Monoclonal antibodies targeting hsp90 and uses thereof

Abstract

This invention discloses a monoclonal antibody targeting HSP90 and its application. The monoclonal antibody targeting HSP90 can effectively inhibit the tumorigenicity and invasiveness of breast cancer cells, lung cancer cells, and osteosarcoma cells, and can significantly inhibit tumor growth in vivo. This invention provides a new targeted drug for diseases or symptoms related to high expression of HSP90, with broad prospects for clinical application.

Description

Technical Field

[0001] This invention belongs to the fields of cell biotechnology and immunology, and relates to a monoclonal antibody targeting HSP90 and its application. Background Technology

[0002] Enhanced synthesis and release of heat shock protein 90 (HSP90) helps maintain cellular homeostasis. Typically, high expression of HSP90 has a positive cytoprotective effect, while inhibition or knockdown of HSP90 reduces cell viability. Therefore, HSP90 is considered a potential therapeutic target for cancers such as colorectal cancer, lung cancer, and glioma.

[0003] The main mechanism by which HSP90 inhibitors act on cancer cells involves inflammation and programmed cell death; similar phenomena are also seen in other diseases such as autoimmune diseases, ischemic diseases, and neurodegenerative diseases.

[0004] Therefore, the development of new HSP90 antibodies is of great significance for the treatment of cancer, autoimmune diseases, ischemic diseases, and neurodegenerative diseases. Summary of the Invention

[0005] In view of this, in order to overcome the shortcomings of the prior art, the present invention is proposed.

[0006] The first aspect of the present invention provides an HSP90 antibody or an antigen-binding fragment thereof, wherein the HSP90 antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3, and the light chain variable region comprises light chain CDR1, light chain CDR2 and light chain CDR3.

[0007] Wherein: the amino acid sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 are as shown in SEQ ID NO.1, 2 and 3 respectively, and the amino acid sequences of the light chain CDR1, light chain CDR2 and light chain CDR3 are as shown in SEQ ID NO.4, 5 and 6 respectively.

[0008] In some embodiments, the heavy chain variable region of the HSP90 antibody or its antigen-binding fragment contains an amino acid sequence that is at least 90% identical to that of SEQ ID NO.7, and the light chain variable region contains an amino acid sequence that is at least 90% identical to that of SEQ ID NO.8.

[0009] In some embodiments, the heavy chain variable region of the HSP90 antibody or its antigen-binding fragment contains an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO. 7, and the light chain variable region contains an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO. 8.

[0010] In one specific embodiment, the heavy chain variable region of the HSP90 antibody or its antigen-binding fragment contains the amino acid sequence shown in SEQ ID NO.7, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO.8.

[0011] As used in this invention, the term "CDR," also known as "complementarity-determining region," "CDR region," or "hypervariant region," refers to a region in the antibody variable region that is highly variable in sequence and forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact sites"). The CDR is primarily responsible for binding to antigen epitopes.

[0012] Based on the variable region amino acid sequence contained in the given HSP90 antibody or its antigen-binding fragment according to this invention, those skilled in the art can routinely determine the CDR contained therein. For example, the Kabat, AbM, Chothia, or Contact protocols can be used to define the CDR in the variable region amino acid sequence.

[0013] When referring to antibodies defined by a specific CDR sequence as defined in this invention, the scope of said antibody also includes antibodies whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundaries differ from those defined in this invention due to the application of different schemes (e.g., different assignment system rules or combinations).

[0014] The boundaries of the CDR of the antibody of the present invention can be determined artificially according to any method or combination thereof in the art. Unless otherwise stated, in this invention, the term "CDR" or "CDR sequence" covers the CDR sequence determined in any of the foregoing methods.

[0015] In some embodiments, functional variants of the HSP90 antibody or its antigen-binding fragment described herein are also included within the scope of protection of this invention. A “functional variant” refers to a protein that has significant or marked sequence identity or similarity to the parent antibody, and that retains the biological activity of the parent antibody. Functional variants encompass, for example, the following variants of the HSP90 antibody or its antigen-binding fragment (parent antibody) described herein, which retain the ability to recognize target cells to a similar, equal, or greater extent than the parent antibody. Referring to the parent antibody, the functional variant may, for example, have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity with the parent antibody in terms of amino acid sequence.

[0016] In some embodiments, the functional variant may, for example, comprise the amino acid sequence of a parent antibody having at least one conserved amino acid substitution. Alternatively or supplementally, the functional variant may comprise the amino acid sequence of a parent antibody having at least one non-conserved amino acid substitution. In this case, the non-conserved amino acid substitution preferably does not interfere with or inhibit the biological activity of the functional variant. The non-conserved amino acid substitution can enhance the biological activity of the functional variant, resulting in increased biological activity of the functional variant compared to the parent antibody.

[0017] In some embodiments, conservative amino acid substitution is known in the art and includes the substitution of one amino acid having a particular physical and / or chemical property with another amino acid having the same or similar chemical or physical property. For example, conservative amino acid substitutions can be: an acidic / negatively charged polar amino acid replacing another acidic / negatively charged polar amino acid (e.g., Asp or Glu); an amino acid with a nonpolar side chain replacing another amino acid with a nonpolar side chain (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.); a basic / positively charged polar amino acid replacing another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.); an uncharged amino acid with a polar side chain replacing another uncharged amino acid with a polar side chain (e.g., Asn, Gin, Ser, Thr, Tyr, etc.); an amino acid with a β-branched side chain replacing another amino acid with a β-branched side chain (e.g., He, Thr, and Val); and an amino acid with an aromatic side chain replacing another amino acid with an aromatic side chain (e.g., His, Phe, Trp, and Tyr).

[0018] Those skilled in the art can readily mutate the nucleotide sequence corresponding to the antibody described in this invention using known methods, such as directed evolution and point mutation. Artificially modified nucleotides that have 90% or more homology to the nucleotide sequence corresponding to the HSP90 antibody or its antigen-binding fragment described in this invention, as long as they encode the aforementioned HSP90 antibody or its antigen-binding fragment, are all derived from and equivalent to the nucleotide sequence of this invention, and are also included within the scope of protection of this invention.

[0019] In this invention, the antibody includes monoclonal antibody, polyclonal antibody, and recombinant antibody.

[0020] As used herein, a "monoclonal antibody" refers to an antibody molecule or antibody formulation that shares a common heavy chain amino acid sequence and a common light chain amino acid sequence, as opposed to a "polyclonal" antibody formulation containing a mixture of antibodies with different amino acid sequences. Monoclonal antibodies can be produced using several known techniques such as phage, bacterial, yeast, or ribosome display, as well as the classic method exemplified by antibodies derived from hybridomas. Therefore, the term "monoclonal" is used to refer to all antibodies derived from a single nucleic acid clone.

[0021] As used in this article, the term "polyclonal antibody" refers to a mixture of antibodies produced by multiple B cell clones after immunization in mammals (such as humans, rabbits, sheep, mice, and horses). Because the antigen contains multiple determinants, it stimulates different B cells to produce antibodies against multiple epitopes.

[0022] As used herein, the term "recombinant antibody" refers to an antibody manufactured, expressed, produced, or isolated by recombinant methods, such as antibodies expressed using a recombinant expression vector transfected into host cells; antibodies isolated from a library of recombinant combination antibodies; or antibodies manufactured, expressed, produced, or isolated by any other means, such as antibodies manufactured, expressed, produced, or isolated by assembling a specific immunoglobulin gene sequence (such as the human immunoglobulin gene sequence) with other DNA sequences.

[0023] In this invention, the antibodies include chimeric antibodies, modified antibodies, SDR transplantation antibodies, antigen epitope-directed selection antibodies, surface remodeling antibodies, framework region replacement antibodies, and fully humanized antibodies.

[0024] As used herein, a “chimeric antibody” refers to an antibody whose variable domain sequence, derived from a non-human mammal species (e.g., mouse), is transplanted onto a constant domain sequence derived from a human antibody.

