human) via DNA recombination technology. See, for example, patent applications PCT / US86 / 02269; EP / 173,494.

[0065] In this article, the term "humanized antibody" refers to an antibody comprising a human immunoglobulin framework region and one or more CDRs from a non-human (e.g., mouse, rat, rabbit or synthetic) immunoglobulin. Except for the CDRs, all other parts in the humanized antibody are substantially identical to the corresponding parts of the natural human immunoglobulin sequence. The method for constructing humanized antibodies by genetic engineering is described in, for example, patent application US / 5,585,089.

[0066] In this article, the term "specific binding" refers to the property of high-affinity complementary binding determined by the spatial conformation of the antigenic determinant and the variable region of the antibody molecule. This high affinity determines that once the antibody molecule binds to the antigen, it can exert its corresponding physiological function, for example, in some embodiments of the present application, the antibody binds to and helps to clear the antigen.

[0067] In this article, the term "human universal framework" is a framework representing the amino acid residues most commonly present in the selection of human immunoglobulin VL or VH framework sequences. In general, human immunoglobulin VL or VH sequences are selected from a subgroup of variable domain sequences. In general, sequence subgroups are such as Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication 91-3242, Bethesda MD (1991), subgroups in Volumes 1-3. In some embodiments, for VL, the subgroup is a subgroup such as Kabat et al. (supra) described. In some embodiments, for VH, the subgroup is a subgroup III such as Kabat et al. (supra) described.

[0068] As used herein, the term "homology" or "identity" refers to the percentage of amino acid residues or nucleotide residues that are identical between two sequences. If the two sequences to be compared are of different lengths, the sequence "homology" or "identity" preferably refers to the percentage of nucleotide residues in the shorter sequence that are identical to the amino acid residues or nucleotide residues in the longer sequence. Sequence identity can be routinely determined using sequence analysis software commonly used in the art, such as the Wisconsin Sequence Analysis Package.

[0069] As used herein, the term "conjugate" (also referred to as "ADC") refers to a conjugate formed by covalently coupling a therapeutic agent to an antibody directly or indirectly via one or more appropriate linkers. ADCs are typically in the form of antibody-linker-therapeutic agent conjugates. Antibody-therapeutic agent conjugates combine the ideal properties of both antibodies and therapeutic agents (cytotoxic molecules or substances with other properties), thereby enhancing their anti-tumor (or other pharmaceutical) activity by targeting powerful therapeutic agents (cytotoxic molecules or substances with other properties) to tumor cells (or other cells / organs) that express antigens. ADCs are designed to distinguish between healthy cells and diseased tissues, such as tumor cells in tumors.

[0070] In this article, the term "therapeutic agent" refers to any cytotoxic molecule having, for example, an anti-tumor effect, an anti-infective or anti-inflammatory effect and having at least one substituent group or a partial structure that allows attachment to a linker structure. Therapeutic agents can kill cells (e.g., cancer cells) and / or inhibit the growth, proliferation or metastasis of cells (e.g., cancer cells), thereby reducing, alleviating or eliminating one or more symptoms of a disease or disorder (e.g., cancer).

[0071] In this article, the term "vector" generally refers to a nucleic acid molecule that can self-replicate in a suitable host, which transfers the inserted nucleic acid molecule into and / or between host cells. The vector may include a vector that is mainly used to insert DNA or RNA into a cell, a vector that is mainly used to replicate DNA or RNA, and a vector that is mainly used to express the transcription and / or translation of DNA or RNA. The vector also includes a vector with a variety of the above functions. The vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector can produce a desired expression product by cultivating a suitable host cell that contains the vector.

[0072] In this article, the term "nucleic acid" or "polynucleotide" or "nucleic acid molecule" generally refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless otherwise specified, the term may include nucleic acids containing analogs of natural nucleotides, which have similar binding properties to reference nucleic acids (e.g., sequence information is shown) and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, the sequence of a nucleic acid may include variants modified in a conservative manner, such as degenerate codon substitutions, alleles, orthologs, SNPs, and complementary sequences, as well as sequences explicitly indicated.

[0073] As used herein, the term "treatment" refers to any intervention that results in any observable beneficial effect of treatment, or any statistically significant indicator of success in treating or ameliorating a disease or condition, such as improving the signs, symptoms, or progression of a disease or pathological condition. For example, a beneficial effect may be demonstrated by reducing the disease in a subject, delaying the onset of the disease, or reducing the severity of clinical symptoms of the disease, reducing the frequency with which a subject experiences symptoms of the disease, slowing the progression of the disease, reducing the number of relapses of the disease, improving the overall health of the subject, or by other parameters specific to a particular disease.

[0074] As used herein, the term "prevention" refers to treatment administered to a subject who does not show signs of a disease or who shows only early signs of the disease, with the goal of reducing the risk of developing the pathology or further progression of the early disease.

[0075] Anti-RAGE antibodies

[0076] The present application provides an anti-RAGE antibody, which comprises three heavy chain complementary determining regions and three light chain complementary determining regions, wherein the three heavy chain complementary determining regions are CDR-H1, CDR-H2 and CDR-H3, and the three light chain complementary determining regions are CDR-L1, CDR-L2 and CDR-L3, wherein:

[0077] The amino acid sequence of the CDR-H1 is shown in SEQ ID NO: 1 (SSYAIS);

[0078] The amino acid sequence of the CDR-H2 is shown in SEQ ID NO: 2 (RIIPILGIAN);

[0079] The amino acid sequence of the CDR-H3 is shown in SEQ ID NO: 3 (QVGAIDY);

[0080] The amino acid sequence of the CDR-L1 is shown in SEQ ID NO: 4 (QSISTYVS);

[0081] The amino acid sequence of the CDR-L2 is shown in SEQ ID NO: 5 (IYYASILKS); and

[0082] The amino acid sequence of the CDR-L3 is shown in SEQ ID NO: 6 (QSYSFPLT).

