CN116063480A - Anti-cytokeratin 18 antibody, preparation method and application thereof - Google Patents
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Abstract
The invention discloses an anti-cytokeratin 18 antibody, a preparation method and application thereof. Wherein the anti-cytokeratin 18 antibody or antigen-binding fragment thereof has the heavy chain variable region and/or the light chain variable region of the amino acid sequences set forth in SEQ ID NO. 1-6. The preparation scheme of the CK18 antibody disclosed by the invention uses the prokaryotic recombinant CK18 full-length antigen, has a relatively complete CK18 dominant epitope, and avoids the problem of less epitope exposure caused by a natural CK18 filiform helical structure. In addition, the immunogen used in the invention has stronger immunogenicity and more exposure of dominant epitopes, so that the affinity of the prepared monoclonal antibody is greatly improved. Meanwhile, the CK18 polypeptide with a specific sequence is selected for further screening and preparing the antibody, so that not only can the omission caused by individual mutation difference be avoided, but also the problem of reduced antibody affinity caused by phosphorylation modification of the CK18 protein can be avoided.
Description
Technical Field
The invention relates to the technical field of biological immunotherapy, in particular to an anti-cytokeratin 18 antibody, a preparation method and application thereof.
Background
Cytokeratin (CK) is the largest, most complex subclass of the intermediate silk protein family of cytoskeleton, with Cytokeratin 18 (CK 18) belonging to one of the members, predominantly distributed in epithelial cells, being the major framework protein of hepatocytes and other epithelial cells. CK18 is an acidic intermediate silk protein of type i that is expressed predominantly in mature epithelial cells, CK8 being its filiform partner. In recent years, with the development of research, CK18 has been increasingly recognized and emphasized not only in the diagnosis, metastasis, recurrence and prognosis of tumors in a plurality of systems, but also in the fields of liver diseases, severe infections and the like. The CK18 full-length recombinant protein is prepared through gene recombination, and monoclonal antibodies are prepared by taking the CK18 full-length recombinant protein as an immunogen, so that a core bioactive raw material can be provided for a CK18 detection kit.
Studies in 1997 by Carlos Cauli n, guy S.Salvesen, and Robert G.Oshima et al have found that structural changes during apoptosis are mediated by proteases of the Caspase (Caspase) family, during which Ck18 IFs recombines into a phosphorylated-rich particle structure. Generating a proteolytic fragment during the drug and uv-induced apoptosis; this fragment binds to K18 cleaved in vitro by caspase-6, -3 and-7. Cleavage of K18 to NH by caspase-6 2 Terminal, 26kd and COOH terminal 22kd fragments; caspase-3 and-7 also cut 22kd fragments into 19kd fragments. The common cleavage site for the three cysteases is the sequence VEVD/A, located in the junction region of CK 18L 1-2. For example, chinese patent application CN111269314a discloses a method for preparing and applying an anti-CK 18 monoclonal antibody, wherein the immunogen is prepared by connecting a CK18 epitope peptide with a carrier protein KLH (keyhole limpet hemocyanin), and the monoclonal antibodies prepared by the method have high epitope homogeneity, but also lose many other CK18 epitopes.
The information in the background section is only for the purpose of illustrating the general background of the invention and is not to be construed as an admission or any form of suggestion that such information forms the prior art that is well known to those of ordinary skill in the art.
Disclosure of Invention
In order to solve the technical problems in the prior art, the inventor prepares and selects specific anti-human CK18 monoclonal antibody sequences through intensive research, and develops in-vitro preparation and production methods of the CK18 monoclonal antibodies. Specifically, the present invention includes the following.
In a first aspect of the invention there is provided an antibody or antigen binding fragment thereof which targets cytokeratin 18, the antibody or antigen binding fragment thereof comprising:
1, wherein the heavy chain comprises the variable regions of the antigen complementarity determining regions CDR1, CDR2, and CDR 3; and/or
2, wherein the light chain comprises the variable regions of the antigen complementarity determining regions CDR1, CDR2, and CDR 3.
In a second aspect of the invention, there is provided an antibody or antigen binding fragment thereof that targets cytokeratin 18, the antibody or antigen binding fragment thereof comprising:
3, wherein the heavy chain comprises the variable regions of the antigen complementarity determining regions CDR1, CDR2, and CDR 3; and/or
4, wherein the light chain comprises the variable regions of the antigen complementarity determining regions CDR1, CDR2, and CDR 3.
In a third aspect of the invention, there is provided an antibody or antigen binding fragment thereof that targets cytokeratin 18, the antibody or antigen binding fragment thereof comprising:
a heavy chain amino acid sequence set forth in SEQ ID No. 5, wherein said heavy chain comprises the variable regions of the antigen complementarity determining regions CDR1, CDR2, and CDR 3; and/or
The light chain amino acid sequence set forth in SEQ ID NO. 6 wherein the light chain comprises the variable regions of the antigen complementarity determining regions CDR1, CDR2 and CDR 3.
