Anti-cytokeratin 18 antibodies, methods of making and uses thereof

Abstract

The application discloses an anti-cellular keratin 18 antibody and a preparation method and application thereof. The anti-cellular keratin 18 antibody or antigen binding fragment thereof has a heavy chain variable region and / or a light chain variable region shown in the amino acid sequences of SEQ ID NO.: 1-6. The preparation scheme of the CK18 antibody in the application uses a prokaryotic recombinant CK18 full-length antigen, has more CK18 dominant epitopes, and avoids the problem of less epitope exposure caused by the filamentous helical structure of natural CK18. In addition, the immunogen used in the application has strong immunogenicity and more dominant epitope exposure, so that the affinity of the prepared monoclonal antibody is greatly improved. Meanwhile, the CK18 polypeptide with a specific sequence is selected to further screen and prepare the antibody, so that the problem of missed detection caused by individual mutation difference can be avoided, and the problem of reduced antibody affinity caused by the phosphorylation modification of the CK18 protein can be avoided.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological immunotherapy, in particular to an anti-cellular keratin 18 antibody and a preparation method and application thereof. BACKGROUND

[0002] Cytokeratin (CK) is the largest and most complex subfamily of the intermediate filament protein family of cytoskeleton, and cytokeratin 18 (CK18) belongs to one of them and is mainly distributed in epithelial cells, which is the main skeletal protein of hepatocytes and other epithelial cells. CK18 is a type I acidic intermediate filament protein mainly expressed in mature epithelial cells, and CK8 is its filamentous partner. In recent years, with the continuous expansion and in-depth research, CK18 not only has significance in the diagnosis, metastasis, recurrence and prognosis of tumors in multiple systems, but also plays an increasingly important role in the fields of liver diseases and severe infections, and is increasingly recognized and valued. By gene recombination to prepare CK18 full-length recombinant protein, and using it as an immunogen to prepare monoclonal antibodies, core biological active raw materials can be provided for CK18 kit detection.

[0003] In 1997, Carlos Caulín, Guy S. Salvesen and Robert G. Oshima et al. found that the structural changes in the process of apoptosis were mediated by the proteases of the caspase family. In the process of apoptosis, Ck18 IFs were reorganized into a granular structure rich in phosphorylation. A proteolytic fragment was produced during drug and ultraviolet light-induced apoptosis; this fragment binds to K18 cleaved by caspase-6, -3 and -7 in vitro. K18 is cleaved by caspase-6 into an NH2-terminal, 26-kd and COOH-terminal 22-kd fragment; caspase-3 and -7 also cleave the 22-kd fragment into a 19-kd fragment. The common cleavage site of the three caspases is the sequence VEVD / A, which is located in the linker region of CK18 L1-2. For example, Chinese patent application CN111269314A discloses a preparation and application method of an anti-CK18 monoclonal antibody, and the immunogen used is prepared by connecting CK18 epitope peptide and carrier protein KLH (keyhole limpet hemocyanin). The monoclonal antibody prepared by this method has high epitope uniformity, but many other epitopes of CK18 are also lost.

[0004] The information in the background section merely serves to explain the general background of the application and should not be considered as admitting or in any form suggesting that this information constitutes prior art common to those skilled in the art. SUMMARY

[0005] In order to solve the technical problems in the prior art, the present inventors have prepared and selected a specific anti-human CK18 monoclonal antibody sequence, and developed an in vitro preparation and production method of the CK18 monoclonal antibody. Specifically, the present application comprises the following contents.

[0006] In a first aspect of the present application, an antibody or an antigen-binding fragment thereof targeting cytokeratin 18 is provided, the antibody or the antigen-binding fragment thereof comprising:

[0007] a heavy chain amino acid sequence shown in SEQ ID NO.: 1, wherein the heavy chain comprises variable regions of antigenic complementarity determining regions CDR1, CDR2 and CDR3; and / or

[0008] a light chain amino acid sequence shown in SEQ ID NO.: 2, wherein the light chain comprises variable regions of antigenic complementarity determining regions CDR1, CDR2 and CDR3.