[0025] As used herein, the “modified antibody” refers to the transplantation of the complementarity-determining region (CDR) of an antibody variable region derived from a non-human mammalian species (e.g., mouse) into the backbone region (FR) of a human antibody variable region.

[0026] The "CDR," also known as the "complementarity-determining region," "CDR region," or "hypervariant region," is a region in the antibody's variable region that is highly variable in sequence and forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact sites"). The CDR is primarily responsible for binding to antigen epitopes.

[0027] "Modified antibodies" include antibodies with complete CDR transplantation and antibodies with partial CDR transplantation.

[0028] Complete CDR transplantation antibody: Six CDRs from a non-human antibody are cloned into the corresponding FRs of a human antibody using methods such as PCR to construct a new antibody. Compared with chimeric antibodies, CDR transplantation further reduces the content of heterologous sequences in the antibody and reduces antibody heterology.

[0029] Partial CDR transplantation antibodies: The properties of six CDRs on non-human antibodies are studied to identify the CDRs necessary for binding antigens in the antibody. These CDRs are then transplanted onto human antibody FR, resulting in lower immunogenicity than fully CDR transplanted antibodies.

[0030] SDR transfer antibodies: Based on partial CDR transplantation, a few residues from specific regions (SDRs) of the CDR that are specifically recognized and bound to the antigen are transplanted to the corresponding positions on the human antibody. These mainly include: SDR residues exposed to the outside of the CDR that are closely related to antigen binding; residues related to maintaining the 4th-order structure of the antibody's V region; amino acid position 71 of the light and heavy chains; and residues at the N-terminus of the antibody.

[0031] Antigen epitope-directed antibody selection: Antigen epitope-directed selection is a humanization strategy developed based on phage antibody library technology. Taking non-human antibodies derived from mice as an example, antigen epitope-directed selection uses the variable regions of light and heavy chain genes as basic units to construct human heavy chain mouse light chain and human light chain mouse heavy chain hybrid antibody libraries. Using a phage surface selection system, human heavy chain mouse light chain genes and human light chain mouse heavy chain genes are screened separately. Then, the human light and heavy chain genes are mixed and screened to obtain fully humanized antibody light and heavy chain genes that recognize the same epitopes as the parent mouse monoclonal antibody.

[0032] Surface-remodeled antibodies: Surface remodeling is a humanization technique directly based on the three-dimensional structure of antibodies. Taking non-human antibodies derived from mice as an example, the immunogenicity of the V region of mouse monoclonal antibodies largely stems from the motility and solution accessibility of surface residues in this region (solution-accessible residues (SAR): amino acid residues that can come into contact with the solvent when the antibody dissolves). Amino acids with high solution accessibility may have strong antigenicity. Surface remodeling emphasizes using the precise or modeled structure of antigen-antibody interactions as a basis, replacing highly surface-accessible amino acid residues in the variable regions of the light and heavy chains of mouse-derived antibodies with corresponding human amino acid residues, while retaining the CDR region and embedded amino acid residues of the mouse-derived antibody. Surface remodeling can maximize the preservation of antibody binding activity while improving its humanization degree, making it a relatively ideal humanization method.

[0033] Frame region replacement antibodies: Unlike CDR transplantation antibodies, the frame region replacement method does not search for human antibody frame regions similar to those from murine sources. Instead, it establishes a human antibody frame region library based on human germline genes, and then transplants the six CDRs (three from the light chain and three from the heavy chain) of the heterologous antibody into the frame regions of the human germline genes. The corresponding antibody combination library can then be screened using antigens. This method can not only be used for humanized antibodies but is also a more general antibody modification method. Through screening, it may even be possible to find human antibodies with stronger affinity than parental murine antibodies.

[0034] In this invention, the antibodies include monospecific antibodies and multispecific antibodies.

[0035] In this invention, the multispecific antibody is a type of antibody that can simultaneously bind to two or more different antigenic epitopes or antigens.

[0036] In some implementations, the multispecific antibody is bispecific, trispecific, quadrispecific, or even more specific.

[0037] In some implementations, the multispecific antibody is bispecific.

[0038] In this invention, the antigen-binding fragments include Fab, Fab′, F(ab′)2, single-chain antibody scFv, and nanobody VHH.

[0039] Fab: Fab is a functional unit of a monoclonal antibody molecule, which contains the heavy chain variable region VH, the heavy chain constant region CH1, the light chain variable region VL, and the light chain constant region CL. It does not contain the Fc fragment and has a molecular weight of approximately 50 kDa.

[0040] scFv: scFv is the variable region of the monoclonal antibody molecule, namely VH+VL, with a molecular weight of approximately 25 kDa.

[0041] VHH: Unlike Fab and scFv, VHH originates from heavy chain antibodies (a type of antibody that naturally lacks a light chain). VHH is the variable region of the heavy chain in a heavy chain antibody, with a molecular weight of approximately 15 kDa. The molecular weight of VHH is only one-tenth that of conventional antibodies, and its size is in the nanometer range, hence it is also called a nanobody (Nano-antibody). Because it has a single structural domain, it is also called a single-domain antibody (SdAbs).

[0042] In some embodiments, the antibody further includes a constant region, and according to the structure of the constant region, the antibody includes IgG antibodies, IgA antibodies, IgM antibodies, IgE antibodies, and IgD antibodies.

[0043] In some embodiments, the constant region is preferably the human IgG constant region, which is divided into four isotypes: IgG1, IgG2, IgG3, and IgG4. They share more than 95% homology in the amino acid sequence of the Fc region, but exhibit major differences in the amino acid composition and structure of the hinge region. The Fc region mediates effector functions such as antibody-dependent cell cytotoxicity (ADCC) and complement-dependent cell cytotoxicity (CDC). In ADCC, the Fc region of the antibody binds to an Fc receptor (FcgR) on the surface of immune effector cells, such as natural killer cells and macrophages, leading to phagocytosis or lysis of the target cell. In CDC, the antibody kills the target cell by triggering a complement cascade reaction on the cell surface. The antibodies described herein include antibodies having the aforementioned features of variable domains combined with any IgG isotype, including those wherein the Fc sequence can be modified to achieve different effector functions.

[0044] For many applications of therapeutic antibodies, Fc-mediated effector function can be detrimental and may pose safety risks by inducing extramechanistic toxicity. Modification of effector function can be achieved by altering the Fc region to weaken its binding to FcgR or complement factors.

[0045] In some implementations, mutations can be introduced into IgG1, IgG2, and IgG4 to reduce or silence Fc function.

[0046] In some embodiments, the antibody comprises an Fc region having one or more of the following characteristics: (a) reduced effector function compared to the parental Fc; (b) reduced affinity for FcgRI, FcgRIIa, FcgRIIb, FcgRIIIb and / or FcgRIIIa; (c) reduced affinity for FcgRI; (d) reduced affinity for FcgRIIa; (e) reduced affinity for FcgRIIb; (f) reduced affinity for FcgRIIIb; or (g) reduced affinity for FcgRIIIa.

[0047] In some implementations, to prevent glycosylation of the antibody, the constant region of human IgG is modified. This modification can be N297A or N297Q.

[0048] In some implementations, modifications are made to the human IgG constant region to alter Fc receptor interaction; such modifications may be L234A and / or L235E or L235A.

[0049] In some implementations, in order to extend the half-life, the human IgG constant region is modified, such as by R435H.

[0050] In some implementations, in order to prevent or reduce chain exchange, the human IgG constant region is modified, such as by S228P.

[0051] In some implementations, heterodimerization can also be induced by T366W modification, and optionally further by introducing disulfide bonds via S354C and Y349C modification on the opposing CH3 domains.

[0052] A second aspect of the present invention provides a polynucleotide molecule or a carrier comprising the polynucleotide molecule, said polynucleotide molecule encoding the HSP90 antibody or an antigen-binding fragment thereof described in the first aspect of the present invention.

[0053] In some embodiments, the sequence of the polynucleotide molecule encoding the heavy chain variable region described in the first aspect of the invention is shown in SEQ ID NO.9.