The antibody or antigen-binding fragment thereof according to the present invention preferably has any one of the amino acid sequences shown in (I) or (II):
(I) An amino acid sequence having at least 90%, preferably at least 95%, still preferably at least 98%, most preferably at least 99% homology to the amino acid sequences shown in SEQ ID No. 1-6;
(II) an amino acid sequence obtained by modifying, substituting, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO. 1-6;
wherein the variant having the amino acid sequence shown in (I) or (II) still retains the activity of targeting cytokeratin 18.
The antibody or antigen-binding fragment thereof according to the present invention, preferably, the antibody comprises at least one of a monoclonal antibody, a chimeric antibody, a humanized antibody, a bispecific antibody or a multispecific antibody; 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.
In a fourth aspect of the invention, there is provided an isolated nucleic acid molecule encoding an antibody or antigen binding fragment thereof of the invention.
In a fifth aspect of the invention, there is provided a vector comprising a nucleic acid molecule according to the invention.
In a sixth aspect of the invention there is provided a host cell comprising a vector according to the invention.
In a seventh aspect of the invention, there is provided a method of preparing an anti-cytokeratin 18 antibody, the method comprising: a step of introducing a polynucleotide encoding the antibody or antigen-binding fragment thereof into the host cell of the present invention, and culturing the host cell under conditions suitable for expression of the polynucleotide, thereby producing the antibody.
In an eighth aspect of the invention, there is provided a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to the invention, or a nucleic acid molecule according to the invention, or a vector according to the invention, or a host cell according to the invention, and optionally a pharmaceutically acceptable carrier.
In a ninth aspect of the invention, there is provided a method of treating and/or preventing cancer or a tumor in a subject by administering to the subject a therapeutically effective amount of a pharmaceutical composition of the invention.
Preferably, the cancer or tumor includes, but is not limited to: acute Myelogenous Leukemia (AML), multiple Myeloma (MM), chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), hodgkin's lymphoma, gastric cancer peritoneal metastasis, liver cancer, leukemia, renal tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal cancer, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), brain glioma, endometrial cancer, or a combination thereof.
In a tenth aspect of the invention there is provided the use of an antibody or antigen binding fragment thereof according to the invention in combination with other medicaments. Other drugs include, but are not limited to: diagnostic, prophylactic and/or therapeutic agents.
In an eleventh aspect of the invention there is provided the use of an antibody or antigen binding fragment thereof according to the invention in the preparation of a CK18 assay kit.
The excellent technical effects of the present invention include, but are not limited to: according to the preparation scheme of the CK18 antibody, prokaryotic recombinant CK18 full-length antigen is used, all the prokaryotic recombinant CK18 dominant epitopes are full, and the CK18 antigen obtained through prokaryotic recombination after inclusion body renaturation cannot be folded into a natural conformation, so that less epitope exposure caused by a filamentous spiral structure of the natural CK18 is avoided. In addition, in the invention, the immunogen is CK18 full-length recombinant antigen, the immunogenicity is strong, the exposure of dominant epitopes is more, and the affinity of monoclonal antibodies prepared by immunization is higher. In addition, two sections of CK18A247-A299 and A323-368 are selected, the two sections of amino acid sequences have no modification such as phosphorylation and glycosylation, are highly conserved, and the prepared antibody is further screened through the synthesized CK18A247-A299 and A323-368 polypeptides, so that not only can the omission caused by individual mutation difference be avoided, but also the reduction of the affinity of the antibody caused by the phosphorylation modification of the CK18 protein can be avoided.
Drawings
FIG. 1 is a plasmid map used in the present invention.
FIG. 2 is a gel diagram of pET28a-CK18 Ni-NTA renaturation purification. Wherein M represents Transgen BlueplusIV;1 represents Ni-NTA renaturation washing; 2 represents Ni-NTA before purification; 3 represents Ni-NTA after purification.
FIG. 3 shows the results of CK18 antibody activity detection.
Figure 4 shows the results of tail blood titer detection after three-phase immunization of mice.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention. The specific techniques or conditions are not noted in the examples and are carried out according to the techniques or conditions described in the literature in the art (for example, refer to J. Sam Brookfield et al, code Huang Peitang et al, molecular cloning Experimental guidelines, third edition, scientific Press) or according to the product specifications. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
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 invention. In addition, for numerical ranges in the present invention, it is understood that the upper and lower limits of the ranges and each intermediate value therebetween are specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
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 invention belongs. Although only 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 invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
The heavy and light chain variable regions of an antibody typically comprise 3 complementarity determining regions CDRs and 4 framework regions FRs. The four FRs are relatively conserved, while the CDR regions (CDR 1, CDR2 and CDR 3) contain hypervariable regions. The complementarity determining regions are linked by a framework region, and when an antibody is recognized, the FR molecules are curled so that the CDR molecules are adjacent to each other. The complementarity determining region is the binding site of an antibody or antigen binding fragment to an antigen, and thus the sequence of the complementarity determining region determines the specificity of the antibody. As understood in the art, an antibody is a glycoprotein or antigen binding portion thereof comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. 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).