[0009] In a second aspect of the present application, an antibody or an antigen-binding fragment thereof targeting cytokeratin 18 is provided, the antibody or the antigen-binding fragment thereof comprising:

[0010] a heavy chain amino acid sequence shown in SEQ ID NO.: 3, wherein the heavy chain comprises variable regions of antigenic complementarity determining regions CDR1, CDR2 and CDR3; and / or

[0011] a light chain amino acid sequence shown in SEQ ID NO.: 4, wherein the light chain comprises variable regions of antigenic complementarity determining regions CDR1, CDR2 and CDR3.

[0012] In a third aspect of the present application, an antibody or an antigen-binding fragment thereof targeting cytokeratin 18 is provided, the antibody or the antigen-binding fragment thereof comprising:

[0013] a heavy chain amino acid sequence shown in SEQ ID NO.: 5, wherein the heavy chain comprises variable regions of antigenic complementarity determining regions CDR1, CDR2 and CDR3; and / or

[0014] a light chain amino acid sequence shown in SEQ ID NO.: 6, wherein the light chain comprises variable regions of antigenic complementarity determining regions CDR1, CDR2 and CDR3.

[0015] According to the antibody or the antigen-binding fragment thereof of the present application, preferably, the antibody has any one of the amino acid sequences shown in (I) or (II):

[0016] (I) an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, most preferably at least 99% homology with the amino acid sequences shown in SEQ ID NO.: 1-6;

[0017] (II) an amino acid sequence modified, substituted, deleted or added with one or more amino acids from the amino acid sequences shown in SEQ ID NO.: 1-6;

[0018] wherein the variant having the amino acid sequence shown in (I) or (II) still retains the activity of targeting cytokeratin 18.

[0019] According to the antibody or the antigen-binding fragment thereof of the present application, preferably, the antibody comprises at least one of a monoclonal antibody, a chimeric antibody, a humanized antibody, a bispecific antibody or a multispecific antibody; and the antigen-binding fragment comprises at least one of a Fab fragment, a Fab', a F(ab')2 fragment, a single-chain variable fragment scFv, a scFv-Fc fragment or a single-chain antibody ScAb.

[0020] In a fourth aspect of the present application, an isolated nucleic acid molecule encoding the antibody or the antigen-binding fragment thereof of the present application is provided.

[0021] In a fifth aspect of the present application, a vector comprising the nucleic acid molecule of the present application is provided.

[0022] In a sixth aspect of the present application, a host cell comprising the vector of the present application is provided.

[0023] In a seventh aspect of the present application, a method for preparing an anti-cytokeratin 18 antibody is provided, the method comprising the steps of: introducing a polynucleotide encoding the antibody or the antigen-binding fragment thereof into the host cell of the present application, and culturing the host cell under conditions suitable for expressing the polynucleotide, thereby producing the antibody.

[0024] In an eighth aspect of the present application, a pharmaceutical composition comprising the antibody or the antigen-binding fragment thereof of the present application, or the nucleic acid molecule of the present application, or the vector of the present application, or the host cell of the present application, and optionally a pharmaceutically acceptable carrier is provided.

[0025] In a ninth aspect of the present application, a method for treating and / or preventing cancer or tumor in a subject by administering to the subject a therapeutically effective amount of the pharmaceutical composition of the present application is provided.

[0026] Preferably, the cancer or tumor includes but is not limited to: acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), Hodgkin's lymphoma, gastric cancer, gastric cancer peritoneal metastasis, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal carcinoma, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), brain glioma, endometrial cancer or a combination thereof.

[0027] In a tenth aspect, the present invention provides use of the antibody or antigen-binding fragment thereof according to the present invention in combination with other drugs, including but not limited to diagnostic agents, preventive agents, and / or therapeutic agents.

[0028] In an eleventh aspect of the present invention, there is provided use of the antibody or antigen-binding fragment thereof according to the present invention in preparing a CK18 detection kit.