[0054] In some embodiments, the sequence of the polynucleotide molecule encoding the light chain variable region described in the first aspect of the invention is shown in SEQ ID NO.10.

[0055] The term "polynucleotide," synonymously referred to as "nucleic acid molecule," "nucleotide," or "nucleic acid," refers to any polynucleotide or polydeoxynucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single-stranded and double-stranded DNA, DNA consisting of a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA consisting of a mixture of single-stranded and double-stranded regions, and hybrid molecules containing DNA and RNA that may be single-stranded or more typically double-stranded, or a mixture of single-stranded and double-stranded regions. Additionally, "polynucleotide" refers to a triple-stranded region containing RNA or DNA, or both RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases, and DNA or RNA with a backbone modified for stability or other reasons.

[0056] In this invention, the polynucleotide molecule may comprise natural, non-natural, or modified nucleotides; and it may comprise natural, non-natural, or modified internucleotide linkages, such as aminophosphate linkages or thiophosphate linkages, instead of phosphodiester linkages present between the nucleotides of the unmodified oligonucleotide. In some embodiments, the nucleotides do not contain any insertions, deletions, inversions, and / or substitutions. However, in some cases, it may be suitable for a nucleotide to contain one or more insertions, deletions, inversions, and / or substitutions, and therefore, nucleotides formed by these insertions, deletions, inversions, and / or substitutions are also within the scope of this invention.

[0057] When applied to polynucleotide molecules, the term "encoding" refers to a polynucleotide that, if in its natural state or when manipulated by methods known to those skilled in the art, can be transcribed and / or translated to produce an mRNA containing a polypeptide and / or fragments thereof, is called "encoding" the polypeptide. The antisense strand is the complement of this nucleic acid, and the coding sequence can be deduced from it.

[0058] In some embodiments, examples of vectors that can be used in this invention include, but are not limited to, plasmids, phage particles, granules, artificial chromosomes, and virus-derived vectors.

[0059] Various vectors known in the art can be used, such as commercially available vectors, and then a polynucleotide encoding the antibody or its antigen-binding fragment can be operatively linked to the expression regulatory sequence to form an expression vector. In some embodiments, the virus-derived vectors include, but are not limited to: lentiviral vectors, retroviral vectors, adenovirus vectors, adeno-associated virus vectors, poxvirus vectors, herpesvirus vectors, baculovirus vectors, papillomavirus vectors, and papillomavirus vectors.

[0060] In some embodiments, the expression vector may contain expression regulatory sequences, such as transcription and translation start and stop codons, which are specific to the type of host cell (e.g., bacteria, fungi, plants, or animals) into which the vector is to be introduced, depending on the circumstances and whether the vector is DNA-based or RNA-based. Recombinant expression vectors may contain restriction sites to facilitate cloning.

[0061] In some embodiments, the vector may also contain one or more marker genes that allow selection of host cells for transformation or transfection. Marker genes include biocidal resistance (e.g., resistance to antibiotics, heavy metals, etc.); prototrophic complementation in auxotrophic hosts, etc. Suitable marker genes for the expression vector of the present invention include, for example, neomycin / G418 resistance genes, hygromycin resistance genes, histidine resistance genes, tetracycline resistance genes, ampicillin resistance genes, kanamycin resistance genes, and puromycin resistance genes.

[0062] A third aspect of the present invention provides a modified host cell or a population of host cells comprising the same, said modified host cell comprising the HSP90 antibody or its antigen-binding fragment as described in the first aspect of the present invention, or the polynucleotide molecule or a vector comprising the same as described in the second aspect of the present invention.

[0063] In some implementations, the host cell population may also include host cells other than the modified host cells.

[0064] In some implementations, the modified host cells include prokaryotic cells and eukaryotic cells.

[0065] In some implementations, the prokaryotic cells include, but are not limited to, bacteria, actinomycetes, cyanobacteria, mycoplasma, chlamydia, and rickettsia.

[0066] In some embodiments, the bacteria include, but are not limited to, Escherichia coli, Bacillus subtilis, Salmonella typhimurium, Pseudomonas, Streptomyces, and Staphylococcus.

[0067] In some embodiments, the eukaryotic cells include, but are not limited to, mammalian cells, insect cells, plant cells, and yeast cells.

[0068] The fourth aspect of the present invention provides an antibody derivative of an HSP90 antibody or an antigen-binding fragment thereof, the antibody derivative comprising a complex formed by directly or indirectly coupling the HSP90 antibody or an antigen-binding fragment thereof described in the first aspect of the present invention to a detectable marker.

[0069] In this invention, the term "detectable marker" refers to a reagent that is detectable, for example, by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Useful detectable markers include, but are not limited to, radionuclides, chemiluminescent agents, bioluminescent agents, paramagnetic ions, enzymes, and photosensitizing diagnostic agents. Detectable markers often produce measurable signals, such as radioactivity, fluorescence, color, or enzyme activity. Examples of suitable radionuclides include, but are not limited to, those mentioned above. 18 F, 52 Fe、 62 Cu、 64 Cu、 67 Cu、 86 Y、 90 Y、 89 Zr、 120 I, 123 I, 124 I, 125 I, 131 I, 13 N、 15 O、 186 Re、 188 Re、 51 Mn, 55 Co、 72 Examples of chemiluminescent agents include, but are not limited to, luminol, isoluminol, aromatic acridine esters, imidazole, acridine salts, and oxalates; examples of bioluminescent agents include, but are not limited to, luciferin, luciferase, and jellyfish luminescent protein; examples of paramagnetic ions include, but are not limited to, chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III), and erbium (III); examples of enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-D-galactosidase, urease, catalase, or glucoamylase; examples of photosensitizing diagnostic agents include, but are not limited to, dihydroxysilylphthalocyanine, methylene blue, protoporphyrin, hematoporphyrin, and photoporphyrin.

[0070] Antibodies conjugated to detectable markers can be used for diagnostic purposes. Detectable markers can be directly linked to or conjugated to antibodies, or indirectly via intermediates such as linkers known in the art, using techniques known in the art.

[0071] The fifth aspect of the present invention provides a detection product comprising the HSP90 antibody or its antigen-binding fragment as described in the first aspect of the present invention, or an antibody derivative of the HSP90 antibody or its antigen-binding fragment as described in the fourth aspect of the present invention.

[0072] In some implementations, the products include, but are not limited to, reagent kits, test strips, reagents, and chips.

[0073] In some embodiments, the kit may also include, but is not limited to, a container for holding its antigen-binding fragments when not in use, instructions for use of its antigen-binding fragments, its antigen-binding fragments attached to a solid support, known standards, and reference samples, or one or more of these.

[0074] The term "known standard" can refer to a solution having a known amount or concentration of HSP90, wherein such solution can be a naturally occurring solution, such as a sample from a patient known to have early, intermediate, late, progressive, or static cancer; or such solution can be a synthetic solution, such as an aqueous buffer solution in which a known amount of HSP90 is diluted. Known standards as described herein may include HSP90 isolated from subjects, recombinant or purified HSP90 protein, or HSP90 concentration values ​​associated with disease symptoms.

[0075] The term "reference sample" is a sample that can be compared with another sample, such as a test sample, to characterize the samples being compared. A reference sample will possess certain characteristic properties that serve as the basis for comparison with the test sample. For example, a reference sample might be used as a benchmark indicating the HSP90 level in a subject to indicate cancer. A reference sample does not necessarily have to be analyzed in parallel with the test sample; therefore, in some cases, a reference sample can be a previously determined value or range used to characterize a given condition, such as the HSP90 level indicating the subject to have cancer. The term also includes samples known to be associated with physiological states or disease conditions (such as HSP90-expressing cancers) but with unknown amounts of HSP90 used for comparative purposes.

[0076] The sixth aspect of the present invention provides a conjugate of an HSP90 antibody or its antigen-binding fragment thereof, the conjugate comprising a complex formed by conjugating the HSP90 antibody or its antigen-binding fragment as described in the first aspect of the present invention with a therapeutic agent.