[0083] In some embodiments, the anti-RAGE antibody comprises a human universal framework region. In some embodiments, the VH comprises a human subgroup III universal framework region (FR). In some embodiments, the VL comprises a human subgroup universal framework. In some embodiments, comprises a human universal framework region (FR) and one or more amino acid substitutions based on a human universal framework region (FR).

[0084] In some embodiments, the framework region is a universal human framework region and comprises one or more (e.g., 1-20, 1-15, 1-10, 1-5, 1-4, 1-3) amino acid substitutions, deletions or insertions. In some embodiments, the framework region is a universal human framework region or has 70%, 80%, 90%, 95%, 97%, 98%, 99% identity with a universal human framework region.

[0085] In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0086] the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:7 (QVQLVQSWAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCASQVGAIDYWGQGTPVTVSS), or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:7;

[0087] The amino acid sequence of the light chain variable region is as shown in SEQ ID NO:8 (NIQLTQSPSSLSASLGDRVTITCRTSQSISTYVSWYQQRSGEAPNLLIYYASILKSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSFPLTFGGGTKVEIK), or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:8.

[0088] In some embodiments, the anti-RAGE antibody is a human antibody, a humanized antibody, or a chimeric antibody.

[0089] In some embodiments, the anti-RAGE antibody is a full-length IgG antibody, preferably a full-length IgG1 antibody.

[0090] In some embodiments, the anti-RAGE antibody is an antigen binding fragment selected from any one of the following: Fv, Fab, F(ab')2, Fab', Fd, dsFv, scFv, sc(Fv)2, dAb, VH-CH1-CH2-CH3, VH-CH1, scFv-Fc, isolated complementarity determining regions, domain-specific antibodies, single domain antibodies, domain-deleted antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies and minibodies.

[0091] In some embodiments, the anti-RAGE antibody comprises a heavy chain and a light chain, wherein the heavy chain configuration is VH-CH (IgG1) and the light chain configuration is VL-CL (Kappa).

[0092] In the above embodiment, the VH sequence is as shown in SEQ ID NO: 7, the CH (IgG1) sequence is as shown in SEQ ID NO: 9 (ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK); the CL (Kappa) sequence is as shown in SEQ ID NO: 10 The VL sequence is shown in SEQ ID NO: 8.

[0093] In some embodiments, the anti-RAGE antibody binds to a RAGE antigen of human, monkey or mouse origin, and the anti-RAGE antibody is a human, mouse and monkey cross molecule, which has wider practicality both in scientific research and clinical practice. In some embodiments, the mouse is a rat or a mouse. In some embodiments, the monkey includes but is not limited to macaques.

[0094] Conjugate

[0095] The present application also provides a conjugate comprising any of the aforementioned anti-RAGE antibodies.

[0096] In some embodiments, the conjugate further comprises a therapeutic agent coupled to the anti-RAGE antibody. In some embodiments, the therapeutic agent is selected from one or more of a cytotoxic agent, a hormone preparation, a targeted small molecule preparation, a proteasome inhibitor, an immunomodulator, an angiogenesis inhibitor, a cell proliferation inhibitor, a pro-apoptotic agent, a cytokine, and an activator of a co-stimulatory molecule.

[0097] Examples of cytotoxic agents include, but are not limited to, anthracyclines, auristatins, CC-1065, dolastatin, duocarmycin, enediynes, geldanamycin, maytansine, puromycin, taxanes, vinca alkaloids, SN-38, tubulysin, hemiasterlin, eribulin, trabectedin, lubinectedin, and stereoisomers, analogs, or derivatives thereof.

[0098] Examples of hormone preparations include, but are not limited to, tamoxifen, onapristone, raloxifene (Evista), 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, flutamide and nilutamide; and pharmaceutically acceptable salts, acids or derivatives thereof.

[0099] Examples of cytokines include, but are not limited to, growth hormones, such as human growth hormone, N-methylsulfonyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones (such as follicle stimulating hormone (FSH); thyrotropin (TSH) and luteinizing hormone (LH)); hepatocyte growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor α and β; Miller inhibitor; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factor (such as NGF-β); platelet growth factor; transforming growth factor (TGF) (such as TGF-α and TGF-β); pancreatic Insulin-like growth factor I and II; erythropoietin (EPO); osteoinductive factor; interferon (such as interferon α; β and γ); colony stimulating factor (CSF) (such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF) and granulocyte-CSF (G-CSF)); interleukin (IL) (such as IL-1; IL-1α; IL-2; IL-3; IL-4; IL-5; IL-6; IL-7; IL-8; IL-9; IL-11; IL-12); tumor necrosis factor such as TNF-α or TNF-β; and other polypeptide factors (including LIF and box ligand (KL, kit ligand)). The term cytokine as used herein includes proteins from natural or recombinant cell culture and biologically active equivalents of natural sequence cytokines.

[0100] Examples of targeted small molecule agents include, but are not limited to, erlotinib, gefitinib, osimertinib, afatinib, icotinib, dacomitinib, sorafenib, imatinib, sunitinib.

[0101] Examples of proteasome inhibitors include, but are not limited to, bortezomib, ixazomib, carfilzomib, oprozomib, dilanzomib, marizomib, MG132.

[0102] Examples of immunomodulators include, but are not limited to, thymosin, levamisole, Toll-like receptor modulators, STING modulators.

[0103] Examples of angiogenesis inhibitors include, but are not limited to, bevacizumab, apatinib, anlotinib, sorafenib, cetuximab.

[0104] A cell proliferation inhibitor refers to an agent that has a cytostatic effect on cells, thereby inhibiting the growth and / or expansion of a specific subset of cells.

[0105] Bispecific or multispecific antibodies, antibodies modified with lipophilic groups, immune cells

[0106] The present application also provides a bispecific or multispecific antibody comprising any of the aforementioned anti-RAGE antibodies.

[0107] The bispecific antibody of the present application comprises an anti-RAGE antibody or an antigen-binding fragment thereof, and another antibody or a fragment thereof, or an antibody analog.