[0029] The excellent technical effects of the present invention include but are not limited to: In the present invention, the preparation scheme of CK18 antibodies uses prokaryotic recombinant CK18 full-length antigen, which has a relatively complete CK18 dominant epitope, and the CK18 antigen after inclusion body renaturation obtained by prokaryotic recombination will not fold into the natural conformation, thereby avoiding the less exposure of epitopes caused by the filamentous helical structure of natural CK18. In addition, in the present invention, the immunogen used is the CK18 full-length recombinant antigen, which has strong immunogenicity, more dominant epitopes are exposed, and the affinity of the monoclonal antibody prepared by immunization will also be higher. In addition, the present invention selected two sequences of CK18A247-A299 and A323-368. These two amino acid sequences have no modifications such as phosphorylation and glycosylation, and are highly conserved. The prepared antibodies are further screened by synthesizing CK18 A247-A299 and A323-368 polypeptides, which can not only avoid missed detection caused by individual mutation differences, but also avoid the phosphorylation modification of CK18 protein causing a decrease in antibody affinity. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The plasmid map used in the present invention.

[0031] Figure 2 This is a gel map of the Ni-NTA refolding purification of pET28a-CK18. M represents TransgenBlueplusIV; 1 represents Ni-NTA refolding and washing; 2 represents before Ni-NTA purification; and 3 represents after Ni-NTA purification.

[0032] Figure 3 The results of CK18 antibody activity detection are shown.

[0033] Figure 4 The results of the detection of the tailed blood titer of the immunized mice after the third immunization are shown. DETAILED DESCRIPTION

[0034] Various illustrative embodiments of the present application are described in detail herein below with reference to the attached drawings. The present application should not be considered limited to the embodiments described herein, but rather those described herein are presented as particular examples of the application. As such, the detailed description includes specific details for the purpose of providing a thorough understanding of the present application. However, it will be apparent to those skilled in the art that the present application can be practiced without these specific details. In some instances, well-known structures and functions have not been described in detail in order to avoid obscuring the concept of the present application. Unless otherwise defined, 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 application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, exemplary methods and materials are described herein. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. In case of conflict, the content of the present specification will control.

[0035] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, the use of the term "about" in relation to a value or a range of values is intended to include the exact value or range of values as well as any intermediate values or ranges that are within the scope of the present application. The upper and lower limits of these smaller ranges can independently be included or excluded in the range.

[0036] 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 application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. In case of conflict, the content of the present specification will control.

[0037] The heavy chain variable region and the light chain variable region of an antibody typically include three complementarity determining regions (CDRs) and four framework regions (FRs). The four FRs are relatively conserved, while the CDR regions (CDR1, CDR2, and CDR3) contain the hypervariable regions. The CDR regions are connected by the framework regions, which molecularly crimp the CDRs next to each other in recognizing the antibody. The CDR regions are the binding sites of the antibody or antigen-binding fragment to the antigen, and thus the sequence of the CDR regions determines the specificity of the antibody. As understood in the art, an antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. The heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region (CH). The light chain comprises a light chain variable region (VL) and a light chain constant region (CL).

[0038] The "antigen-binding fragment" herein refers to a polypeptide fragment comprising a portion of an intact antibody, such as an antigen-binding region or variable region of an intact antibody, and having the property of being able to specifically target CK18. Preferably, it contains at least one CDR of the variable region of the heavy chain and / or the variable region of the light chain; more preferably, it can contain CDR1-3 of the variable region of the heavy chain and / or CDR1-3 of the variable region of the light chain. The antigen-binding fragment can be prepared by various techniques, including but not limited to hybridoma technology, or proteolytic digestion of an intact antibody protein, or expression production by a host cell containing an antigen-binding fragment.

[0039] The present application provides the above-mentioned antibody or antigen-binding fragment thereof targeting CK18, which has good safety and targeting property and is capable of specifically binding to human CK18.