[0077] In some embodiments, the therapeutic agent includes, but is not limited to, cytotoxic agents, hormonal preparations, targeted small molecule preparations, proteasome inhibitors, chemotherapeutic agents, oncolytic drugs, cytokines, activators of co-stimulatory molecules, or inhibitors of inhibitory molecules.

[0078] In some implementations, the therapeutic agent is selected from chemotherapeutic agents.

[0079] In this invention, "chemotherapeutic agent" is used to refer to compounds or compositions administered in the treatment of cancer. These agents or drugs are classified according to their intracellular activity patterns (e.g., whether they affect the cell cycle and at what stage). Alternatively, agents may be characterized based on their ability to directly crosslink DNA, embed in DNA, or induce chromosomal and mitotic abnormalities by influencing nucleic acid synthesis.

[0080] In some embodiments, examples of the chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, indomethacin, and piperazine; aziridines such as benzodopa, carbaquinone, metopepa, and uredopa; ethyleneimine and methylmelamine, including hexamethylmelamine, triethylene melamine, triethylphosphamide, triethylthiophosphamide, and trimethylol melamine; anechoic acid lactones (especially anocaryopicrin and bratasinone); camptothecin (including the synthetic analogue topotecan); lichenin; callystatin; CC-1065 (including its synthetic analogues adolaxine, calcetin, and bizelinine); cryptophytic acid (especially macrolide 1 and macrolide 8); dolalastatin; docalamicin (including the synthetic analogue KW-2189). And CB1-TM1); eleutherobin; sarcodictyin; spongistatin; nitrogen mustard, such as chlorambucil, naphthalenemustine, chophosphatamide, estradiol, ifosfamide, nitrogen mustard, oxynitric acid mustard hydrochloride, melphalan, neo-enpichin, cholesterol phenylacetic acid mustard, prednimustine, trofosfamide, and uracil mustard; nitrosourea, such as Carmustine, chloramphenicol, formustine, lomustine, nimustine, and ranola mustard; antibiotics, such as enematic antibiotics (e.g., calcipomycin, especially calcipomycin γI and calcipomycin ωI1); danendomycin, including danendomycin A; bisphosphonates, such as clophosphonates; esporomycin; and new carcinogen chromophores and related chromogens, enematic antibiotic chromophores, aclacinomysins, actinomycins, azoserine, bleomycin, and actinomycin. Carabicin C, erythromycin, carcinomycin, chromomycinis, daunorubicin, detoxin, 6-diazo-5-oxo-L-leucine, doxorubicin (including morpholino-doxorubicin, cyanomolino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, isorubicin, idarubicin, mesorcinol, mitomycin such as mitomycin C, mycophenolic acid, nogamycin, olivomycin, pepromycin, pofibromycin, purine Doxorubicin, triamcinolone acetonide, rodobisacin, streptomycin, strepzotocin, tuberculin, ubenmexicoside, fentostatin, and fentostatin; anti-metabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as folate, pteroxate, and trimethyltroxa; purine analogs, such as fludarabine, 6-mercaptopurine, thioimidazoline, and thioguanine; pyrimidine analogs, such as azacitidine, azacitidine, 6-azouridine, carmoflurane, cytarabine, dideoxyuridine, deoxyfluorouridine, enoxabin, and fluorouridine;Androgens, such as capetestosterone, drotalbutone propionate, cyclothiosterol, meandrazan, and testosterone lactone; anti-adrenergics, such as mitotane and trilosterone; folic acid supplements, such as folinic acid; acetoglucuronolactone; aldehydephosphoramide glycoside; aminolevulinic acid; enuracil; acridine; bestrabucil; dianthraxetine; edaraxazole; desphosphonamide; desmecoxine; desaccharin; epoxetine; edaraxetine; ethoxyphenidate Gallium nitrate; Hydroxyurea; Lentinan; Lolitainine; Maytansine, such as Maytansine and Ansorcinol; Mitoguanidine hydrazone; Mitoanthraquinone; Mopidanmol; Nitraerine; Pensistatin; Methionine mustard; Pirarubicin; Loxoanthraquinone; Podophyllotoxin; 2-Ethylhydrazide; Methylbenzylhydrazide; PSK polysaccharide complex; Razosen; Rhizobium; Cizonan; Germanium spiroamine; Fine crosslinking Spore-forming acid; triaminoquinone; 2,2',2”-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verrucosporin A, baculosporin A, and serpentin); urethane; vincristine; dacarbazine; mannomustine; dibromomannitol; dibromoeusol; piperbbromoethane; gacytosine; arabinoside (Ara-C); cyclophosphamide; taxanes, such as paclitaxel and docetaxel gemcitabine. 6-Thioguanine; mercaptopurine; platinum coordination complexes such as cisplatin, oxaliplatin, and carboplatin; vincristine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edaraxazole; daunorubicin; aminopterin; capecitabine; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); visual pigments such as retinoic acid; capecitabine; carboplatin, procarbazine, procamycin, gemcitabine, novibenten, farnesyltransferase inhibitors, farnesyl-transferase inhibitors, transplatinum, and one or more pharmaceutically acceptable salts, acids, or derivatives of any of the above substances.

[0081] The seventh aspect of the present invention provides a pharmaceutical composition for treating diseases or conditions related to HSP90 high expression, the pharmaceutical composition comprising the HSP90 antibody or antigen-binding fragment thereof described in the first aspect of the present invention, or a conjugate of the HSP90 antibody or antigen-binding fragment thereof described in the sixth aspect of the present invention.

[0082] In this invention, the term "treatment" refers to the improvement, prevention, relief, or reversal of a disease or condition or at least one identifiable symptom thereof, including treating patients at risk of or suspected of having the disease, and patients who have or have been diagnosed with the disease or condition, and includes suppressing clinical relapse. The term "prevention" as used herein refers to the prevention of the occurrence of a disease or its symptoms. The term "relief" as used herein refers to the reduction of the severity or duration of disease symptoms. Relief includes, but does not require, complete recovery from or complete prevention of the disease or its symptoms.

[0083] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0084] As used herein, the term "pharmaceutically acceptable" means a molecular entity and composition that, when administered to a subject such as a human, does not produce an adverse reaction, allergic reaction, or other adverse reaction, as appropriate. According to this disclosure, the preparation of pharmaceutical compositions comprising antibodies or additional active ingredients is known to those skilled in the art. Furthermore, for administration to a subject (e.g., a human), it should be understood that the pharmaceutical composition should meet standards of sterility, pyrogenicity, general safety, and purity as required by the FDA Office of Biostandards.

[0085] In this invention, the subject can be any mammal, including but not limited to: rodent mammals, such as mice and hamsters; and lagomorph mammals, such as rabbits. The mammal can be from the order Carnivora, including Felidae (cats) and Canidae (dogs). The mammal can be from the order Artiodactyla, including Bovidae (cattle) and Suidae (pigs); or Perissodactylus, including Equidae (horses). The mammal can be from the orders Primates, Cebooids, or Simoids (monkeys); or Anthropoids (humans and apes). In a specific embodiment of this invention, the subject is preferably a human.

[0086] As used herein, “pharmaceutically acceptable carriers and / or excipients” include, but are not limited to, aqueous solvents (e.g., water, alcohol / aqueous solutions, saline solutions, parenteral media such as sodium chloride, Ringer's glucose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils and injectable organic esters such as ethyl oleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents and inert gases), isotonic agents, adsorption delay agents, salts, pharmaceuticals, pharmaceutical stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, liquids, nutritional supplements, and similar materials and combinations thereof, as is known to those skilled in the art. The pH and exact concentration of various components in a pharmaceutical composition are adjusted according to known parameters.

[0087] In some embodiments, the pharmaceutical compositions of the invention are advantageously administered in the form of injectable compositions (as liquid solutions or suspensions); solid forms suitable for reconstitution into solutions or suspensions in liquids prior to injection can also be prepared. These formulations can also be emulsified.

[0088] In some implementations, the diseases or conditions associated with high HSP90 expression include, but are not limited to, cancer, autoimmune diseases, ischemic diseases, and neurodegenerative diseases.