[0108] In some embodiments, the multispecific antibody is a trispecific antibody or a tetraspecific antibody.

[0109] The multispecific antibody of the present application comprises an anti-RAGE antibody or an antigen-binding fragment thereof, and two or three additional antibodies or fragments thereof, or antibody analogs.

[0110] The antibody belonging to the bispecific antibody or multispecific antibody can be an antibody based on scFv, an antibody based on Fab or an antibody based on IgG. Bispecific antibodies or multispecific antibodies can inhibit or amplify two or more signals at the same time, and are therefore more effective than single antibodies that inhibit / amplify one signal. Compared with treating each signal with each signal inhibitor, it can be administered at a low dose, and can inhibit / amplify two or more signals at the same time and space.

[0111] Methods for producing bispecific or multispecific antibodies are well known. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two or more immunoglobulin heavy chain / light chain pairs under conditions in which the two or more heavy chains have different specificities.

[0112] The present application also provides an immune cell comprising any of the aforementioned anti-RAGE antibodies or nucleic acids encoding them.

[0113] In some embodiments, the immune cells may be selected from one or more of T cells, NK cells, cytokine-induced killer cells (CIK), activated cytotoxic T lymphocytes (CTL), macrophages, tumor infiltrating lymphocytes (TIL), and dendritic cells.

[0114] Fusion Protein

[0115] The present application also provides a fusion protein comprising any of the aforementioned anti-RAGE antibodies and one or more polypeptides or proteins.

[0116] In some embodiments, the polypeptide or protein is a scaffold protein or a truncation or variant thereof, an antibody, or a cytokine.

[0117] Herein, "truncated form of scaffold protein" refers to any fragment of the original sequence of scaffold protein.

[0118] In this article, "variant of scaffold protein" refers to a protein that is distinguished from the original scaffold protein by the addition, deletion or substitution of at least one amino acid residue. In some embodiments, the variant of the scaffold protein is distinguished from the original scaffold protein by one or more substitutions that may be conservative or non-conservative. In some embodiments, the variant of the scaffold protein comprises conservative substitutions. Variants of scaffold proteins also include scaffold proteins in which one or more amino acids are added or deleted, or replaced by other amino acid residues.

[0119] In some embodiments, the antibody is selected from any one or more of Fv, Fab, F(ab')2, Fab', Fd, dsFv, scFv, sc(Fv)2, dAb, isolated complementarity determining region, domain-specific antibody, single domain antibody, domain-deleted antibody, CDR-grafted antibody, diabody, triabody, tetrabody and minibody.

[0120] Nucleic acid molecules, expression vectors, cells

[0121] The present application also provides a nucleic acid molecule encoding any of the aforementioned anti-RAGE antibodies.

[0122] In some embodiments, the sequence of the nucleic acid molecule encoding the heavy chain of the anti-RAGE antibody is as shown in SEQ ID NO: 11, or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% similarity to SEQ ID NO: 11; the sequence of the nucleic acid molecule encoding the light chain of the anti-RAGE antibody is as shown in SEQ ID NO: 12, or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% similarity to SEQ ID NO: 12.

[0123] Wherein, SEQ ID NO: 11 is:

[0124] CAAGTGCAGCTGGTCCAGAGCTGGGCCGAGGTGAAGAAGCCAGGATCTAGCGTGAAGGTGTCTTGTAAAGCCTCTGGCGGCACATTCAGCTCCTACGCCATCAGCTGGGTGCGGCAGGCCCCTGGCCAGGGCCTGGAATGGATGGGCAGAATCATCCCCATCCTGGGCATCGCC AACTACGCCCAGAAGTTCCAGGGAAGAGTGACCATCACCGCTGATAAGAGCACCAGCACAGCTTATATGGAACTGAGCAGCCTGAGAAGCGAGGACACCGCCGTGTACTACTGCGCCAGCCAGGTTGGAGCCATTGACTACTGGGGCCAGGGCACCCCTGTGACAGTGTCCTCC

[0125] SEQ ID NO: 12 is:

[0126] AACATCCAGCTGACCCAGTCTCCATCTAGCCTGAGCGCTTCTCTGGGCGACCGGGTGACCATCACCTGTAGAACCAGCCAGTCCATCTCAACATACGTGTCCTGGTATCAGCAAAGAAGCGGCGAGGCCCCTAACCTGCTGATCTACTACGCCAGCATCC TGAAAAGCGGCGTCCCTAGCAGATTCAGCGGCAGCGGATCTGGCACAGATTTTACCCTGACAATTAGCAGCCTCCAGCCTGAGGACTTCGCCACCTACTACTGCCAGCAGTCCTACAGCTTCCCCCTGACCTTCGGCGGCGGAACAAAGGTGGAAATCAAG

[0127] The present application also provides an expression vector comprising any of the aforementioned nucleic acid molecules.

[0128] In some embodiments, the expression vector can be any type of plasmid, which refers to a circular double-stranded DNA loop into which additional DNA segments can be inserted, for example, by standard molecular cloning techniques.

[0129] The present application also provides a cell comprising any one of the aforementioned nucleic acid molecules or expression vectors.

[0130] The antibodies of the present application can be displayed on any delivery vector.

[0131] In some embodiments, other drug molecules are loaded into the delivery vector of the present application.

[0132] Pharmaceutical uses and methods for diagnosing, treating and / or preventing disease

[0133] The present application also provides a use of any of the aforementioned anti-RAGE antibodies, conjugates, bispecific or multispecific antibodies, immune cells or fusion proteins in the preparation of drugs for diagnosing, treating and / or preventing diseases.

[0134] For example, the disease is an immune disease or cancer, and the cancer may be lung cancer.

[0135] Example

[0136] Example 1 Construction of RAGE antigen protein and antigen cells

[0137] 1.1 Synthesize RAGE antigen proteins from human, monkey and mouse for antibody screening.