[0040] Without being limited or bound by theory, the heavy chain amino acid sequence containing variable region CDR1, CDR2, CDR3 and the light chain amino acid sequence containing variable region CDR1, CDR2, CDR3 can be randomly selected within the following ranges:

[0041] (1) having the heavy chain amino acid sequence shown in SEQ ID NO: 1, 3, 5;

[0042] (2) having the light chain amino acid sequence shown in SEQ ID NO: 2, 4, 6.

[0043] In the present application, the antibody or antigen-binding fragment thereof has any one of the amino acid sequences shown in (I), (II) or (III): (I) the heavy chain amino acid sequence shown in SEQ ID NO: 1, 3, 5 and the light chain amino acid sequence shown in SEQ ID NO: 2, 4, 6; (II) an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, most preferably at least 99% homology with the amino acid sequence shown in SEQ ID NO: 1-6; (III) an amino acid sequence obtained by modifying, substituting, deleting or adding one or more than one amino acid to the amino acid sequence shown in SEQ ID NO: 1-6, it should be noted that the above-mentioned homology (sometimes also referred to as "identity" herein) sequence does not change the antigen-binding property of the antibody, i.e. the amino acid sequence selected from the above still retains the activity of the antibody against tumor surface antigen CK18.

[0044] The amino acid sequence in the present application can be obtained by expression after the coding sequence of the mouse-derived antibody is modified according to the host codon bias. In the present application, the modification according to the host codon bias refers to base substitution of the base sequence according to the degenerate codon in order to adapt to the need of expression in different hosts, and the modification of codon bias generally does not change the sequence of the product protein or polypeptide.

[0045] In the present application, the antibody includes at least one of a monoclonal antibody, a humanized antibody, a chimeric antibody, and a bispecific antibody; and the antigen binding fragment is at least one of Fab, F(ab'), F(ab')2, Fd, a single-chain antibody scFv, a disulfide-bonded Fv (sdFv), or a single-domain antibody.

[0046] Preferably, the antibody further includes an antibody constant region; and more preferably, the antibody constant region is selected from the constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.

[0047] Preferably, the heavy chain constant region of the antibody constant region is selected from the heavy chain constant region of any one of IgG1, IgG2, IgG3, and IgG4, and is preferably the heavy chain constant region of IgG4; and the light chain constant region of the antibody constant region is kappa or lambda.

[0048] The antibody of the present application can include an Fc region, which is from IgG, such as IgG1, IgG2, IgG3, or IgG4.

[0049] The term "monoclonal antibody", sometimes also referred to as "monos" or mAb, as used herein refers to an immunoglobulin obtained from a population of identical cells, having identical structural and chemical properties, and specific for a single antigenic determinant. Monoclonal antibodies differ from conventional polyclonal antibody preparations, which typically include different antibodies directed against different determinants. Each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are obtained by culture of hybridoma or recombinant engineered cells, and are not mixed with other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a population of identical antibodies, but this is not to be construed as requiring use of any particular method for production of the antibody.

[0050] Variant antibodies are also included within the scope of the present application. The sequence of the variant is not particularly limited in the present application, as long as it is an antibody having a binding property to target CK18 antigen, or having an improved affinity, and other variants having such a sequence can be obtained using methods known in the art, and are included within the scope of the present application. The skilled person can modify the amino acid sequence of the polypeptide using recombinant methods for producing variant polypeptides and / or synthetic chemical techniques. For example, amino acid substitutions can be used to obtain antibodies having further improved affinity. Alternatively, codon optimization of the nucleotide sequence can be used to improve translation efficiency in the expression system used to produce the antibody. Such variant antibody sequences have 80% or more (i.e. 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater) sequence identity to the sequences recited in the present application. The sequence identity is calculated with respect to the sequences recited in the present application, or when optimally aligned, as by the program GAP or BESTFIT using default gap weights.

