CN109705219B - Monoclonal antibody combined with mesothelin and preparation method thereof - Google Patents

Abstract

The present disclosure provides a monoclonal antibody that binds to mesothelin, the monoclonal antibody comprising a variable region comprising Complementarity Determining Regions (CDRs) having amino acid sequences of at least two selected from the following sets of amino acid sequences: 4, 6, 8, 12, 14 and 16 of SEQ ID NO. In the present disclosure, since the monoclonal antibody can specifically recognize mesothelin protein in tissues, cell surfaces and body fluids, can block mesothelin receptor and mesothelin action, it is particularly useful for the detection of mesothelin and the treatment of antibody-dependent diseases.

Description

Monoclonal antibody combined with mesothelin and preparation method thereof

Technical Field

The present disclosure relates to a monoclonal antibody that binds to mesothelin and a method for preparing the same.

Background

In recent years, researchers have found that the Mesothelin (Mesothelin) cDNA contains a 1884bp open reading frame, encoding a 69kDa precursor protein (628 amino acids). After glycosylation, the precursor is cleaved by furan at amino acids 288-293, resulting in a 40kDa protein and a smaller 32kDa fragment that is released from the cell. This 32kDa shed-off fragment is called Megakaryocyte Potentiator (MPF). The 40kDa protein is present on the cell surface and is released after treatment with phosphatidylinositol-specific phospholipase C. Since malignant mesotheliomas and ovarian adenocarcinomas originate from normal mesothelial cells, these malignant diseases express mesothelin.

The most common form of mesothelin is membrane-bound, but 2 variants are found: variant 1 with an 8 amino acid insertion is also membrane bound. Due to the lack of the GPI-anchor signal sequence, variant 2 is an exfoliated, soluble form. Soluble mesothelin protein can be detected from the serum of patients with ovarian cancer and can be used as a useful new marker for diagnosing ovarian cancer and/or monitoring therapeutic response together with CA125 (cancer antigen-125). In addition, soluble mesothelin is elevated in blood and exudate from patients with many tumors (mesothelioma, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, colorectal cancer, gastric cancer, soft tissue tumors, etc.). Determination of mesothelin levels in these fluids has been primarily approved by the FDA in the united states as a tool for monitoring patient response and progression.

Besides, mesothelin is also involved in the development of tumors and serves as a new target for tumor treatment. Many novel therapeutic antibody drugs targeting mesothelin have been developed and applied to clinical trials of solid tumors. They include the unconjugated monoclonal antibody amatuximab supplied by Morphotek/NCI, the recombinant immunotoxins SS1P and RG7787 supplied by NCI/Roche, and the different antibody-drug conjugates (ADCs) supplied by Bayer, Roche/Genentech and BMS/Metarex. Therefore, the establishment of a novel high-specificity recognition and high-affinity binding mesothelin antibody has important values for tumor diagnosis, treatment response monitoring and tumor treatment.

Since mesothelin is a protein with a long amino acid sequence and has more epitopes, more than one antibody (including monoclonal or polyclonal) can be obtained for mesothelin, and the binding properties (e.g., specificity, etc.) of these antibodies to the antigen may be different. Therefore, it is often difficult for those skilled in the art to find a particularly desirable monoclonal antibody with a significant blocking effect in the research.

Disclosure of Invention

The present disclosure has been made in view of the above-mentioned state of the art, and an object thereof is to provide a mesothelin monoclonal antibody that recognizes with high specificity and has high affinity, a method for producing the same, and an application thereof.

To this end, a first aspect of the present disclosure provides a monoclonal antibody that binds to mesothelin, optionally the antibody comprises a variable region comprising Complementarity Determining Regions (CDRs) having amino acid sequences of at least two selected from the following sets of amino acid sequences: 4, 6, 8, 12, 14 and 16 of SEQ ID NO.

In the disclosure, the CDR in the monoclonal antibody is a completely new CDR compared to the CDR of the existing mesothelin monoclonal antibody, and has high specificity and high affinity, and has an obvious effect of blocking the mesothelin-mesothelin receptor interaction, thereby blocking the mesothelin receptor activation function. Therefore, the monoclonal antibody can specifically recognize mesothelin protein on the tissue, cell surface and body fluid, can block the actions of mesothelin receptor and mesothelin, and is particularly suitable for mesothelin detection and antibody-dependent disease treatment.

In addition, in the monoclonal antibody relating to the first aspect of the present disclosure, the variable region may include a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region may be SEQ ID No. 2, and the amino acid sequence of the light chain variable region may be SEQ ID No. 10.

In addition, in the monoclonal antibody related to the first aspect of the present disclosure, optionally, SEQ ID NOs 4, 6, or 8 are CDRs of the heavy chain variable region, and SEQ ID NOs 12, 14, or 16 are CDRs of the light chain variable region.