[0138] Experimental methods:

[0139] (1) Six RAGE antigen protein extracellular domains (ECD) from different sources and with different tags were expressed and synthesized. The antigen design information and expression synthesis results are shown in Table 1 below. The amino acid sequence of Human-RAGE-ECD-Fc (IgG1) is shown in SEQ ID NO: 13 ().The amino acid sequence of Cyno-RAGE-ECD-Fc(IgG1) is as shown in SEQ ID NO: 14 (AQNITARIGEPLVLKCKGAPKKPPQQLEWKLNTGRTEAWKVLSPQGGPWDSVARVLPNGSLFLPAVGIQDEGIFRCQAMNRNGKETKSNYRVRVYQIPGKPEIIDSASELTAGVPNKVGTCVSEGSYPAGTLSWHLDGKPLVPNEKGVSVKEETRRHPETGLFTLQSELMVTPARGGNPHPTFSCSFSPGLPRRRALHTAPIQPRVWEPVPLEEVQLVVEPEGGAVAPGGTVTLTCEVPAQPSPQIHWMKDGVPLPLSPSPVLILPEIGPQDQGTYRCVATHPSHGPQESRAVSISIIEPGEEGPTAGSVGGSGPGTLAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK),The amino acid sequence of Mouse-RAGE-ECD-Fc(IgG1) is shown in SEQ ID NO: 15 (GQNITARIGEPLVLSCKGAPKKPPQQLEWKLNTGRTEAWKVLSPQGGPWDSVARILPNGSLLLPATGIVDEGTFRCRATNRRGKEVKSNYRVRVYQIPGKPEIVDPASELTASVPNKVGTCVSEGSYPAGTLSWHLDGKLLIPDGKETLVKEETRRHPETGLFTLRSELTVIPTQGGTHPTFSCSFSLGLPRRRPLNTAPIQLRVREPGPPEGIQLLVEPEGGIVAPGGTVTLTCAISAQPPPQVHWIKDGAPLPLAPSPVLLLPEVGHEDEGTYSCVATHPSHGPQESPPVSIRVTETGDEGPAEGSVGESGLGTLAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK), and the amino acid sequence of Human-RAGE-ECD-His is shown in SEQ ID NO: 16 (AQNITARIGEPLVLKCKGAPKKPPQRLEWKLNTGRTEAWKVLSPQGGGPWDSVARVLPNGSLFLPAVGIQDEGIFRCQAMNRNGKETKSNYRVRVYQIPGKPEIVDSASELTAGVPNKVGTCVSEGSYPAGTLSWHLDGKPLVPNEKGVSVKEQTRRHPETGLFTLQSELMVTPARGGDPRPTFSCSFSPGLPRHRALRTAPIQPRVWEPVPLEEVQLVVEPEGGAVAPGGTVTLTCEVPAQPSPQIHWMKDGVPLPLPPSPVLILPEIGPQDQGTYSCVATHSSHGPQESRAVSISIIEPGEEGPTAGSVGGSGLGTLAGGSGGSHHHHHHHH).The amino acid sequence of Cyno-RAGE-ECD-His is shown in SEQ ID NO: 17 (AQNITARIGEPLVLKCKGAPKKPPQQLEWKLNTGRTEAWKVLSPQGGPWDSVARVLPNGSLFLPAVGIQDEGIFRCQAMNRNGKETKSNYRVRVYQIPGKPEIIDSASELTAGVPNKVGTCVSEGSYPAGTLSWHLDGKPLVPNEKGVSVKEETRRHPETGLFTLQSELMVTPARGGNPHPTFSCSFSPGLPRRRALHTAPIQPRVWEPVPLEEVQLVVEPEGGAVAPGGTVTLTCEVPAQPSPQIHWMKDGVPLPLSPSPVLILPEIGPQDQGTYRCVATHPSHGPQESRAVSISIIEPGEEGPTAGSVGGSGPGTLAGGSGGSHHHHHHHH). The amino acid sequence of Mouse-RAGE-ECD-His is shown in SEQ ID NO: 18 NO:18(GQNITARIGEPLVLSCKGAPKKPPQQLEWKLNTGRTEAWKVLSPQGGPWDSVARILPNGSLLLPATGIVDEGTFRCRATNRRGKEVKSNYRVRVYQIPGKPEIVDPASELTASVPNKVGTCVSEGSYPAGTLSWHLDGKLLIPDGKETLVKEETRRHPETGLFTL RSELTVIPTQGGTHPTFSCSFSLGLPRRRPLNTAPIQLRVREPGPPEGIQLLVEPEGGIVAPGGTVTLTCAISAQPPPQVHWIKDGAPLPLAPSPVLLLPEVGHEDEGTYSCVATHPSHGPQESPPVSIRVTETGDEGPAEGSVGESGLGTLAGGSGGSHHHHHHHH). ,

[0140] Table 1

[0141]

[0142] (2) The six RAGE antigens were subjected to SDS-PAGE-Coomassie Brilliant Blue staining. Figure 1A and 1B As shown, the purity is estimated by the grayscale of the strips, and the results are shown in Tables 2 and 3:

[0143] Table 2

[0144]

[0145] Table 3

[0146]

[0147] Experimental results: All six RAGE antigens were successfully expressed, and the purity of all of them was greater than 70% in reducing SDS-PAGE.

[0148] 1.2 Anti-RAGE antibody expression detection is used to evaluate antigen activity as a control for antibody screening.

[0149] Experimental methods:

[0150] (1) Two control anti-RAGE antibodies were expressed and synthesized. The relevant information and expression and synthesis results are shown in Table 4 below. The antibody h11E6.16 can be synthesized according to the method in Patent ES2577718T3. The amino acid sequence of the heavy chain of the antibody h11E6.16 is shown in SEQ ID NO: 19 (EIQLVQSGAEVKKPGASVKVSCKASGYTFTNFGMNWVRQAPGQGLEWMGYINTNTGESIYSEEFKGRFTFTLDTSTSTAYMELSSLRSEDTAVYFCARSRMVTAYGMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK), the light chain is as SEQ ID NO:20(EIVMTQSPATLSLSPGERATLSCKASQNVGTAVAWYQQKPGQSPRLLIFSASNRYTGVPARFSGSGSGTDFTLTISSLQSEDFAVYFCQQYSSYPLTFGQGTKLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC).