[0051] The term "modification" as used herein means that the amino acid modification does not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such modifications include substitutions, additions and deletions of amino acids. Preferably, non-identical residues are different because of conservative amino acid substitutions. The antibodies of the present application can include glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or non-naturally occurring amino acid modifications, etc.

[0052] Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, the group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; the group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; the group of amino acids having amide-containing side chains is asparagine and glutamine; the group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; the group of amino acids having basic side chains is lysine, arginine, and histidine; and the group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acid substitutions groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine valine, glutamate-aspartate, and asparagine-glutamine. Thus, one or more amino acid residues in the CDR regions of the antibodies of the present application can be replaced with other amino acid residues from the same side chain family.

[0053] Another type of variable region modification that can be present is mutation of amino acid residues in the CDR1, CDR2, and / or CDR3 regions of the VH and / or VL to improve one or more binding properties (e.g., affinity) of the antibody of interest. Mutations can be introduced by site-directed mutagenesis or PCR-mediated mutagenesis. Preferably, conservative modifications (as described above) are introduced. The mutations can be substitutions, additions or deletions of amino acids, but are preferably substitutions. Furthermore, the changes in residues in the CDR regions are typically no more than one, two, three, four, or five.

[0054] The present application provides an isolated nucleic acid encoding the antibody or antigen binding fragment thereof as described above.

[0055] The present application provides a vector comprising the isolated nucleic acid described in the present application. The vector can be an expression vector or a cloning vector, examples of which include but are not limited to pET28a, pET32a, pET22b, pET30a, etc., preferably pET28a.

[0056] The present application provides a host cell comprising the vector described above. Suitable host cells for cloning or expression of DNA include prokaryotic cells, yeast cells, or higher eukaryotic cells. Examples of commonly used prokaryotic host cells include but are not limited to Escherichia coli, Bacillus subtilis, etc. Commonly used eukaryotic host cells include yeast cells, insect cells, mammalian cells, etc.

[0057] The present application provides a method for preparing an anti-cytokeratin 18 antibody, the method comprising the steps of introducing a polynucleotide encoding the antibody or antigen binding fragment thereof into the host cell described in the present application, and culturing the host cell under conditions suitable for expression of the polynucleotide, thereby producing the antibody.

[0058] In certain embodiments, the method for preparing an anti-cytokeratin 18 antibody of the present application comprises the following steps:

[0059] (1) preparing a recombinant CK18 antigen;

[0060] (2) inoculating the antigen obtained in step (1) into mice to prepare monoclonal antibodies;

[0061] (3) screening the antigen obtained in step (1) for positive hybridoma cells.

[0062] In step (1), the host bacteria used for expressing the recombinant antigen include Rosetta (DE3) or BL21 (DE3), and the CK18 full-length gene is inserted into a vector. Preferably, the CK18 antigen in the form of denatured inclusion body is denatured using a surfactant such as Triton X-100, Tween-20, Sarkosyl, NP40, and the like, and more preferably, Sarkosyl is used as the denaturant. Further preferably, the optimal concentration of the denaturant is between 0.5% and 1.5%. The renaturation solution of the denatured recombinant CK18 protein contains 10% glycerol, Tris, NaCl, Sarkosyl, EDTA.

[0063] In step (2), the amount of antigen inoculated into the experimental mouse is 0-500 ug, preferably 100-300 ug, and more preferably 200 ug. The immunoadjuvant used for the antigen is Freund's complete adjuvant.

[0064] The present application further provides a medicament or a pharmaceutical composition comprising at least one of the antibody or antigen-binding fragment thereof specifically binding to CK18, the isolated nucleic acid, the vector, or the antibody against human CK18 prepared by the preparation method.

[0065] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0066] The pharmaceutical composition can be prepared by mixing the active agent having the desired purity with an optional pharmaceutically acceptable carrier in the form of a lyophilized preparation or an aqueous solution. The pharmaceutically acceptable carrier is non-toxic to the recipient at the used dose and concentration, and can include at least one of a buffer, an antioxidant, a preservative, an isotonic agent, a stabilizer, and a surfactant. In addition, in order for the pharmaceutical composition to be useful for in vivo administration, they must be sterile. The pharmaceutical composition can be rendered sterile by filtration through a sterile filtration membrane.