[0089] In some implementations, the cancers include, but are not limited to, breast cancer, lung cancer, osteosarcoma, liver cancer, stomach cancer, bile duct cancer, ovarian cancer, cervical cancer, esophageal cancer, pancreatic cancer, kidney cancer, colorectal cancer, glioma, rheumatoid arthritis, Parkinson's disease, and Alzheimer's disease.

[0090] In some implementations, the lung cancer includes lung adenocarcinoma.

[0091] In some implementations, the diseases or conditions associated with high HSP90 expression are selected from breast cancer, lung adenocarcinoma, and osteosarcoma.

[0092] In some implementations, the autoimmune disease includes, but is not limited to, rheumatoid arthritis.

[0093] In some implementations, the ischemic disease is cerebral cardiovascular ischemic disease.

[0094] In some implementations, the neurodegenerative diseases include, but are not limited to, Alzheimer's disease and Parkinson's disease.

[0095] The eighth aspect of the present invention provides any one of the following methods, the method comprising:

[0096] (1) A method for preparing the HSP90 antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the method comprising the following steps: culturing the modified host cell or host cell population containing the third aspect of the present invention, and isolating the HSP90 antibody or antigen-binding fragment thereof according to the first aspect of the present invention from the culture.

[0097] (2) A method for detecting HSP90 or a fragment thereof in a test sample, the method comprising the following steps: contacting the test sample with the HSP90 antibody or its antigen-binding fragment as described in the first aspect of the present invention, or contacting the test sample with an antibody derivative of the HSP90 antibody or its antigen-binding fragment as described in the fourth aspect of the present invention, or contacting the test sample with a detection product as described in the fifth aspect of the present invention, and detecting the formation of a complex of the HSP90 antibody or its antigen-binding fragment or the antibody derivative of the HSP90 antibody or its antigen-binding fragment with HSP90 or a fragment thereof;

[0098] (3) A method for preparing the modified host cell or host cell population containing the present invention according to the third aspect of the present invention, the method comprising the following steps: introducing the polynucleotide molecule or the vector containing the present invention according to the second aspect of the present invention into the host cell.

[0099] In some implementations, the vector is introduced into the host cell by means including but not limited to physical methods, chemical methods, and biological methods.

[0100] In some implementations, the physical methods include, but are not limited to, the use of calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection, and electroporation.

[0101] In some implementations, the chemical method includes, but is not limited to, the use of colloidal dispersion systems and lipid-based systems.

[0102] In some implementations, the biological method includes, but is not limited to, the use of DNA vectors, lentiviral vectors, poxvirus vectors, herpes simplex virus vectors, adenovirus vectors, and adeno-associated virus vectors.

[0103] The ninth aspect of the present invention provides any of the following applications:

[0104] (1) The use of the HSP90 antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the antibody derivative of the HSP90 antibody or antigen-binding fragment thereof described in the fourth aspect of the present invention, and the detection product described in the fifth aspect of the present invention in the preparation of products for detecting HSP90 or fragment thereof;

[0105] (2) The application of the HSP90 antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the antibody derivative of the HSP90 antibody or antigen-binding fragment thereof described in the fourth aspect of the present invention, and the detection product described in the fifth aspect of the present invention in the preparation of products for diagnosing whether a subject suffers from a disease or condition related to high expression of HSP90.

[0106] In some implementations, the products include, but are not limited to, reagent kits, test strips, reagents, and chips;

[0107] In some implementations, the kits include, but are not limited to, ELISA detection kits, immunofluorescence detection kits, flow cytometry kits, and IHC detection kits;

[0108] (3) The use of the HSP90 antibody or antigen-binding fragment thereof described in the first aspect of the present invention, and the conjugate of the HSP90 antibody or antigen-binding fragment thereof described in the sixth aspect of the present invention, in the preparation of a pharmaceutical composition for treating diseases or symptoms related to high expression of HSP90.

[0109] In some implementations, the diseases or conditions associated with high HSP90 expression include, but are not limited to, cancer, autoimmune diseases, ischemic diseases, and neurodegenerative diseases.

[0110] In some implementations, the cancers include, but are not limited to, breast cancer, lung cancer, osteosarcoma, liver cancer, stomach cancer, bile duct cancer, ovarian cancer, cervical cancer, esophageal cancer, pancreatic cancer, kidney cancer, colorectal cancer, glioma, rheumatoid arthritis, Parkinson's disease, and Alzheimer's disease.

[0111] In some implementations, the lung cancer includes lung adenocarcinoma.

[0112] In some implementations, the diseases or conditions associated with high HSP90 expression are selected from breast cancer, lung adenocarcinoma, and osteosarcoma.

[0113] In some implementations, the autoimmune disease includes, but is not limited to, rheumatoid arthritis.

[0114] In some implementations, the ischemic disease is cerebral cardiovascular ischemic disease.

[0115] In some implementations, the neurodegenerative diseases include, but are not limited to, Alzheimer's disease and Parkinson's disease.

[0116] The tenth aspect of the present invention provides a method for diagnosing whether a subject suffers from a disease or condition related to high HSP90 expression. The method includes contacting a test sample with an HSP90 antibody or its antigen-binding fragment as described in the first aspect of the present invention, or contacting the test sample with an antibody derivative of an HSP90 antibody or its antigen-binding fragment as described in the fourth aspect of the present invention, or contacting the test sample with a detection product as described in the fifth aspect of the present invention, detecting the formation of a complex between the HSP90 antibody or its antigen-binding fragment or the antibody derivative of the HSP90 antibody or its antigen-binding fragment and HSP90 or its fragment, and if the formation of the complex is detected, diagnosing the subject with a disease or condition related to high HSP90 expression.

[0117] In some embodiments, the term "sample" refers to similar fluids, cells, or tissues separated from the subject (e.g., surgically removed tumor tissue, biopsy sections, including fine-needle aspirated tissue) and collections of fluids, cells, or tissues present in the subject. In some embodiments, the sample is a biological fluid. Biological fluids are generally liquids at physiological temperatures and may include naturally occurring fluids present in, extracted from, expressed from, or otherwise extracted from the subject or biological source. Some biological fluids originate from a specific tissue, organ, or localized area, and some other biological fluids may be more systemic or present in the subject or biological source. Examples of biological fluids include blood, serum, and serous fluid, plasma, lymph, urine, saliva, cystic fluid, tears, feces, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites such as those associated with non-solid tumors, fluids from the pleura, pericardium, peritoneum, abdomen, and other body cavities, fluids collected via bronchial lavage, etc. Biofluids may also include liquid solutions that come into contact with the subject or biological sources, such as cell and organ culture media, including cell or organ conditioned media, lavage solutions, etc. As used herein, the term "sample" encompasses substances taken from or present in the subject.

[0118] The eleventh aspect of the present invention provides a method for treating diseases or conditions related to HSP90 high expression, the method comprising administering the HSP90 antibody or antigen-binding fragment thereof described in the first aspect of the present invention, a conjugate of the HSP90 antibody or antigen-binding fragment thereof described in the sixth aspect of the present invention, or a pharmaceutical composition described in the seventh aspect of the present invention to a subject.

[0119] The advantages and beneficial effects of this invention are as follows:

[0120] This invention discloses a monoclonal antibody targeting HSP90 and its application. The monoclonal antibody targeting HSP90 can effectively inhibit the tumorigenicity and invasiveness of breast cancer cells, lung cancer cells, and osteosarcoma cells, and can significantly inhibit tumor growth in vivo. This invention provides a new targeted drug for diseases or symptoms related to high expression of HSP90, with broad prospects for clinical application. Attached Figure Description

[0121] Figure 1 This is an image of the SDS-PAGE results for the HSP90 monoclonal antibody.

[0122] Figure 2 This is a graph showing the results of an HSP90 monoclonal antibody ELISA test.

[0123] Figure 3 This is a graph showing the inhibitory effect of 4H7 monoclonal antibody on the stem cell self-renewal capacity of MCF-7.