Further, in the monoclonal antibody relating to the first aspect of the present disclosure, optionally, the SEQ ID No. 4 is CRD1 of the heavy chain variable region; 6 is CRD2 of the heavy chain variable region; the SEQ ID NO. 8 is CRD3 of the heavy chain variable region.

Additionally, in the monoclonal antibody relating to the first aspect of the present disclosure, the SEQ ID No. 12 may be CRD 1; 14 can be CRD2 of the light chain variable region; 16 can be CRD3 of the light chain variable region.

In addition, in the monoclonal antibody according to the first aspect of the present disclosure, the monoclonal antibody may further include a heavy chain constant region and a light chain constant region, wherein the heavy chain type of the heavy chain constant region may be IgGl, IgG2a, IgG2b, or IgG3, and the light chain type of the light chain constant region may be κ chain or λ chain.

In addition, in the monoclonal antibody relating to the first aspect of the present disclosure, the monoclonal antibody may be an immunoglobulin molecule or an antigen-binding fragment. In this case, it is capable of binding to mesothelin.

A second aspect of the disclosure provides a functional variant of a monoclonal antibody that binds to mesothelin, optionally having 50% to 99% amino acid sequence homology to the variable region of any one of the above.

In the present disclosure, functional variants have 50% to 99% amino acid sequence homology with the variable regions of monoclonal antibodies to which the present disclosure relates. In this case, the monoclonal antibody has the same or different, higher or lower binding affinity than the functional variant, which is still capable of recognizing and binding to mesothelin or a fragment thereof.

A third aspect of the present disclosure provides an immunoconjugate, which may comprise at least the monoclonal antibody of any one of the above. In this case, the immunoconjugate can be used for in vitro immunoassay or purification of mesothelin protein or a fragment thereof.

A fourth aspect of the present disclosure provides a nucleic acid molecule, optionally encoding a monoclonal antibody of any one of the above. In this case, the nucleic acid molecule may be used as an intermediate in the cloning process.

A fifth aspect of the present disclosure provides an expression vector, which may comprise the nucleic acid molecule described above. In this case, the expression vector can be transferred into an appropriate cell so that it can express a monoclonal antibody that binds to mesothelin.

A sixth aspect of the present disclosure provides a host cell, optionally comprising an expression vector as described above. In this case, the host cell is capable of expressing a monoclonal antibody that binds to mesothelin.

A seventh aspect of the present disclosure provides a method for producing a monoclonal antibody that binds to mesothelin, which may comprise culturing the host cell described above. In this case, the monoclonal antibody binding to mesothelin can be isolated and purified by various isolation methods.

An eighth aspect of the present disclosure provides a pharmaceutical composition, which may include a monoclonal antibody of any one of the above. In this case, the compound can be used for diagnosing, treating and/or preventing mesothelin-positive tumors and other related diseases.

A ninth aspect of the present disclosure provides a kit comprising a monoclonal antibody of any one of the above. Under the condition, the mesothelin or the content thereof in the sample to be detected can be conveniently, quickly and accurately detected, so that whether the mesothelin receptor-mesothelin excessive interaction exists in the sample to be detected or not can be known.

Drawings

Fig. 1 shows a schematic structural diagram of a monoclonal antibody that binds to mesothelin according to an embodiment of the present disclosure.

Fig. 2 is a graph showing the results of LK19 specifically recognizing mesothelin protein of tumor cells.

Fig. 3 is a graph showing the results of three antibodies, LK19, LK20 and LK423, detecting mesothelin expression on the surface of pancreatic cancer cells.

Fig. 4 is a graph showing the results of LK19 detection of mesothelin expression in tumor tissues and paracarcinoma.

Detailed Description

Hereinafter, preferred embodiments of the present disclosure will be described in detail with reference to the accompanying drawings. In the following description, the same components are denoted by the same reference numerals, and redundant description thereof is omitted. The drawings are schematic and the ratio of the dimensions of the components and the shapes of the components may be different from the actual ones.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

The term "monoclonal antibody" as used herein refers to an antibody that exhibits a single binding specificity for only a particular antigenic determinant. In addition, antibodies include proteins comprising heavy (H) chains and light (L) chains and antigen-binding portions. Wherein the heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region (CH) comprising 3 domains CH1, CH2 and CH 3; the light chain comprises a light chain variable region (VL) and a light chain constant region (CL), which comprises one domain CL. In addition, the VH and VL regions can be subdivided into hypervariable regions, termed Complementarity Determining Regions (CDRs), between which more conserved regions, termed Framework Regions (FRs), are inserted. Each VH and VL comprises 3 CDRs and 4 FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The variable regions of the heavy and light chains comprise binding domains that interact with an antigen.

The term "antigen-binding portion" of an antibody refers to one or more fragments of an antibody that are capable of specifically binding to an antigen.

The term "Fab fragment" refers to a monovalent fragment consisting of the VL, VH, CL and CH1 domains; the term "F (ab') 2" fragment refers to a bivalent fragment of two Fab fragments connected by a disulfide bridge at the hinge region; the term "Fd fragment" refers to a fragment consisting of the VH and CH1 domains; the term "Fv fragment" refers to a fragment minimal antibody fragment consisting only of the VL and VH domains.