[0067] In some embodiments, the pharmaceutical composition can contain at least one additive of a cytotoxic agent, a chemotherapeutic agent, a cytokine, an immunosuppressant, a growth inhibitor, and an active agent required for the specific indication to be treated. The specific amount of additive added can be adjusted according to the actual need.

[0068] The present application also provides the use of a reagent in the preparation of a medicament or preparation for treating or ameliorating cancer, the treatment and / or prevention being achieved by administering a therapeutically effective amount of the medicament or preparation to the subject, thereby treating and / or preventing cancer or tumor, wherein the reagent includes the antibody or antigen-binding fragment thereof according to the present application, or the nucleic acid molecule according to the present application, or the vector according to the present application, or the host cell according to the present application.

[0069] Preferably, the treatment or amelioration of cancer means the ability to stimulate or enhance immune function in a cancer patient.

[0070] Preferably, the cancer means a cancer associated with CK18 expression.

[0071] The present application also provides a method of treating / preventing cancer or tumor comprising the step of administering to a subject in need thereof a therapeutically effective amount of a medicament, wherein the medicament comprises at least one of: an antibody or antigen-binding fragment thereof according to the present application, or a nucleic acid molecule according to the present application, or a vector according to the present application, or a host cell according to the present application.

[0072] The terms "subject" and "patient" are used interchangeably and refer here to any animal that can be treated with the antibody-related preparations or medicaments, treatments described herein. Subjects and patients therefore include, but are not limited to, primates (including humans), canine, feline, murine, and other mammalian subjects. Preferably, the subject is a human.

[0073] In the present application, the term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the progress of an autoimmune disease. Beneficial or desired results include, but are not limited to, one or more of the following: alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those in which the condition or disorder is to be prevented.

[0074] The term "effective amount" as used herein means that amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal, or human that is being sought by a researcher, veterinarian, medical doctor or other clinician. Furthermore, the term "therapeutically effective amount" means that amount of a drug or pharmaceutical agent that, when administered to a subject in need thereof, will cure or cause the regression of the disease, condition, or disorder, or will prevent or lessen the disease, condition, or disorder, or will decrease the rate of progression of the disease or condition. The term also includes within its scope amounts effective to enhance normal physiological function. In general, the effective amount herein will vary according to factors such as the given drug or compound, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the subject to be treated, and the like, but can nevertheless be routinely determined by one of ordinary skill in the art. Effective amounts of the compounds of the present application can be readily determined by one of ordinary skill in the art by routine methods known in the art.

[0075] The present application also provides the use of the antibody or antigen binding fragment thereof according to the present application in combination with other drugs. Preferably, the other drugs include diagnostic agents, prophylactic agents and / or therapeutic agents.

[0076] The antibody provided by the present application can be further applied to the preparation of immune chimeric antigen receptor and immune chimeric antigen receptor modified T cells.

[0077] Example 1

[0078] This example is the preparation of CK18 monoclonal antibody.

[0079] 1. Preparation of immunogen

[0080] The pET28a-CK18-Rosetta(DE3) strain correctly sequenced was inoculated into 10ml LB+kan medium (plasmid as shown in Figure 1 After overnight culture, 1:100 was inoculated into 1L LB+Kan medium, and cultured to OD600≈0.6, then 0.5mM IPTG was added, and the bacteria were cultured at 220rpm and 20℃ overnight, then the bacteria were collected, the fermentation product was completely resuspended in 250ml 50mM Tris-HCl pH8.0+500mM NaCl, and pre-cooled on ice for 10min, then ultrasonic lysis was performed, the ultrasonic power was 300w, the ultrasonic time was 1s, the interval was 1s, and the total ultrasonic time was 30min. It was identified that CK18 was expressed in the form of inclusion body in prokaryotic expression.