[0124] Figure 4 This is a graph showing the inhibitory effect of 4H7 monoclonal antibody on the invasive ability of MCF-7 stem cells;

[0125] Figure 5 This is a graph showing the results of an in vivo treatment experiment with 4H7 monoclonal antibody in nude mice;

[0126] Figure 6 This is a graph showing the inhibitory effect of 4H7 monoclonal antibody on the self-renewal capacity of SPCA-1 stem cells in lung cancer.

[0127] Figure 7 This is a graph showing the results of the analysis of the ability of 4H7 monoclonal antibody to inhibit the invasion of lung cancer stem cells;

[0128] Figure 8 This is a graph showing the results of an in vivo treatment experiment using monoclonal antibody 4H7 in nude mice;

[0129] Figure 9 This is a graph showing the inhibitory effect of 4H7 monoclonal antibody on the self-renewal capacity of osteosarcoma stem cells;

[0130] Figure 10 This is a graph showing the results of the analysis of the ability of the 4H7 monoclonal antibody to inhibit the invasion of osteosarcoma stem cells. Detailed Implementation

[0131] The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the invention are all within the protection scope of the present invention.

[0132] Example 1: Preparation of HSP90 monoclonal antibody

[0133] 1) Animal immunization

[0134] HSP90 protein (100 μg / ml) was mixed with an equal volume of Freund's complete adjuvant and injected subcutaneously at multiple sites on the back of female BALB / c mice. Three weeks later, a second immunization was performed: HSP90 protein (200 μg / ml) was mixed with an equal volume of Freund's incomplete adjuvant and injected in the same manner. A third immunization was performed three weeks later, following the same protocol as the second. A fourth immunization was performed three weeks later, following the same protocol as the third. Blood was collected from the mice, and serum antibody titers were measured using indirect ELISA. A pulse immunization was performed 3-4 days later, consisting of HSP90 protein (500 μg / ml) injected via the tail vein.

[0135] (2) BALB / c mouse thymocytes (trophoblasts)

[0136] After removing the mouse from 75% alcohol, the abdominal skin was cut open with ophthalmic scissors to expose the peritoneum and remove the thymus. Surrounding fat or tissue was removed and photographed. The thymus was gently ground on a 100-mesh sieve with toothless forceps. The cells were washed with culture medium and sieved into a large petri dish to obtain a cell suspension. The suspension was transferred to a 50 ml centrifuge tube, centrifuged at 1200 rpm for 5 min, the supernatant was discarded, and the cells were gently resuspended in 20 ml of IMDM culture medium.

[0137] (3) Spleen cells of mice after immunization

[0138] Using ophthalmic scissors, incise the abdominal skin and peritoneum, remove the intact spleen, and remove any fat or tissue. Gently grind the spleen cells on a 100-mesh sieve with toothless forceps, rinse with culture medium, and sieve into a large petri dish to obtain a spleen cell suspension. Transfer to a 50 ml centrifuge tube and take 20 μl for counting.

[0139] (4) SP2 / 0 cell culture

[0140] Discard the DMEM-H culture medium, add 10 ml of IMDM for soaking, gently tap the flask wall to detach cells, collect them into a 50 ml centrifuge tube, and take 20 μl for counting. Centrifuge at 1200 rpm for 5 min, discard the supernatant, and resuspend in 20 ml of culture medium.

[0141] (5) Mixing spleen cells with SP2 / 0 cells

[0142] Transfer the SP2 / 0 suspension to a spleen cell tube (supernatant discarded) and gently mix. Centrifuge at 1200 rpm for 5 min, discard the supernatant, and rub the bottom of the tube in your palm to mix thoroughly. Prepare 38°C warm water, place the tube in the water, slowly add 1 ml of PEG and shake well, then add 10 ml of IMDM, for a total of 5 min. Centrifuge and discard the supernatant, add the thymocyte suspension and mix, centrifuge and discard the supernatant, then resuspend in 24 ml of IMDM. Prepare the culture medium: 2 ml cell suspension + 1 ml HAT + 2 ml IMDM + 10 ml HyClone serum + 25 ml 2.5% methylcellulose, invert the tube for 20 min to mix thoroughly.

[0143] (6) Screening and cloning of positive clones

[0144] Positive fusion cells can be transferred to 24-well plates for expansion and cloning, or directly sampled from 96-well plates for cloning. Cell populations are pipetted into a suspension and accurately counted. One drop of suspension is taken from each well of a 96-well plate and diluted 10-fold with 9 drops of serum-free culture medium for counting. HT culture medium is used to dilute to 40 ml each of 5, 30, and 50 cells per ml. Each hybridoma is processed using 96-well plates containing feeder cells, with 32 wells per dilution and 0.1 ml of suspension added to each well. Remaining cells after cloning are transferred to 24-well plates for expansion. Cells are incubated at 37°C and 5%–7% CO2 for 7 days, and colony counts are examined under a microscope. Antibody detection is performed, and single-cloned positive cells are transferred to 24-well plates and flasks for expansion and cryopreservation. Four hybridomas were obtained: HSP90 mAb (3D2, 5F8, 4H7, 15F5), which were subsequently cryopreserved.

[0145] (7) Culture of hybridoma cells

[0146] Hybridomas were cultured in DMEM-H medium containing 12% FBS, 2 mmol / L L-glutamine, 100 U / ml penicillin, and 100 μg / ml streptomycin at 37°C and 5% CO2. When collecting supernatant from large-scale cultures, new cells were revived after 15-20 stable passages. Typically, cells were passaged at a 1:2 ratio, with the medium changed after 24 hours, and passaged again after 48 hours of confluence.

[0147] (8) Collection of serum-free hybridoma supernatant

[0148] Expand to approximately 30 T175 flasks. Change the medium on the second day after subculturing. When the cell count reaches 75-90% confluence after 48 hours, discard the complete medium and replace it with 20 ml of serum-free medium per flask. Starting the next day, adjust the pH to 7.0-7.5 daily with 1 mol / L NaOH, adding 2-3 ml of fresh serum-free medium. When cell viability drops to 40%-50%, centrifuge at 3000 rpm for 10 minutes and collect the supernatant.

[0149] (9) Antibody purification

[0150] Unscrew the pre-packed column cap, use a peristaltic pump to drip PBS to remove air bubbles, and equilibrate to 20 column volumes. After loading the sample, take 1 ml for flow-through, wash with PBS (flow rate 1 ml / min, at least 5 column volumes), and measure OD280. Add 75 μl of 1M Tris-HCl (pH 9.0) to neutralize. Elute with 0.1 M Glycine-HCl (pH 2.7) (flow rate 1 ml / min), immediately mix the eluent to restore pH to 7.0, and measure OD280. Stop when OD < 0.1. Mix the eluted antibody and retain 10 μl as a sample. Dialyze the concentrated antibody in 20 times pre-chilled PBS at 4°C (4 h each time, 3 times). After dialysis, centrifuge at 12000 rpm for 15 min, filter sterilize at 0.22 μm, and aliquot to avoid freeze-thaw cycles. Analyze the HSP90 mAb concentration and perform SDS-PAGE analysis. The results show good purification, no impurities, and conformity to the heavy chain / light chain size (…). Figure 1 ).

[0151] (10) Non-competitive ELISA assay for the affinity of anti-HSP90 monoclonal antibody for recombinant HSP90.

[0152] The EC50 of each monoclonal antibody can be determined using a non-competitive ELISA method (Beatty method). 50 First, the gradient concentrations of HSP90 antigen coating and antibody serial dilutions were optimized, and the final experimental conditions were determined as follows: HSP90 antigen (2ug / ml) was used for coating, and monoclonal antibody dilutions were 32000, 16000, 8000, 4000, 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.8125, 3.90625, 1.953125, 0.9765625, 0.48828125, 0.244140625, 0.122070313, 0.061035156, 0.030517578, and 0.015258789 ng / ml. Under these conditions, assays were performed on unlabeled HSP90 monoclonal antibodies. Reaction curves were plotted with antibody OD values ​​on the ordinate and the logarithm of antibody concentration on the abscissa. Good typical S-shaped reaction curves were obtained at various HSP90 coating concentrations. The EC values ​​of the four tested antibodies were calculated. 50 ( Figure 2 The affinity of anti-HSP90 monoclonal antibodies for recombinant HSP90 is: 4H7 > 15F5 > 5F8 > 3D2.