The term "single chain antibody (scFv)" refers to a monovalent molecule formed by pairing of VL and VH regions; the term "three chain antibody" refers to a trimer formed by covalently linking after the ScFv is shortened and paired with VH and VL between different molecules.

The term "phage antibody" refers to a phage antibody formed by the fusion of an antibody molecule fragment to a phage coat protein and expressing it on the surface of a phage particle. The term "domain antibody" refers to a fragment consisting of a VH domain.

The term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody comprising the amino acid sequence.

The term "mesothelin receptor (mesothelin L)" refers to a biological macromolecule in a cell that is capable of binding to mesothelin and causing a change in cell function.

The term "functional variant" refers to an amino acid (nucleotide) sequence that is altered by one or more amino acids (nucleotides) compared to the amino acid (nucleotide) sequence of the parent binding molecule and still competes with the parent binding molecule for binding to a binding partner.

The term "mg/kg body weight" refers to the amount administered (mg) per 1kg body weight of a human or animal.

Fig. 1 shows a schematic structural diagram of a monoclonal antibody that binds to mesothelin according to an embodiment of the present disclosure.

A monoclonal antibody that binds to mesothelin according to the first aspect of the present embodiment may include a variable region that may include Complementarity Determining Regions (CDRs) having amino acid sequences selected from at least two of the following sets of amino acid sequences: 4, 6, 8, 12, 14 and 16 of SEQ ID NO.

In this embodiment, the monoclonal antibody may comprise a variable region, which may comprise at least the CDRs of two of the amino acid sequences of SEQ ID NOs 4, 6, 8, 12, 14, 16. Compared with the CDR of the existing mesothelin monoclonal antibody, the CDR in the monoclonal antibody is a brand new and different CDR, is a novel mesothelin monoclonal antibody with high specificity and high affinity, and has an obvious effect of blocking the interaction of mesothelin-mesothelin receptor (mesothelin L) so as to block the activation function of mesothelin L.

In addition, in this embodiment, the variable region may include a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region may be SEQ ID NO. 2, and the amino acid sequence of the light chain variable region may be SEQ ID NO. 10.

In addition, in some examples, SEQ ID NOs 4, 6, or 8 can be CDRs of a heavy chain variable region and SEQ ID NOs 12, 14, or 16 can be CDRs of a light chain variable region.

In addition, in this embodiment, SEQ ID NO 4 may be CRD1 of the heavy chain variable region; SEQ ID NO 6 may be CRD2 of the heavy chain variable region; SEQ ID NO 8 may be CRD3 of the heavy chain variable region.

Additionally, in some examples, SEQ ID NO 12 can be CRD1 of the light chain variable region; 14 can be CRD2 of the light chain variable region; SEQ ID NO 16 may be CRD3 of the light chain variable region.

In addition, in this embodiment, the monoclonal antibody may further include a heavy chain constant region and a light chain constant region, wherein the heavy chain type of the heavy chain constant region may be IgGl, IgG2a, IgG2b, or IgG3, and the light chain type of the light chain constant region may be kappa chain or lambda chain.

In addition, in this embodiment, the antibody may be an immunoglobulin molecule or an antigen-binding fragment. In this case, it is capable of binding to mesothelin. Antigen-binding fragments include, but are not limited to, Fab fragments, Fd fragments, Fv fragments, F (ab') 2 fragments, Complementarity Determining Region (CDR) fragments, single chain antibodies (scFv), domain antibodies, phage antibodies, triabodies, and the like.

A functional variant of a monoclonal antibody that binds to mesothelin according to the second aspect of the present embodiment may have 50% to 99% amino acid sequence homology with the variable region according to the present disclosure. For example, it has 50% to 99% amino acid sequence homology with SEQ ID NO. 2, 50% to 99% amino acid sequence homology with SEQ ID NO. 10, and the like.

In this embodiment, the functional variant has 50% to 99% amino acid sequence homology with the variable region of a monoclonal antibody to which the disclosure relates. In this case, the monoclonal antibody has the same or different, higher or lower binding affinity than the functional variant, which is still capable of recognizing and binding to mesothelin or a fragment thereof.

In addition, in some instances, functional variants can be obtained by altering a parent monoclonal antibody or a portion thereof using common molecular biology methods known in the art, including but not limited to error-prone PCR, oligonucleotide-directed mutagenesis, site-directed mutagenesis, and heavy or light chain shuffling.

In addition, in some instances, it may be preferred that the functional variant have 50% to 99% amino acid sequence homology with the variable region of the monoclonal antibody; more preferably, the functional variant may have 60% to 99% amino acid sequence homology with the variable region of the monoclonal antibody; more preferably, the functional variant may have 70% to 99% amino acid sequence homology with the variable region of the monoclonal antibody; more preferably, the functional variant may have 80% to 99% amino acid sequence homology with the variable region of the monoclonal antibody; more preferably, the functional variant may have 90% to 99% amino acid sequence homology with the variable region of the monoclonal antibody; more preferably, the functional variant may have 95% to 99% amino acid sequence homology with the variable region of the monoclonal antibody; most preferably, the functional variant may have 97% to 99% amino acid sequence homology with the variable region of a monoclonal antibody.