[0151] Table 4

[0152]

[0153] (2) SDS-PAGE-Coomassie Brilliant Blue staining was performed on two control anti-RAGE antibodies. The experimental results are shown in Figure 2A and 2B As shown, the purity is estimated by the grayscale of the strips, and the results are shown in Tables 5 and 6:

[0154] Table 5

[0155]

[0156] Table 6

[0157]

[0158] (3) HPLC-SEC detection of h11E6.16 antibody, the results are shown in Table 7 and Figure 3 shown.

[0159] Table 7

[0160]

[0161] Experimental results: h11E6.16 antibody was successfully expressed, with a monomer rate of 100%.

[0162] 1.3 Detect the binding activity of different antigens with h11E6.16 antibody (P38411).

[0163] Experimental methods:

[0164] (1) Different antigens (the six RAGE antigens synthesized in Example 1, Ipilimumab (Sanyou Bio / CHA032) (negative control), and 1% skim milk (blank control)) were coated on Elisa plates. The antigens were diluted with PBS to a working concentration of 2 μg / mL, 30 μL was added to each well, and incubated at 4°C overnight.

[0165] (2) Wash three times with PBST (PBS plus 0.05% TWEEN-20) and block with 5% skim milk (prepared with PBS) for 2 h at room temperature.

[0166] (3) Wash three times with PBST, add 30 µL of h11E6.16(P38411) diluted with 1% skim milk (prepared with PBS) to each well, and incubate at room temperature for 60 min.

[0167] (4) Wash three times with PBST, add Goat-Anti-human-κ+λ-HRP (Millipore / AP502P, AP506P) diluted with 1% skim milk (dilution ratio 1:4000), and incubate at room temperature for 50 min.

[0168] (5) Add tetramethylbenzidine (TMB, HRP chromogenic substrate), stop the reaction with 2M HRP reaction stop solution, and read the OD450. The result is as follows: Figure 4A shown.

[0169] Experimental results: h11E6.16 antibody (P38411) has better binding ability with human and monkey antigens, but weaker binding ability with mouse antigens.

[0170] 1.4 Detect the binding activity of different antigens with Anti-hRAGE (P46085) (Cat. No.: R&D systems / MAB11451).

[0171] Experimental methods:

[0172] (1) Elisa plates were coated with different antigens. The antigen was diluted with PBS to a working concentration of 4 μg / mL. 30 μL was added to each well and incubated at 4°C overnight.

[0173] (2) Wash three times with PBST and block with 5% skim milk (prepared with PBS) at room temperature for 2 h.

[0174] (3) Wash three times with PBST, add 30 µL of Anti-hRAGE (P46085) diluted in 1% skim milk (prepared with PBS) to each well, and incubate at room temperature for 60 min.

[0175] (4) Wash three times with PBST, add Goat-Anti-mouse-IgG (1+2a+2b+3)-HRP (Jackson / 115-035-164) diluted with 1% skim milk (dilution ratio 1:4000), and incubate at room temperature for 50 min.

[0176] (5) Add TMB, stop the reaction with stop solution, and read the OD450. Figure 4B and Figure 4C shown.

[0177] Experimental results: Anti-hRAGE (P46085) has a strong binding ability to human antigens, but a very weak binding ability to monkey and mouse antigens.

[0178] 1.5 Construction of biotin-labeled RAGE antigen.

[0179] Experimental methods:

[0180] (1) Antigen protein was labeled with biotin N-hydroxysulfosuccinimide (NHS) ester (Thermo Scientific / 20217).

[0181] (2) Dilute the biotin-labeled antigen with PBS and coat the Elisa plate. Add 30 μL to each well and incubate at 4°C overnight.

[0182] (3) Wash three times with PBST and block with 5% skim milk (prepared with PBS) at room temperature for 2 h.

[0183] (4) Wash three times with PBST, add NeutrAvidin-HRP (ThermoFisher / 31001) diluted with 1% skim milk (dilution ratio 1:2000), and incubate at room temperature for 60 min.

[0184] (5) Wash 12 times with PBST.

[0185] (6) Add 30 μL TMB to each well for color development, then add stop solution to stop the reaction and read the OD450. The result is as follows: Figure 5 shown.

[0186] Experimental results: All antigen proteins were successfully labeled with biotin.

[0187] 1.6 Verify that Biotin labeling does not affect the antigen-binding antibody activity.

[0188] Experimental methods:

[0189] (1) Elisa plates were coated with the above biotin N-hydroxysulfosuccinimide (NHS) ester-labeled antigens. The antigens were diluted with PBS to a working concentration of 4 μg / mL. 30 μL was added to each well and incubated at 4°C overnight.

[0190] (2) Wash three times with PBST and block with 5% skim milk (prepared with PBS) at room temperature for 2 h.

[0191] (3) Wash three times with PBST, add 30 µL of h11E6.16(P38411) diluted with 1% skim milk (prepared with PBS) to each well, and incubate at room temperature for 60 min.

[0192] (4) Wash three times with PBST, add Goat-Anti-human-κ+λ-HRP (Millipore / AP502P, AP506P) diluted with 1% skim milk (1:4000) and incubate at room temperature for 50 min. Alternatively, add Goat-Anti-human-IgG-Fc-HRP (abcam, ab97225) diluted with 1% skim milk (1:8000) and incubate at room temperature for 50 min.

[0193] (5) Add TMB, stop the reaction with stop solution, and read the OD450. Fig. 6A and 6B shown.

[0194] Experimental results: Before and after Biotin labeling, the RAGE antigen binding antibodies from different sources had comparable activities.

[0195] 1.7 Plasmids were stably transformed into 293F suspension cells to construct a cell line that highly expresses RAGE antigen protein.