[0153] Example 2: HSP90 monoclonal antibody 4H7 sequencing

[0154] RNA was extracted using a total RNA extraction kit for 4H7 hybridoma cells, and the extracted RNA solution was stored at -70℃. The extracted RNA underwent reverse transcription, end repair, adapter ligation, and PCR amplification purification to obtain NGS libraries. After passing library quality checks, the libraries were used for sequencing. High-throughput sequencing was performed on the constructed libraries, with 6GB of data per sample. The raw image data obtained from the second-generation high-throughput sequencing instrument was converted into sequence data (raw data or raw reads) through base calling, and the results were stored in FASTQ file format, including the sequence of reads and sequencing quality. The raw data was filtered to obtain clean data for subsequent analysis. Parameter-free assembly of clean reads was performed using Trinity-v2.11.0 software. Alignment analysis was performed using igblast, with the mouse immune database from IMGT. The fmt7 files were then processed and filtered. Analysis was performed using the Change-O submodels MakeDb.py, ParseDb.py, and ConvertDb.py. The data were analyzed and organized to obtain the sequences of the light and heavy chains of the monoclonal antibody, and the amino acid sequences of the variable region were obtained based on the sequences (specific sequence information is shown in Table 1 and Table 2).

[0155] Table 1. Amino acid sequence of HSP90 monoclonal antibody 4H7

[0156] Serial Number amino acid sequence name SEQ ID NO.1 GYTFSSYW Heavy chain CDR1 SEQ ID NO.2 ILPGSGTS Heavy chain CDR2 SEQ ID NO.3 ARGDYRDSWFAY Heavy chain CDR3 SEQ ID NO.4 TGAVTTSNY Light chain CDR1 SEQ ID NO.5 GIN Light chain CDR2 SEQ ID NO.6 ALWYSNHLV Light chain CDR3 SEQ ID NO.7 QVQLQQSGAELMKPGASVKISCKATGYTFSSYWIEWVKQRPGHGLEWIGEILPGSGTSNYNEKFKGKATFTADTSSNTAFMQLSSSLTSEDSAVYYCARGDYRDSWPAYWGQGTLVTVSA Heavy chain variable region SEQ ID NO.8 QAVVTQESALTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGINNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHLVFGGGTKLTVL Light chain variable region

[0157] Table 2. Nucleotide sequence of HSP90 monoclonal antibody 4H7

[0158] Serial Number nucleotide sequence name SEQ ID NO.9 caggttcagctgcagcagtctggagctgagctgatgaagcctggggcctcagtgaagatctcctgcaaggctactggctacacattcagtagctactggatagagtgggtaaagcagaggcctggacatggccttgagtggattggagagattttacctggaagtggaacttctaatta taatgagaagttcaagggcaaggccacattcactgcagacacatcctccaacacagccttcatgcaactcagcagcctgacatctgaggactctgccgtctattactgtgcaagaggggactatagggactcctggtttgcttactggggccaagggactctggtcactgtctctgcag Heavy chain variable region SEQ ID NO.10 caggctgttgtgactcaggaatctgcactcaccacatcacctggtgaaacagtcacactcacttgtcgctcaagtactggggctgttacaactagtaactatgccaactgggtccaggaaaaaccagatcatttattcactggtctaataggtggtatcaacaaccgagctccaggtgttcctgccagattctcaggctccctgattggagacaaggctgccctcaccatcacaggggcacagactgaggatgaggcaatatatttctgtgctctatggtacagcaaccatttggtgttcggtggaggaaccaaactgactgtcctag Light chain variable region

[0159] Example 3: In vitro and in vivo therapeutic study of HSP90 monoclonal antibody 4H7 on breast cancer stem cells and breast cancer treatment.

[0160] After digesting MCF-7 cells cultured for 48 h, purified monoclonal antibody 4H7 (1 mg / ml) was used as the experimental group. Cells were incubated with 4H7 at 37℃ for 2 h. 500 cells were then seeded in sphere medium. After 14 days, the number and size of cell spheres in both groups were observed. The results showed that monoclonal antibody 4H7 inhibited MCF-7 cell sphere formation by 30.1% (P < 0.001), indicating a statistically significant difference. Figure 3 The results suggest that monoclonal antibody 4H7 exerts its inhibitory effect on breast cancer stem cells by acting on them.

[0161] Invasion is a crucial functional characteristic of tumor stem cells. This study analyzed the inhibitory effect of monoclonal antibody 4H7 on the invasion of breast cancer stem cells. The experimental group used monoclonal antibody 4H7 at 1 mg / ml. MCF-7 sphere cells were incubated with 4H7 at 37°C for 2 hours, then added to the upper chamber of a Matrigel-lined Transwell container. Complete culture medium was added to the lower chamber. After 24 hours of culture, the cells were fixed, stained, and observed. The results showed that the invasive ability of MCF-7 sphere cells treated with monoclonal antibody 4H7 was significantly reduced, with an inhibition rate of 27.2% (P < 0.01), indicating a statistically significant difference. Figure 4 ).

[0162] MCF-7 sphere cells were inoculated into nude mice for in vivo treatment with the monoclonal antibody 4H7. The experiment consisted of four groups: a PBS group, a low-dose antibody group (2.5 mg / kg), a medium-dose antibody group (10 mg / kg), and a high-dose antibody group (40 mg / kg). Each mouse received an intraperitoneal injection of 0.4 ml. Treatment began after cell inoculation and was administered twice weekly for two months. The inhibition rate of the transplanted tumor gradually increased with increasing antibody dosage. Two months after the end of treatment, the inhibition rates of low, medium, and high-dose 4H7 antibodies on transplanted tumor volume reached 9.18%, 22.32%, and 27.09%, respectively. Therefore, the results of this experiment (…) Figure 5 This indicates that monoclonal antibody 4H7 can effectively inhibit tumor growth.

[0163] Example 4: In vitro and in vivo study of the effects of HSP90 monoclonal antibody 4H7 on lung cancer stem cells and lung cancer treatment.

[0164] The purified monoclonal antibody 4H7 (1 mg / ml) was used as the experimental group. SPCA-1 Sphere cells were incubated with it at 37°C for 2 hours. 500 cells were then seeded into SPCA-1 Sphere culture medium. After 14 days, the number and size of cell spheres in both groups were observed. The results showed that monoclonal antibody 4H7 inhibited cell sphere formation in SPCA-1 Sphere cells by 33.5% (…). Figure 6 The results showed that the monoclonal antibody 4H7 exerted its inhibitory effect on lung cancer stem cells by acting on them, with P < 0.001 indicating a statistically significant difference.

[0165] Analysis of the inhibitory effect of monoclonal antibody 4H7 on lung cancer stem cell invasion: Monoclonal antibody 4H7 1 mg / ml was used as the experimental group. Cells were incubated with 4H7 at 37℃ for 2 h, then added to the upper chamber of a Transwell containing Matrigel, with complete culture medium added to the lower chamber. After culturing for 24 h, the cells were removed, fixed, stained, and observed. The results showed that the invasive ability of cells treated with monoclonal antibody 4H7 was significantly reduced, with an inhibition rate of 42.4% against SPCA-1 sphere cell invasion.Figure 7 (P < 0.001, indicating a statistically significant difference).