In addition, in some examples, functional variants may have conservative sequence modifications, including substitutions, additions or deletions of conserved amino acids (nucleotides). These modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and random PCR-mediated mutagenesis, and can comprise natural as well as non-natural nucleotides and amino acids. Among them, conservative amino acid substitution includes substitution in which an amino acid residue is replaced with another amino acid residue having similar structure or chemical properties. Conservative sequence modifications amino acid modifications do not significantly affect or alter the binding characteristics of an antibody containing the amino acid sequence.

Additionally, in some examples, families of amino acid residues with similar side chains can include amino acids with basic side chains, such as lysine, arginine, histidine; acidic side chain amino acids such as aspartic acid, glutamic acid; uncharged polar side chain amino acids, such as, for example, aspartic acid, glutamic acid, serine, threonine, tyrosine, cysteine, tryptophan; nonpolar side chain amino acids such as glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine; branched side chain amino acids such as threonine, valine, isoleucine; and aromatic side chain amino acids such as tyrosine, phenylalanine, tryptophan. In addition, other amino acid residue family classifications besides the above-described families may also be used.

In addition, in some instances, a functional variant may also have a non-conservative amino acid substitution, e.g., an amino acid is replaced with another amino acid residue having a different structural or chemical property. Similar minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted or deleted without abolishing immunological activity can be found using computer programs well known in the art.

In addition, in some instances, functional variants may include, but are not limited to, derivatives that are substantially similar in primary structural sequence, but contain in vitro or in vivo chemical and/or biochemical modifications not found in the parent monoclonal antibody.

Additionally, in some examples, chemical and/or biochemical modifications may include phthalylation, covalent attachment of nucleotides or nucleotide derivatives, covalent attachment of lipids or lipid derivatives, cross-linking, disulfide bond formation, glycosylation, hydroxylation, methylation, oxidation, pegylation, proteolytic processing, phosphorylation, and the like.

An immunoconjugate according to a third aspect of this embodiment may comprise at least a monoclonal antibody according to the present disclosure. In this case, the immunoconjugate can be used for in vitro immunoassay or purification of mesothelin protein or a fragment thereof.

In some examples, the immunoconjugate may comprise at least one monoclonal antibody and comprise at least one functional molecule, where the functional molecule may be a detectable label. Alternatively, the monoclonal antibody and functional molecule may be conjugated, attached, crosslinked, etc., by, for example, covalent attachment, coupling, etc.

Additionally, in some examples, the functional molecule may be a label, a marker sequence, or other antibody. In addition, markers may include, but are not limited to: fluorescent markers, chromogenic markers, such as: enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals, nonradioactive paramagnetic metal ions, and the like. In other examples, the functional molecule may be a mixture of multiple labels.

In addition, in some examples, the monoclonal antibodies or immunoconjugates of the disclosure can also be attached to a solid support, which is particularly useful for in vitro immunoassays or purification of mesothelin protein or fragments thereof.

Additionally, in some examples, such solid supports can be porous or non-porous, planar or non-planar. Monoclonal antibodies of the present disclosure may be fused to tag sequences for purification, wherein the tag sequences include, but are not limited to, a hexahistidine tag, a Hemagglutinin (HA) tag, a myc tag, a flag tag.

In addition, in some instances, a monoclonal antibody of the present disclosure may also be conjugated to another antibody to form an antibody heteroconjugate (heteroconjugate).

The nucleic acid molecule according to the fourth aspect of the present embodiment may encode a monoclonal antibody according to the present disclosure. In this case, the nucleic acid molecule may be used as an intermediate in the cloning process. In some examples, the nucleic acid molecule may encode a functional variant to which the disclosure relates. In other examples, the nucleic acid molecule may encode an immunoconjugate of interest.

In addition, in some examples, the DNA sequence of the nucleic acid molecule can be obtained using conventional techniques or using hybridoma technology. For example, the DNA sequence of a nucleic acid molecule can be obtained by artificial synthesis, and particularly when the length of the fragment is short, a fragment with a long sequence can be obtained by synthesizing a plurality of small fragments and then ligating the small fragments. At present, the skilled person can obtain the DNA sequence encoding the monoclonal antibody (or fragment thereof, or derivative thereof) in the present disclosure completely by chemical synthesis, and can introduce mutations into the sequence of the monoclonal antibody in the present disclosure by chemical synthesis.