[0081] The inclusion body precipitate after the bacteria were broken was resuspended using 25mM Tris-HCl pH8.0, 125mM Nacl, 0.5% Tween-20, 1mM EDTA, 1M UREA, vertically mixed for 30min, centrifuged at 12000rpm for 10min, the supernatant was discarded, and the washing was repeated twice. The precipitate was washed using 25mM Tris-HCl pH8.0, 125mM Nacl, and resuspended completely, then centrifuged at 12000rpm for 10min to completely remove the residual UREA.

[0082] After the inclusion body was washed, 25mM Tris-HCl pH8.0, 125mM Nacl, 1.5% SLS solubilization buffer was used for solubilization, and after complete solubilization, 12000rpm, 20min, centrifugation was performed to remove unsolubilized impurities.

[0083] The supernatant after dissolution was columned on Ni-NTA for purification and renaturation, and renaturation was achieved by slowly reducing the SLS concentration. The final optimal washing concentration was 40 mM, and the optimal elution imidazole concentration was 200 mM. The eluted product of the target protein was dialyzed into 25 mM Tris-HCl pH 8.0, 125 mM Nacl, 0.1% Tween-20, 50% glycerol, and the purity of the purified recombinant CK18 protein was about 90%. Figure 2

[0084] 2. Mouse immunization

[0085] Purebred BALB / C mice were selected, and 50 μg of CK18 antigen was used for the first immunization. After emulsification by syringe mutual pushing with Freund's complete adjuvant, it was injected subcutaneously in multiple points.

[0086] After 3 weeks

[0087] The second immunization dose was the same as above, and Freund's incomplete adjuvant was added for subcutaneous injection.

[0088] After 3 weeks

[0089] The third immunization dose was the same as above, and no adjuvant was added for intraperitoneal injection. Blood was collected 5-7 days later to measure the titer.

[0090] After 2-3 weeks

[0091] Booster immunization, dose 100 μg CK18 antigen, intraperitoneal injection,

[0092] After 3 days

[0093] Spleen fusion

[0094] 3. Cell fusion

[0095] Myeloma cells SP2 / 0 were mixed with spleen cells at a ratio of 1:10 in a 50 ml centrifuge tube, washed once with serum-free incomplete culture medium, centrifuged at 1200 rpm for 8 min, the supernatant was discarded, and the residual liquid was sucked up with a pipette. The bottom of the centrifuge tube was lightly tapped to slightly loosen the cell sediment. 1 ml of 37°C pre-warmed 45% PEG (molecular weight 3250) solution was added within 90 s, and the mixture was gently shaken. 37°C water bath for 90 s.

[0096] 37°C pre-warmed incomplete culture medium was added to terminate the PEG effect, and 1 ml, 2 ml, 3 ml, 4 ml, 5 ml and 6 ml were added every 2 min, respectively. Centrifugation, 800 rpm, 6 min. Supplement the supernatant, resuspend with HAT selection medium containing 20% calf serum.

[0097] ​The above cells were added to a 96-well plate with a feeder cell layer, 100 μl per well. The plate was incubated at 37°C in a 5% CO2 incubator.

[0098] 4. Cloning and screening of hybridoma cells

[0099] The hybridoma cells were monoclonalized by limited dilution, and the reaction activity of the supernatant of each clone with CK18 recombinant antigen was detected. The cells in the positive well were transferred to a 24-well plate for expansion culture.

[0100] 5. Epitope screening of CK18 antibody

[0101] BALB / C mice were injected intraperitoneally with 0.5 ml of liquid paraffin, and 1-2 weeks later, 1 x 10 6 The ascites were collected 7-10 days after inoculation of the cells, and the antibody was purified.

[0102] The CK18 A247-A299 and A323-368 polypeptides were used for antibody epitope screening, and the antibody with the target epitope was screened. The CK18 A247-299 amino acid sequence is: IMADIRAQYDELARKNREELDKYWSQQIEESTTVVTTQSAEVGAAETTLTELR (SEQ ID NO.: 7); and the CK18 A323-368 amino acid sequence is: LREVEARYALQMEQLNGILLHLESELAQTRAEGQRQAQEYEALLNI (SEQ ID NO.: 8).