An "antigen binding fragment" as used herein refers to a polypeptide fragment that comprises a portion of an intact antibody, such as an antigen binding or variable region of an intact antibody, and has the property of being able to specifically target CK18. Preferably, it contains at least one CDR of an antibody heavy chain variable region and/or light chain variable region; also preferably, it may contain CDRs 1-3 of the heavy chain variable region and/or CDRs 1-3 of the light chain variable region. Antigen binding fragments may be prepared by a variety of techniques including, but not limited to, hybridoma techniques, or by proteolytic digestion of the intact antibody, or by expression from a host cell containing the antigen binding fragment.
The invention provides the antibody or the antigen binding fragment thereof for targeting CK18, which has good safety and targeting property and can specifically bind human CK18.
Without being bound by or theoretical to, the heavy chain amino acid sequence comprising variable regions CDR1, CDR2, CDR3 and the light chain amino acid sequence comprising variable regions CDR1, CDR2, CDR3 may be randomly selected within the following ranges:
(1) Has the sequence of SEQ ID NO: 1. 3, 5;
(2) Has the sequence of SEQ ID NO: 2. 4, 6.
In the present invention, the antibody or antigen-binding fragment thereof has any one of the amino acid sequences shown in (I), (II) or (III): (I) SEQ ID NO: 1. 3, 5 and the heavy chain amino acid sequence shown in SEQ ID NO: 2. 4, 6; (II) with SEQ ID No.:1-6 has an amino acid sequence having at least 90%, preferably at least 95%, still preferably at least 98%, most preferably at least 99% homology; (III) amino acid sequence obtained by modification, substitution, deletion or addition of one or more amino acids to the amino acid sequence shown in SEQ ID No. 1-6, it being noted that the above-mentioned sequences of homology (sometimes referred to herein as "identity") do not alter the antigen-antibody binding properties, i.e.the amino acid sequences selected from the above still retain the activity of the antibody against the tumor surface antigen CK18.
The amino acid sequence of the present invention may be a sequence obtained by modifying the sequence according to the codon preference of the host according to the coding sequence of a murine antibody. In the present invention, by host codon bias modification is meant that the base sequence is base-substituted according to degenerate codons in order to adapt to the needs of different host expression, the codon bias modification generally does not alter the sequence of the product protein or polypeptide.
In the present invention, the antibody includes at least one of monoclonal antibody, humanized antibody, chimeric antibody and bispecific antibody; the antigen binding fragment is at least one of Fab, F (ab') 2, fd, single chain antibody scFv, disulfide linked Fv (sdFv) or single domain antibody.
Preferably, the antibody further comprises an antibody constant region; also preferably, the antibody constant region is selected from the group consisting of: a constant region of any of IgG1, igG2, igG3, igG4, igA, igM, igE, and IgD.
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, igG4, preferably the heavy chain constant region of IgG 4; the light chain constant region of the antibody constant region is kappa or lambda.
Antibodies of the invention may comprise an Fc region from IgG, e.g., igG1, igG2, igG3, or IgG4.
The term "monoclonal antibody", sometimes referred to herein as "mAb" or "mAb," refers to an immunoglobulin derived from a pure line of cells, having the same structural and chemical properties, specific for a single epitope. Monoclonal antibodies are directed against a single determinant on an antigen, unlike conventional polyclonal antibody preparations (typically with different antibodies directed against different determinants). In addition to their specificity, monoclonal antibodies are beneficial in that they are obtained by hybridoma or recombinant engineering cell culture without intermixing with other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a homogeneous population of antibodies, but this should not be construed as requiring any particular or particular method of producing the antibody.
Variant antibodies are also included within the scope of the invention. The sequence of the variant is not particularly limited in the present invention as long as it has binding properties targeting the CK18 antigen, or an antibody having improved affinity, and other variants having such a sequence can be obtained using methods known in the art and are included in the scope of the present invention. The amino acid sequence of a polypeptide can be modified by one skilled in the art using recombinant methods and/or synthetic chemical techniques for producing variant polypeptides. For example, amino acid substitutions may be used to obtain antibodies with further improved affinity. Alternatively, codon optimization of the nucleotide sequence may be used to improve translational efficiency in expression systems for the production of antibodies. Such variant antibody sequences have 80% or greater (i.e., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater) sequence identity to the sequences recited in the present invention. The sequence identity is calculated relative to the sequences listed in the present invention. Or for optimal alignment, such as by program GAP or BESTFIT using default GAP weights.