An expression vector according to the fifth aspect of the present embodiment may include a nucleic acid molecule according to the present disclosure. In this case, the expression vector can be transferred into an appropriate cell, so that the cell can express the monoclonal antibody binding to mesothelin. In addition, in some instances, the expression vector can be transferred into an appropriate cell, such that the cell can express the functional variant contemplated by the present disclosure. In other examples, the expression vector can be transferred into an appropriate cell, such that the cell can express an immunoconjugate according to the present disclosure.

In addition, in this embodiment, the expression vector may further include the nucleic acid molecule of the present disclosure as well as a promoter or control sequence. In addition, in some instances, expression vectors can be constructed by combining the DNA sequences of the nucleic acid molecules of the present disclosure as the gene of interest with a carrier (e.g., a plasmid, phage, etc.) that serves as a vehicle for the nucleic acid molecules of the present disclosure, must be capable of mass replication, and has a restriction enzyme site as well as a marker gene. The expression vector can thus be used to transform an appropriate host cell so that it can express the protein.

The host cell according to the sixth aspect of the present embodiment may include the expression vector according to the present disclosure. In this case, the host cell is capable of expressing a monoclonal antibody that binds to mesothelin. In addition, in some examples, the host cell is capable of expressing the functional variants contemplated by the present disclosure. In other examples, the host cell is capable of expressing an immunoconjugate to which the disclosure relates.

In some examples, the host cell can be a prokaryotic cell, such as a bacterial cell (e.g., escherichia coli, streptomyces, salmonella typhimurium, etc.); or may be eukaryotic cells including yeast cells, plant cells, insect cells, animal cells, and the like, such as COS7, NSO or Bowes melanoma cells, mammalian cells (e.g., CHO cells, 293 cells), and the like. Preferably, the host cell may be a eukaryotic cell. Further preferably, the host cell may be a mammalian cell.

The method for producing a monoclonal antibody that binds to mesothelin according to the seventh aspect of the present embodiment can be performed by culturing the host cell according to the present disclosure. In this case, the monoclonal antibody binding to mesothelin can be isolated and purified by various isolation methods.

In addition, in some examples, functional variants contemplated by the present disclosure can be obtained by culturing a host cell contemplated by the present disclosure. In other examples, the immunoconjugates contemplated by the present disclosure can be obtained by culturing a host cell contemplated by the present disclosure.

In some examples, a monoclonal antibody can be isolated from a nucleic acid molecule encoding at least one monoclonal antibody, functional variant, or immunoconjugate of the disclosure by using the nucleic acid molecule as an intermediate to cloning, cloning into a vector, transferring into a cell, and culturing the host cell.

In addition, in some instances, physical, chemical, or other properties may be exploited to isolate and purify recombinant monoclonal antibodies by various separation methods. These methods are well known to those skilled in the art. For example, conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, a combination of these methods, and the like.

The pharmaceutical composition according to the eighth aspect of the present embodiment may include a monoclonal antibody according to the present disclosure. In this case, the compound can be used for diagnosing, treating and/or preventing mesothelin-positive tumors and other related diseases. Meanwhile, since diseases caused by the disturbance of the interaction between mesothelin and its ligand are known in the art, it can be expected that the monoclonal antibody capable of blocking the interaction between mesothelin and its ligand in the present disclosure has a therapeutic effect on diseases associated with the disturbance of the action of mesothelin and its ligand.

In addition, in the present embodiment, mesothelin-positive tumors may include mesothelin-expressing tumors. In some examples, mesothelin-expressing tumors may include mesothelioma, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, colorectal cancer, gastric cancer, soft tissue tumors, and the like.

In addition, in some examples, an effective amount of a monoclonal antibody of the disclosure can be included in a pharmaceutical composition along with a pharmaceutically acceptable carrier. A therapeutically effective dose means the amount of monoclonal antibody or immunoconjugate that can be used to effectively treat the disease. Therapeutically effective doses can be estimated initially in animal models (e.g., mice, monkeys) and the route of administration can be determined. Furthermore, the term "pharmaceutically acceptable" as used herein means that the molecular entities and compositions do not produce adverse, allergic, or other untoward reactions when properly administered to an animal or human. As used herein, a "pharmaceutically acceptable carrier" should be compatible with, i.e., capable of being blended with, the monoclonal antibodies of the present disclosure without substantially reducing the effectiveness of the composition as is often the case.

In addition, in some examples, suitable dosage ranges for monoclonal antibodies can be 0.001-100mg/kg body weight, e.g., 0.001mg/kg body weight, 0.005mg/kg body weight, 0.01mg/kg body weight, 0.02mg/kg body weight, 0.05mg/kg body weight, 0.1mg/kg body weight, 0.2mg/kg body weight, 0.5mg/kg body weight, 1mg/kg body weight, 2mg/kg body weight, 5mg/kg body weight, 8mg/kg body weight, 10mg/kg body weight, 12mg/kg body weight, 15mg/kg body weight, 20mg/kg body weight, 50mg/kg body weight, 100mg/kg body weight, and the like. Preferably, a suitable dose of monoclonal antibody may be 0.01 to 15mg/kg body weight.