[0103] Example 2

[0104] This example is for antibody activity detection. The coating conditions are as follows: cold packaging and hot sealing, CK18-full length, ED, coating concentration: 1 ug / ml 10 ug / plate, coating solution: 0.05 M CB 100 ul / well, blocking solution: CEA blocking solution 130 ul / well, reactant concentration: see the table below, enzyme concentration: 1:1000, reaction mode: sample addition 30 min + secondary antibody 30 min + color development 10 min + termination.

[0105]

[0106]

[0107] The detection results show that the antibodies 18#-26# all have activity.

[0108] Figure 3The CK18-25# antibody activity detection Logistic curve fitting results are shown, wherein the equation is: y = (A-D) / [(1+(x / C)^B)]^n+D, A = 2.91389, B = -8.32167, C = 3.60835, D = 0.07198, n = 0.88425, r^2 = 0.99918.

[0109] Example 3

[0110] This example is a CK18 full-length experimental mouse titer detection, and the experimental mouse serum is detected, wherein the coating conditions are: cold packaging and hot sealing, CK18 full-length-Ag, coating concentration: 1 ug / ml 10 ug / plate, coating solution: 0.05M CB 100 ul / well, blocking solution: CEA blocking solution 130 ul / well, reactant concentration: see the table below, enzyme concentration: 1:1000, reaction mode: sample addition 30 min + secondary antibody 30 min + color development 10 min + termination.

[0111]

[0112] The experimental results show that the average mouse titer of 1-4# is about 800 million, and the high titer of 5# mouse is 163 million compared with other mice.

[0113] Figure 4 Exemplary immunized mouse tail blood titer detection results after three immunizations are shown, wherein the Logistic curve fitting equation is: y = (A-D) / [(1+(x / C)^B)]^n+D, wherein A = 2.38848, B = -22.05521, C = 5.93496, D = 0.03741, n = 0.56833, r^2 = 0.99183.

[0114] Although the present application has been described with reference to exemplary embodiments, it is understood that the present application is not limited to the disclosed exemplary embodiments. Various adjustments or changes can be made to the exemplary embodiments of the present application without departing from the scope or spirit of the present application. The scope of the claims should be based on the broadest interpretation to encompass all modifications and equivalent structures and functions.

Claims

1. An antibody or antigen-binding fragment thereof, characterized in that: It targets cytokeratin 18, and the antibody or antigen-binding fragment thereof comprises: The heavy chain variable region amino acid sequence shown in SEQ ID NO.: 1, wherein the heavy chain variable region comprises antigen complementarity determining regions CDR1, CDR2 and CDR3; and The amino acid sequence of the light chain variable region shown in SEQ ID NO.: 2, wherein the light chain variable region comprises antigen complementarity determining regions CDR1, CDR2 and CDR3.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody includes at least one of a monoclonal antibody, a chimeric antibody, a bispecific antibody or a multispecific antibody; the antigen-binding fragment includes at least one of a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a single-chain variable fragment scFv or a scFv-Fc fragment.

3. An isolated nucleic acid molecule, characterized in that It encodes the antibody or antigen-binding fragment thereof according to claim 1.

4. A carrier, characterized in that It comprises the nucleic acid molecule according to claim 3.

5. A host cell, characterized in that It comprises the vector according to claim 4.

6. A method for preparing an anti-cytokeratin 18 antibody, characterized in that: The method comprises the step of culturing the host cell according to claim 5 to produce the antibody.

7. Use of the antibody or antigen-binding fragment thereof according to claim 1 or 2 in the preparation of a CK18 detection kit.

8. A CK18 detection kit, characterized in that: The method comprises the antibody or antigen-binding fragment thereof according to claim 1 or 2.

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