[0196] Experimental methods:

[0197] (1) Construction of plasmid p318 (RAGE(1-404)-3xflag-mCherry), the sequence of which is shown in SEQ ID NO: 21 ().

[0198] (2) Use a chemical transfection reagent to transiently transfect the plasmid into 293F suspension cells and collect the cells for later use.

[0199] (3) Take the cells collected in (2) and detect mCherry fluorescence using Attune™ NxT flow cytometer (Thermo Fisher, A24860). The experimental results are as follows: Fig. 7A The experimental results showed that p318 transiently transfected 293F cells highly expressed RAGE-3xflag-mCherry.

[0200] (4) The cells collected in (2) were lysed and the coupled mCherry fluorescent protein was detected by Western blotting (WB). The experimental results are shown in Figure 7B~7D The experimental results showed that p318 transiently transfected 293F cells highly expressed RAGE-3xflag-mCherry.

[0201] (5) Add 0.1 mg / mL poly-lysine (PDL) to the confocal culture dish and place it in an incubator (37°C 5% CO2) for 30 min. Then wash the dish three times with ultrapure water, add the cells collected in (2), and culture them in an incubator overnight to allow the cells to adhere to the wall. Add 4% PFA to the dish to fix the cells, wash them with PBS, and then add 0.5 μg / mL DAPI stain to stain at room temperature for 30 min. Wash them with PBS, add 70% glycerol (prepared with PBS), and transfer the dish to a confocal microscope for observation and photography. The photos are shown in Figure 2. Fig. 7E The experimental results showed that p318 transiently transfected 293F cells highly expressed RAGE-3xflag-mCherry (scale bar: 100 μm).

[0202] 1.8 Plasmid transiently transfected HepG2 adherent cells to construct a cell line that highly expresses RAGE antigen.

[0203] Experimental methods:

[0204] (1) Construction of plasmid p605 (RAGE(1-404)-3xflag-eBFP), the sequence of which is shown in SEQ ID NO: 22 ().

[0205] (2) Use a chemical transfection reagent to transiently transfect the plasmid into HepG2 cells and collect the cells for later use.

[0206] (3) Take the cells collected in (2) and detect the eBFP fluorescence by flow cytometry. The experimental results are as follows: Fig. 8A The experimental results showed that p605 transiently transfected HepG2 cells highly expressed RAGE-3xflag-eBFP.

[0207] (4) The cells collected in (2) were lysed and the coupled flag tag was detected by WB. The experimental results are as follows: Figures 8B to 8DThe experimental results showed that p605 transiently transfected HepG2 cells highly expressed RAGE-3xflag-eBFP.

[0208] Example 2 Antibody library screening

[0209] 2.1 Phage antibody library screening

[0210] Experimental methods:

[0211] Three rounds of screening were performed on antigen proteins coupled with different labels from different sources and phage antibody libraries (as shown in Tables 8 and 9 below) to screen out human, mouse and monkey cross-antibodies that can specifically bind and have good binding to antigen-expressing cells.

[0212] Table 8

[0213]

[0214] Table 9

[0215]

[0216] 2.2 Detection of the specific binding ability of candidate phage antibody pools to human RAGE antigen-expressing cells

[0217] Experimental methods:

[0218] (1) According to the screening strategy in section “2.1 Phage antibody library screening”, the RAGE antigen high-expressing cells p318-293F and 293F cells (blank cells) constructed in section “1.7 Plasmid stably transformed 293F suspension cells to construct a cell line that highly expresses RAGE antigen protein” were plated at 1E5 cells / well and the cells were washed with FACS buffer.

[0219] (2) Add 100 μL of antibody dilution to the cells. Incubate at 4°C for 60 minutes. Centrifuge and wash the cells twice with FACS buffer.

[0220] (3) Add 100 μL of M13 (Sino Biological 11973-MM05T-P 1:500) or Anti-human Fc (PE) (abcam ab98596 1:300) or anti-Mouse Fc (abcam 98742 1:300) to the cells and incubate at 4°C for 30 min.

[0221] (4) Resuspend the cells in FACS buffer and perform flow cytometry detection to show the cell average fluorescence intensity results of the specific binding ability of each candidate phage antibody group to humanRAGE antigen-expressing cells p318-293F. Fig. 9A and Fig. 9B The results of the average fluorescence intensity of cells showing the specific binding ability of each candidate phage antibody group to 293F cells are shown in Tables 10 and 11, wherein PC represents positive bacteria infected with phages, and NC represents negative bacteria without the presence of phages.

[0222] Table 10

[0223]

[0224] Table 11

[0225]

[0226] Experimental results analysis:

[0227] (1) By Fig. 9A and Fig. 9B It can be seen that phage library 5(0802-43(2nd)), 6(0802-44(2nd)), 7(0802-45(2nd)), 9(0802-47(2nd)), 10(0802-48(2nd)), 11(0802-49(2nd)), 13(0802-51(2nd)), 14(0802-52(2nd)), 17(0802-55(2nd)), 18(0802-56(2nd)) (0807-43(3rd)), 22(0807-44(3rd)), 23(0807-45(3rd)), 24(0807-46(3rd)), 25(0807-47(3rd)), 26(0807-48(3rd)), 27(0807-49(3rd)), 28(0807-50(3rd)), 29(0807-51(3rd)), 30(0807-52(3rd)), 31(0807-53(3rd)), 33(0807-55(3rd)), 34(0807-56(3rd)), 35(0807-57(3rd)), 36(0807-58(3rd)) had better binding on p318-293F cells.

[0228] (2) As shown in Tables 10 and 11, all phages did not bind to 293F cells.

[0229] 2.3 Detect the specific binding ability of candidate phage antibody groups (pools) to RAGE antigens of different species and screen human, mouse and monkey cross-pools.