In addition, in some examples, some of the substances of the pharmaceutically acceptable carrier or component thereof may be sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and methyl cellulose; powdered gum tragacanth; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyhydric alcohols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as Tween; wetting agents, such as sodium lauryl sulfate; a colorant; a flavoring agent; tabletting agents, stabilizers; an antioxidant; a preservative; pyrogen-free water; isotonic saline solution; and phosphate buffer, and the like.

In addition, in some instances, the pharmaceutical compositions may be formulated into various dosage forms as desired, and the dosage that is beneficial to the patient may be determined by the physician, depending on such factors as the type, age, weight, and general condition of the patient, the mode of administration, and the like. Administration may be by injection or other therapeutic means, for example. Furthermore, a bolus may be administered, for example, using an injection therapy, multiple divided doses may be administered over time, or the dose may be scaled down or up depending on the urgency of the therapeutic situation.

In addition, in some examples, the pharmaceutical composition may be administered alone, or in combination with other molecules. The other molecules and pharmaceutical composition compositions herein are preferably sterile, such that methods of rendering such molecules and compositions sterile are well known in the art. For other molecules used for diagnosis, prevention or treatment, administration can be according to the monoclonal antibody similar dosing scheme. If other molecules are administered alone, they can be administered to the patient before, concurrently with, or after administration of one or more monoclonal antibodies or pharmaceutical compositions of the disclosure. The precise dosing regimen for a human patient is typically selected during a clinical trial.

In addition, in some examples, monoclonal antibodies may also be used in combination, for example, as a pharmaceutical composition comprising two or more monoclonal antibodies, functional variants, or antigen binding fragments of the present disclosure. For example, monoclonal antibodies having different but complementary activities can be combined in a therapeutic regimen to achieve a desired prophylactic, therapeutic or diagnostic effect, but alternatively monoclonal antibodies having the same activity can be combined in a therapeutic regimen to achieve a desired prophylactic, therapeutic or diagnostic effect.

A kit according to a ninth aspect of the present embodiment may include the monoclonal antibody of the present disclosure. Under the condition, the mesothelin or the content thereof in the sample to be detected can be conveniently, quickly and accurately detected, so that whether the mesothelin receptor-mesothelin excessive interaction exists in the sample to be detected or not can be known. As used herein, the term "test sample" encompasses a variety of sample types, including blood and other bodily fluid samples of biological origin, solid tissue samples such as biopsy tissue samples or tissue cultures, or cells derived therefrom or progeny thereof. And the term also includes samples that have been treated by any means after they have been obtained, for example by treating with reagents, solubilizing, or enriching certain components such as proteins or polynucleotides.

In some examples, the kit may contain the monoclonal antibody or immunoconjugate of the disclosure, and may also contain other detection or auxiliary reagents, such as those conventionally used in ELISA kits, the identity of which, as well as their formulation methods, are well known to those skilled in the art, including chromogenic agents, labels, secondary antibodies, anti-antibodies, sensitizers, and the like. Furthermore, it will be understood by those skilled in the art that various variations of the detection kit are encompassed by the present disclosure, as long as the monoclonal antibody of the present disclosure is utilized therein as a reagent for recognizing mesothelin, and fall within the scope of the present disclosure.

Fig. 2 is a graph showing the results of LK19 specifically recognizing mesothelin protein of tumor cells. Fig. 3 is a graph showing the results of three antibodies, LK19, LK20 and LK423, detecting mesothelin expression on the surface of pancreatic cancer cells. Fig. 4 is a graph showing the results of LK19 detection of mesothelin expression in tumor tissues and paracancer, wherein in fig. 4, the left graph is normal breast tissue, the middle graph is breast cancer tissue, and the right graph is pancreatic cancer tissue.

To further illustrate the present disclosure, the monoclonal antibodies provided by the present disclosure that bind to mesothelin and methods for making the same are described in detail below with reference to the examples, and the beneficial effects achieved by the present disclosure are fully illustrated. The experimental procedures, for which specific conditions are not noted in the following examples, are generally carried out under conventional conditions such as those described in J. SammBruk et al, molecular cloning, A laboratory Manual, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.

[ example 1]

(preparation of mesothelin monoclonal antibody)

(1) Firstly, selecting a mesothelin protein segment sequence, wherein a specific coding sequence is SEQ ID NO: 17; then, the synthetic sequence is SEQ ID NO 18: DELYPQGYPESV, the mesothelin antigen peptide; the antigen peptide is coupled with Bovine Serum Albumin (BSA) to prepare the mesothelin polypeptide.