[0166] To further demonstrate that monoclonal antibody 4H7 is a functional monoclonal antibody targeting lung cancer stem cells, an in vivo function study of monoclonal antibody 4H7 was conducted. SPCA-1 Sphere cells were inoculated into nude mice for in vivo treatment experiments with monoclonal antibody 4H7. The experiment consisted of five groups: a PBS group, a control antibody group (mouse IgG), a low-dose antibody group (2.5 mg / kg), a medium-dose antibody group (10 mg / kg), and a high-dose antibody group (40 mg / kg). Treatment began 3 days after cell inoculation, with 0.4 ml of antibody injected intraperitoneally twice a week for a total of 4 weeks. After tumors began to appear in the nude mice, the tumor volume was recorded twice a week. At day 33, the mice were sacrificed, and the tumors were removed and weighed. The experimental results showed (…). Figure 8 The monoclonal antibody 4H7 significantly inhibited the growth of transplanted tumors, effectively suppressing tumor volume increase. Furthermore, tumor weight measurements revealed that the low, medium, and high doses of the antibody inhibited tumor weight by 24.45%, 33.56%, and 40.36%, respectively, indicating a dose-dependent inhibitory effect.

[0167] Example 5: In vitro efficacy study of HSP90 monoclonal antibody 4H7 against osteosarcoma stem cells.

[0168] The purified monoclonal antibody 4H7 (1 mg / ml) was used as the experimental group. Cells were incubated with it at 37°C for 2 hours, and 500 cells were seeded in Saos-2 sphere medium. After 14 days, the number and size of cell spheres in both groups were observed. The results showed that monoclonal antibody 4H7 inhibited cell sphere formation by 62.88% (…). Figure 9 The results showed that monoclonal antibody 4H7 exerted its inhibitory effect on osteosarcoma stem cells by acting on them, with P < 0.01 indicating a statistically significant difference.

[0169] Analysis of the inhibitory effect of monoclonal antibody 4H7 on osteosarcoma stem cell invasion: Monoclonal antibody 4H7 1 mg / ml was used as the experimental group. Cells were incubated with 4H7 at 37℃ for 2 h, then added to the upper chamber of a Transwell containing Matrigel, with complete culture medium added to the lower chamber. After culturing for 24 h, the cells were removed, fixed, stained, and observed. The results showed that the invasive ability of Saos-2 sphere cells was significantly reduced after treatment with monoclonal antibody 4H7, with an inhibition rate of 22.2% (…). Figure 10 (P < 0.001, indicating a statistically significant difference).

[0170] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. An HSP90 antibody or antigen-binding fragment thereof, characterized in that, The HSP90 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3, the light chain variable region comprising a light chain CDR1, a light chain CDR2 and a light chain CDR3; The amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 are respectively shown as SEQ ID NO. 1, 2, 3, and the amino acid sequences of the light chain CDR1, the light chain CDR2 and the light chain CDR3 are respectively shown as SEQ ID NO. 4, 5, 6.

2. The HSP90 antibody or antigen-binding fragment thereof of claim 1, wherein, The heavy chain variable region of the HSP90 antibody or antigen-binding fragment thereof contains an amino acid sequence shown as SEQ ID NO. 7 or an amino acid sequence having at least 90% identity with SEQ ID NO. 7, and the light chain variable region contains an amino acid sequence shown as SEQ ID NO. 8 or an amino acid sequence having at least 90% identity with SEQ ID NO.

8.

3. A polynucleotide molecule or a vector comprising the same, characterized in that, The polynucleotide molecule encodes the HSP90 antibody or antigen-binding fragment thereof of any one of claims 1-2.

4. The polynucleotide or a vector comprising the same according to claim 3, wherein, The sequence of the polynucleotide molecule encoding the heavy chain variable region of any one of claims 1-2 is shown as SEQ ID NO.

9.

5. The polynucleotide or a vector comprising the same according to claim 3, wherein, The sequence of the polynucleotide molecule encoding the light chain variable region of any one of claims 1-2 is shown as SEQ ID NO.

10.

6. An engineered host cell or a population of host cells comprising the same, wherein, The engineered host cell comprises the HSP90 antibody or antigen-binding fragment thereof of any one of claims 1-2, or the polynucleotide molecule of any one of claims 3-5 or a vector comprising the same.

7. An antibody derivative of an HSP90 antibody or an antigen-binding fragment thereof, characterized in that, The antibody derivative includes a complex formed by directly or indirectly conjugating the HSP90 antibody or antigen-binding fragment thereof of any one of claims 1-2 to a detectable marker.

8. The antibody derivative of an HSP90 antibody or antigen-binding fragment thereof according to claim 7, characterized in that, The detectable marker includes a radionuclide, a chemiluminescent agent, a bioluminescent agent, a paramagnetic ion, an enzyme, a photosensitive diagnostic agent.

9. The antibody derivative of an HSP90 antibody or antigen-binding fragment thereof according to claim 8, characterized in that, The radionuclides include 18 F, 52 Fe, 62 Cu, 64 Cu, 67 Cu, 86 Y, 90 Y, 89 Zr, 120 I, 123 I, 124 I, 125 I, 131 I, 13 N, 15 O, 186 Re, 188 Re, 51 Mn, 55 Co, 72 As.

10. The antibody derivative of an HSP90 antibody or antigen-binding fragment thereof according to claim 8, characterized in that, The chemiluminescent agent includes luminol, isoluminol, aromatic acridinium ester, imidazole, acridinium salt, oxalate ester.

11. The antibody derivative of an HSP90 antibody or antigen-binding fragment thereof according to claim 8, characterized in that, The bioluminescent agent includes fluorescein, luciferase, aequorin.

12. The antibody derivative of an HSP90 antibody or antigen-binding fragment thereof according to claim 8, characterized in that, The paramagnetic ion includes chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III), erbium (III).

13. The antibody derivative of an HSP90 antibody or antigen-binding fragment thereof according to claim 8, characterized in that, The enzyme includes horseradish peroxidase, alkaline phosphatase, glucose oxidase, beta-D-galactosidase, urease, catalase, or glucoamylase.

14. The antibody derivative of an HSP90 antibody or antigen-binding fragment thereof according to claim 8, characterized in that, The photosensitive diagnostic agent includes dihydroxy silicon phthalocyanine, methylene blue, protoporphyrin, hematoporphyrin, photoporphyrin.

15. A testing product, characterized in that, The detection product includes the HSP90 antibody or antigen-binding fragment thereof of any one of claims 1-2 or the antibody derivative of the HSP90 antibody or antigen-binding fragment thereof of any one of claims 7-14.

16. The test product according to claim 15, characterized in that The product includes a kit, a test paper, a reagent, a chip.

17. A pharmaceutical composition comprising, in combination, a compound of any one of claims 1-16 and a pharmaceutically acceptable carrier. The pharmaceutical composition comprises the HSP90 antibody or antigen-binding fragment thereof of any one of claims 1-2.

18. A method of producing the HSP90 antibody or antigen-binding fragment thereof of any one of claims 1-2, comprising, The method comprises the following steps: culturing the modified host cell of claim 6 or a host cell population comprising the same, and isolating the HSP90 antibody or antigen-binding fragment thereof of any one of claims 1-2 from the culture.

19. A method of making the engineered host cell of claim 6 or a population of host cells comprising the same, wherein, The method comprises the following steps: introducing the polynucleotide molecule of any one of claims 3-5 or a vector comprising the same into a host cell.

20. Use of the HSP90 antibody or antigen-binding fragment thereof of any one of claims 1-2, the antibody derivative of the HSP90 antibody or antigen-binding fragment thereof of any one of claims 7-14, or the detection product of any one of claims 15-16 in the preparation of a product for detecting HSP90.

21. Use of the HSP90 antibody or antigen-binding fragment thereof of any one of claims 1-2, the antibody derivative of the HSP90 antibody or antigen-binding fragment thereof of any one of claims 7-14, or the detection product of any one of claims 15-16 in the preparation of a product for diagnosing whether a subject has breast cancer, lung cancer or osteosarcoma.

22. The use according to claim 21, characterized in that, The product comprises a kit, a test paper, a reagent, a chip.

23. The use according to claim 22, characterized in that, The kit comprises an ELISA detection kit, an immunofluorescence detection kit, a flow sorting kit, an IHC detection kit.

24. Use of the HSP90 antibody or antigen-binding fragment thereof of any one of claims 1-2 in the preparation of a pharmaceutical composition for treating breast cancer, lung cancer or osteosarcoma.

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