[0230] Experimental method (see Sections 1.3 and 1.4 of Example 1 for methods):

[0231] (1) Different antigens Human-RAGE-ECD-His (P38405), Cyno-RAGE-ECD-His (P38407), Mouse-RAGE-ECD-His (P38409) and PBS (blank control) were coated on Elisa plates, respectively. Antigens were diluted with PBS to a working concentration of 4 μg / mL, 30 μL was added to each well, and incubated at 4°C overnight.

[0232] (2) Wash three times with PBST and block with 5% skim milk (prepared with PBS) at room temperature for 2 h.

[0233] (3) Wash three times with PBST, add phage antibody pool or control phage antibody (H11E6.16) and blank control (NC), 30 µL per well, and incubate at room temperature for 60 min.

[0234] (4) Wash three times with PBST, add Mouse Anti-M13 (Sinobiological / 11973-MM05T-H) diluted in 1% skim milk (1:8000), and incubate at room temperature for 50 min.

[0235] (5) Add TMB, stop the reaction with stop solution, and read the OD450. The experimental results are as follows: Figure 10A to Figure 10L shown.

[0236] The experimental results show that phage antibody pool 0802-43, 0802-44, 0802-45, 0802-47, 0802-48, 0802-49, 0802-51, 0802-52, 0802-53, 0802-55, 0802-56, 0802-57, 0807-43, 0807-44, 0807-45, 0807-46, 0807-47, 0807-48, 0807-49, 0807-50, 0807-51, 0807-52, 0807-53, 0807-54, 0807-55, 0807-56, 0807-57, 0807-58 have human, mouse and monkey cross enrichment, and the other pools have human, mouse and monkey cross enrichment.

[0237] Example 3 Identification of positive monoclonal

[0238] 3.1 Monoclonal identification and screening of positive monoclonal sequencing

[0239] Experimental methods:

[0240] (1) The phage antibody-positive bacteria screened in Section 2.3 of Example 2 were sequenced.

[0241] (2) Each monoclonal clone in (1) was subjected to Elisa validation (see Section 2.3 of Example 2 for the method). The experimental results are summarized in Table 12 below:

[0242] Table 12

[0243] ;

[0244] .

[0245] The experimental results showed that among the 744 clones selected, 542 positive clones that bound to humans were screened, of which 263 had human-monkey cross-activity and 184 had human-rat-monkey cross-activity. 264 positive clones were sent for testing, and 112 unique clones were obtained after sequence analysis, of which 1 only bound to humans, 46 only had human-monkey cross-activity, and 65 had human-rat-monkey cross-activity.

[0246] Example 4 Activity verification of eukaryotic expressed antibodies and antibody fragments

[0247] 4.1 Detection of the binding ability of candidate antibodies on p318 cells

[0248] (1) Among the antibodies screened in Section 3.1 of Example 3, 31 candidate monoclonal antibodies with human, mouse and monkey cross-reactivity were selected for eukaryotic expression.

[0249] (2) According to the screening strategy in Section 2.1 of Example 2, 1E5 cells / well of p318-293F and 293F cells highly expressing RAGE antigen constructed in Section 1.7 of Example 1 were plated and the cells were washed with FACS buffer.

[0250] (3) Add 100 μL of antibody dilution to the cells. Incubate at 4°C for 60 minutes. Centrifuge and wash the cells twice with FACS buffer.

[0251] (4) Add 100 μL Anti-human Fc (PE) (abcam ab98596 1:300) to the cells and incubate at 4°C for 30 min.

[0252] (5) Resuspend the cells in FACS buffer and perform flow cytometry to detect the binding ability of each candidate antibody on p318-293F cells. The results of the cell average fluorescence intensity are as follows: Fig.11A and Fig. 11B The results of the cell average fluorescence intensity showing the binding ability of each candidate antibody on 293F cells are shown in Table 13.

[0253] Table 13

[0254]

[0255] The experimental results showed that except for NL-A69-VH (P57676), NL-A190-VH (P57694) molecules did not bind to p318-293F cells, while other samples all bound to the cells.

[0256] 4.2 Detection of the binding activity of eukaryotic expressed antibody supernatant with antigens Human-RAGE-Fc (P38406), Cyno-RAGE-Fc (P38408), and Mouse-RAGE-Fc (P38410)

[0257] (1) Among the antibodies screened in Section 3.1 of Example 3, 31 candidate monoclonal antibodies with human, mouse and monkey cross-reactivity were selected for eukaryotic expression.

[0258] (2) Different antigens Human-RAGE-Fc (P38406), Cyno-RAGE-Fc (P38408), and Mouse-RAGE-Fc (P38410) were coated on Elisa plates respectively. The antigens were diluted with PBS to a working concentration of 2 μg / mL, 30 μL was added to each well, and the plates were incubated at 4°C overnight.

[0259] (3) Wash three times with PBST and block with 5% skim milk (prepared with PBS) at room temperature for 2 h.

[0260] (4) Wash three times with PBST, add phage antibody pool, 30 µL per well, and incubate at room temperature for 60 min.

[0261] (5) Wash three times with PBST, add Goat-Anti-mouse-IgG-Fc-HRP (abcam; ab97265) (diluted at 1:8000) or Goat-Anti-human-κ+λ-HRP (Millipore / AP502P, AP506P) (diluted at 1:4000) diluted in 1% skim milk, and incubate at room temperature for 50 min.

[0262] (6) Add TMB, stop the reaction with stop solution, read OD450, and analyze the binding activity of eukaryotic antibody supernatant with Human-RAGE-Fc (P38406) as shown in the following figure: Figures 12A to 12D As shown in the figure, the binding activity analysis results of the eukaryotic expression antibody supernatant and Cyno-RAGE-Fc (P38408) are as follows Figures 12E to 12H As shown in the figure, the binding activity analysis results of the eukaryotic expression antibody supernatant and Mouse-RAGE-Fc (P38410) are as follows Figure 12I~12L shown.

[0263] The experimental results showed that all 31 candidate antibodies screened by eukaryotic expression had cross-reactivity between humans, mice and monkeys.