(2) Intraperitoneal injection of the mesothelin polypeptide prepared in step (1) (about 150 micrograms/mouse) and 30 micrograms of Freund's complete adjuvant into 6-8 weeks old BalB/c female mice (first immunization); a second immunization was performed 2 weeks later; a third immunization was performed 3 weeks later; a fourth immunization was performed after 5 weeks; the fifth immunization was performed 8 weeks. Spleen cells were obtained after 9 weeks under sterile conditions. Then, the spleen cells and myeloma cells are fused under aseptic conditions to obtain fused cells. The inventors performed repeated screening and validation for the large number of fused cells obtained, and further performed antibody screening by ELISA method, thereby selecting 9 antibodies: LK160, LK19, LK20, LK417, LK420, LK423, LK426, LK427, LK428, an antibody, LK19, was selected for sequencing and further analysis. And then culturing the hybridoma cell strain capable of secreting the mesothelin antibody to a logarithmic growth phase, and freezing and storing the hybridoma cell strain in liquid nitrogen for later use.

(3) Liquid paraffin or pristane is pretreated for 6-8 weeks to obtain large BALB/C femaleA mouse. And (3) simultaneously culturing the cell strains obtained in the step (2) in a large amount. 1-2 weeks later, the mice were inoculated with hybridoma cells (1-2X 10)⁷Only). The hybridoma cells proliferate in the mouse abdominal cavity and produce and secrete monoclonal antibodies. The abdomen of the mouse was enlarged about 1 to 2 weeks. The ascites is extracted by a syringe, and a large amount of monoclonal antibodies can be obtained.

[ example 2]

(monoclonal antibody LK19 sequence sequencing)

For the monoclonal antibody LK19, the inventors cloned and sequenced the V region gene of the antibody by TA cloning method, and the sequences of the heavy chain and the light chain are as follows:

nucleotide sequence of heavy chain V region of LK 19: 1, SEQ ID NO;

amino acid sequence of heavy chain V region of LK 19: 2, SEQ ID NO;

the nucleotide sequence of the CDR1 of the heavy chain V region of LK19 is SEQ ID NO. 3;

amino acid sequence of heavy chain V region CDR1 of LK 19: 4, SEQ ID NO;

the LK19 heavy chain V region CDR2 nucleotide sequence is SEQ ID NO 5;

amino acid sequence of heavy chain V region CDR2 of LK 19: 6, SEQ ID NO;

LK19 heavy chain V region CDR3 nucleotide sequence: 7 in SEQ ID NO;

amino acid sequence of heavy chain V region CDR3 of LK 19: 8 in SEQ ID NO;

the nucleotide sequence of the V region of the LK19 light chain: 9, SEQ ID NO;

amino acid sequence of light chain V region of LK 19: 10 in SEQ ID NO;

the light chain V region CDR1 nucleotide sequence of LK19 SEQ ID NO. 11;

amino acid sequence of light chain V region CDR1 of LK 19: 12 is SEQ ID NO;

LK19 light chain V region CDR2 nucleotide sequence: 13 in SEQ ID NO;

amino acid sequence of light chain V region CDR2 of LK 19: 14, SEQ ID NO;

LK19 light chain V region CDR3 nucleotide sequence: 15, SEQ ID NO;

amino acid sequence of light chain V region CDR3 of LK 19: 16 in SEQ ID NO.

[ example 3]

(monoclonal antibody affinity assay)

Loading 1-2 micrograms of mesothelin protein per hole on SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) glue, adding an electrode buffer solution, carrying out 80V electrophoresis for a period of time, and adjusting the voltage to 120V constant voltage electrophoresis value when a protein Marker strip comes out, wherein bromophenol blue just leaves the PAGE glue quickly; after the running of the glue, cutting off the upper concentrated glue, and transferring the SDS-PAGE glue into a film transfer device; on ice, 100V voltage and less than 400 mA current are carried out, and the film is rotated for 1 hour; washing the transferred nitrocellulose membrane once by using TBST, adding a certain amount of 10% skimmed milk-TBST, and sealing for 1 hour; washing with TBST for three times, each time for 5 minutes, shearing a target strip along a protein Marker, adding LK19 monoclonal antibody, and incubating overnight in a shaking table at 4 ℃; and (5) recovering primary antibodies, and developing the darkroom punching sheet.

The results are shown in FIG. 2, and the results indicate that LK19 can be used as a Western Blot primary antibody, and the detection signal of LK19 is strong.

[ example 4]

(monoclonal antibody binding soluble mesothelin)

Adding the antibody into the sample to be detected, adding the anti-mouse IgG-HRP at room temperature for 1 hour, and developing. The results are shown in table 1, and indicate that LK19 can be used as a primary antibody in ELISA assay, and can effectively recognize mesothelin expressed in mammals and mesothelin in plasma.

Table 1: shows that the prepared 9-strain antibody can be used for ELISA (enzyme-linked immunosorbent assay) to detect the content of mesothelin

Color development results	LK160	LK19	LK20	LK417	LK420	LK423	LK426	LK427	LK428
										Self-made mesothelin	4	10	10	10	5	10	9	10	4
Commercial mesothelin	0.5	4	7	8	0.8	7	1	0	0

[ example 5]

(monoclonal antibody binding to Membrane surface mesothelin)

Collecting cells, washing the cells once with PBS, diluting the antibody according to a certain proportion according to each sample of 1 × 106 cells, resuspending each sample with 100 microliters of antibody diluent (2% calf serum), dyeing for 15 minutes in the dark at room temperature, centrifuging for 3 minutes at 300g, washing once with PBS, resuspending 300 microliters of antibody diluent (2% calf serum), and detecting on a machine.

Among them, the inventors used mesothelin monoclonal cell culture supernatant as a primary antibody, and detected mesothelin expression on the cell membrane surface using flow cytometry using a fluorescently labeled secondary antibody.

The results are shown in fig. 3, and indicate that LK19 can be used as a detection primary antibody for FACS, detecting mesothelin expression on the cell surface.

[ example 6]

After fully baking the slices in a 65 ℃ oven, carrying out xylene treatment for three times, each time for 15 minutes; treating with anhydrous ethanol for 3 times, each for 5 min; respectively treating with 90% ethanol, 80% ethanol and 70% ethanol for 5 min, washing with tap water for 15 min, and soaking in double distilled water for 30 min. Treating in citric acid buffer solution at 92-98 deg.C for 15 min, and cooling to room temperature; washing with tap water for 5 minutes; treating with 0.3% hydrogen peroxide for 15 min; PBS wash 4 times, each for 5 minutes; blocking with 10% antiserum for 30 min; one PBS wash followed by four degrees overnight.

Taking out the slices the next day, washing with PBS for 4 times, 5 minutes each time; incubating the secondary antibody for 30 minutes; PBS wash 5 times, each for 5 minutes; preparing ABC staining solution (A: B: PBS 1: 1: 50), and treating for 45 minutes at room temperature; PBS wash 5 times, each for 5 minutes; DAB is developed for 3 to 10 minutes, and the dyeing condition is observed under a microscope; washing with tap water for 5 minutes, after washing with double distilled water once, dyeing cell nuclei with hematoxylin for 5 minutes, washing with tap water for 5 minutes, and washing with double distilled water once; 70%, 80%, 90% ethanol treatment, respectively, once, 100% ethanol treatment 3 times (shaking up and down several times); the dimethylbenzene is respectively treated for three times, and each time lasts for 10 minutes; and sealing and taking a picture.

Among them, the inventors used mesothelin monoclonal cell culture supernatant as a primary antibody, and detected mesothelin expression in tissue cells using immunohistochemistry with HRP-labeled secondary antibody.

The results are shown in fig. 4, and indicate that LK19 can be used as a detection primary antibody for immunohistochemistry to detect mesothelin expression in tissue cells, and the detection signal of LK19 is not expressed in normal breast tissues, but is strongly expressed on tumor cells such as breast cancer and pancreatic cancer.

While the present disclosure has been described in detail above with reference to the drawings and the embodiments, it should be understood that the above description does not limit the present disclosure in any way. Those skilled in the art can make modifications and variations to the present disclosure as needed without departing from the true spirit and scope of the disclosure, which fall within the scope of the disclosure.

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Claims (11)

1. A monoclonal antibody that binds to mesothelin,

the antibody comprises a variable region comprising a heavy chain variable region and a light chain variable region, wherein SEQ ID NO 4 is CDR1 of the heavy chain variable region, SEQ ID NO 6 is CDR2 of the heavy chain variable region, SEQ ID NO 8 is CDR3 of the heavy chain variable region, SEQ ID NO 12 is CDR1 of the light chain variable region, SEQ ID NO 14 is CDR2 of the light chain variable region, and SEQ ID NO 16 is CDR3 of the light chain variable region.

2. The monoclonal antibody of claim 1,

the amino acid sequence of the heavy chain variable region is SEQ ID NO:2,

the amino acid sequence of the light chain variable region is SEQ ID NO. 10.

3. The monoclonal antibody of claim 1 or 2,

also included are heavy chain constant regions and light chain constant regions, wherein,

the heavy chain type of the heavy chain constant region is IgGl, IgG2a, IgG2b or IgG3,

the light chain type of the light chain constant region is a kappa chain or a lambda chain.

4. The monoclonal antibody of claim 1,

the monoclonal antibody is an immunoglobulin molecule or an antigen binding fragment.

5. An immunoconjugate, characterized in that,

comprising the monoclonal antibody of any one of claims 1 to 4.

6. A nucleic acid molecule comprising a nucleic acid sequence,

the nucleic acid molecule encodes the monoclonal antibody of any one of claims 1 to 4.

7. An expression vector characterized in that,

comprising the nucleic acid molecule of claim 6.

8. A host cell characterized by a host cell,

comprising the expression vector of claim 7.

9. A method for preparing a monoclonal antibody which binds to mesothelin,

comprising culturing the host cell of claim 8.

10. A pharmaceutical composition characterized by comprising, in combination,

comprising the monoclonal antibody of any one of claims 1 to 4.

11. A kit of parts, characterized in that,

comprising the monoclonal antibody of any one of claims 1 to 4.

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