[0264] 4.3 Anti-RAGE-Fc antibody (A201) can specifically bind to p605 transiently transfected HepG2 cells

[0265] Experimental methods:

[0266] (1) Synthetic expression: 6xHis-antiRAGE(A201VH)-G4S-HA and antiRAGE(A201VL) were expressed in pairs, and the His-tagged protein was purified using a nickel column to obtain antiRAGE(A201)-Fc-G4S-HA; in the embodiment of the present application, the antiRAGE-Fc configuration was expressed by pairing the heavy chain with the light chain to obtain antiRAGE-Fc-G4S-HA, wherein the configuration of antiRAGE(A201VH) was 6xHis-VH-CH1-CH2-CH3-G4S-HA, and the amino acid sequence was as shown in SEQ ID NO: 23 (HHHHHQVQLVQSWAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCASQVGAIDYWGQGTPVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPELLGGPSVFLFPPKPKDT The configuration of antiRAGE (A201 VL) is VL-CL (Kappa), the amino acid sequence of VL is shown in SEQ ID NO: 8, and the sequence of CL (Kappa) is shown in SEQ ID NO: 10. After SDS-PAGE detection, the purity of antiRAGE (A201)-Fc-G4S-HA was >95%, and the results were as follows: Fig.13Ashown.

[0267] (2) Using the p605-HepG2 cells constructed in Section 1.8 of Example 1, wash and resuspend the cells with PBS.

[0268] (3) Fix the cells with 4% PFA for 20 min at room temperature, then wash and resuspend the cells in PBS.

[0269] (4) Block the cells with 5% BSA for 30 min at room temperature.

[0270] (5) Add 1 μg and 5 μg of antiRAGE(A201)-Fc to 100 μL of the incubation system, incubate at 4°C for 2 h, and then wash and resuspend the cells in 0.5% BSA diluted in PBS.

[0271] (6) Add 1 μL of FITC-conjugated anti-HA fluorescent antibody to cells without A201-Fc antibody and cells incubated with A201-Fc antibody, incubate at room temperature for 1 h, wash the cells twice with PBS, and resuspend the cells.

[0272] (7) Flow cytometry was used for detection. The experimental results are as follows Fig. 13B shown.

[0273] The experimental results showed that compared with the group without A201-Fc, A201-Fc had a stronger binding force to p605 cells.

[0274] The above is only the preferred embodiment of the present application, and does not limit the present application in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment based on the technical essence of the present application without departing from the technical solution of the present application still falls within the protection scope of the technical solution of the present application.

Claims

1. An anti-RAGE antibody, wherein: The antibody comprises three heavy chain complementary determining regions and three light chain complementary determining regions, wherein the three heavy chain complementary determining regions are CDR-H1, CDR-H2 and CDR-H3, and the three light chain complementary determining regions are CDR-L1, CDR-L2 and CDR-L3, wherein: The amino acid sequence of the CDR-H1 is shown in SEQ ID NO: 1, SEQ ID NO: 1 is SSYAIS; The amino acid sequence of the CDR-H2 is shown in SEQ ID NO: 2, SEQ ID NO: 2 is RIIPILGIAN; The amino acid sequence of the CDR-H3 is shown in SEQ ID NO: 3, and SEQ ID NO: 3 is QVGAIDY; The amino acid sequence of the CDR-L1 is shown in SEQ ID NO: 4, and SEQ ID NO: 4 is QSISTYVS; The amino acid sequence of the CDR-L2 is shown in SEQ ID NO: 5, which is IYYASILKS; and The amino acid sequence of the CDR-L3 is shown in SEQ ID NO: 6, and SEQ ID NO: 6 is QSYSFPLT.

2. The anti-RAGE antibody according to claim 1, comprising a human universal framework region.

3. The anti-RAGE antibody according to claim 1, wherein The antibody comprises a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO:7, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:7, wherein SEQ ID NO:7 is QVQLVQSWAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCASQVGAIDYWGQGTPVTVSS; The amino acid sequence contained in the light chain variable region is as shown in SEQ ID NO:8, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:8, wherein SEQ ID NO:8 is NIQLTQSPSSLSASLGDRVTITCRTSQSISTYVSWYQQRSGEAPNLLIYYASILKSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSFPLTFGGGTKVEIK. 4 . The anti-RAGE antibody according to claim 1 , which is a human antibody, a humanized antibody or a chimeric antibody. 5 . The anti-RAGE antibody according to claim 1 , which is a full-length IgG antibody. The anti-RAGE antibody according to any one of claims 1 to 3, which is a full-length IgG1 antibody.

7. The anti-RAGE antibody according to any one of claims 1 to 3, which is an antigen-binding fragment selected from any one of the following: Fv, Fab, F(ab')2, Fab', dsFv, scFv, sc(Fv)2.

8. The anti-RAGE antibody according to any one of claims 1 to 3, which binds to RAGE antigens derived from humans, monkeys and mice.

9. A conjugate comprising the anti-RAGE antibody according to any one of claims 1 to 8.

10. The conjugate of claim 9, comprising a therapeutic agent conjugated to the anti-RAGE antibody.

11. The conjugate according to claim 10, wherein the therapeutic agent is selected from one or more of a cytotoxic agent, a hormone preparation, a targeted small molecule preparation, a proteasome inhibitor, an immunomodulator, an angiogenesis inhibitor, a cell proliferation inhibitor, a pro-apoptotic agent, a cytokine, and an activator of a co-stimulatory molecule.

12. A nucleic acid molecule encoding the anti-RAGE antibody according to any one of claims 1 to 8. An expression vector comprising the nucleic acid molecule of claim 12 .

14. A cell comprising the nucleic acid molecule of claim 12 or the expression vector of claim 13.

15. Use of the anti-RAGE antibody according to any one of claims 1 to 8 or the conjugate according to any one of claims 9 to 11 in the preparation of a medicament for treating and / or preventing Alzheimer's disease, diabetes, inflammation or cancer.

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