The term "modification" as used herein means that amino acid modification does not significantly affect or alter the binding characteristics of an antibody comprising the amino acid sequence. Such modifications include amino acid substitutions, additions and deletions. Preferably, the different residue positions differ by conservative amino acid substitutions. Antibodies of the invention may include glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting/blocking groups, proteolytic cleavage, or non-naturally occurring amino acid modification, and the like.
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 amino acid group with aliphatic-hydroxyl side chains is serine and threonine; the group of amino acids having amide-containing side chains is asparagine and glutamine; amino acids having aromatic side chains are phenylalanine, tyrosine and tryptophan; amino acid groups with basic side chains are lysine, arginine and histidine; and the group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine valine, glutamic acid-aspartic acid and asparagine-glutamine. Thus, one or more amino acid residues in the CDR regions of the antibodies of the invention may be replaced with other amino acid residues from the same side chain family.
Another class of variable region modifications that may be present are mutations of amino acid residues in the CDR1, CDR2 and/or CDR3 regions of VH and/or VL to improve one or more binding characteristics (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 mutation may be a substitution, addition or deletion of an amino acid, but is preferably a substitution. Furthermore, residues in the CDR regions typically do not vary by more than one, two, three, four or five.
The present invention provides an isolated nucleic acid encoding an antibody or antigen-binding fragment thereof as described above.
The present invention provides a vector comprising an isolated nucleic acid according to the present invention. The vector may be an expression vector or a cloning vector, examples of which include, but are not limited to, pET28a, pET32a, pET22b, pET30a, etc., preferably pET28a.
The present invention provides a host cell comprising the vector described above. Suitable host cells for cloning or expressing the DNA include prokaryotic cells, yeast cells, or higher eukaryotic cells. Examples of commonly used prokaryotic host cells include, but are not limited to, E.coli, bacillus subtilis, and the like. Common eukaryotic host cells include yeast cells, insect cells, mammalian cells, and the like.
The present invention provides a method of producing an anti-cytokeratin 18 antibody, comprising the steps of introducing a polynucleotide encoding the antibody or antigen-binding fragment thereof into a host cell of the invention, and culturing the host cell under conditions suitable for expression of the polynucleotide, thereby producing the antibody.
In certain embodiments, the methods of making anti-cytokeratin 18 antibodies of the present invention comprise the steps of:
(1) Preparing recombinant CK18 antigen;
(2) Inoculating the antigen obtained in the step (1) into a mouse to prepare a monoclonal antibody;
(3) Screening positive hybridoma cells for the antigen obtained in the step (1).
Wherein, in step (1), the host bacterium used for expressing the recombinant antigen includes Rosetta (DE 3) or BL21 (DE 3), and the CK18 full-length gene is inserted into the vector. Preferably, the CK18 antigen in the form of inclusion bodies is denatured using a surfactant such as Triton X-100, tween-20, sarkosyl, NP40, etc., more preferably Sarkosyl is used as a denaturing agent. Further preferably, the optimum concentration of denaturing agent is between 0.5% and 1.5%. The recombinant CK18 protein renaturation solution in denatured form contained 10% glycerol, tris, naCl, sarkosyl, EDTA.
In step (2), the amount of antigen inoculated into the laboratory mice is 0-500ug, preferably 100-300ug, and more preferably 200ug. Wherein, the immunological adjuvant used by the antigen is Freund's complete adjuvant.
The invention further provides a medicament or pharmaceutical composition comprising: at least one of said antibody or antigen binding fragment thereof that specifically binds CK18, said isolated nucleic acid, said vector, or said antibody against human CK18 prepared by said method of preparation.
In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
The pharmaceutical compositions may be prepared in the form of lyophilized formulations or aqueous solutions by mixing the active agent of the desired purity with an optional pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is non-toxic to the recipient at the dosage and concentration employed and may include at least one of buffers, antioxidants, preservatives, isotonic agents, stabilizers and surfactants. Furthermore, in order for pharmaceutical compositions to be useful for in vivo administration, they must be sterile. The pharmaceutical composition may be rendered sterile by filtration through a sterile filtration membrane.
In some embodiments, the pharmaceutical composition may contain: at least one additive of cytotoxic agents, chemotherapeutic agents, cytokines, immunosuppressants, growth inhibitors, active agents required for the particular indication to be treated. The specific addition amount of the additive can be adjusted according to actual needs.
The invention also provides the use of an agent comprising an antibody or antigen binding fragment thereof according to the invention, or a nucleic acid molecule according to the invention, or a vector according to the invention, or a host cell according to the invention, in the manufacture of a medicament or formulation for the treatment or amelioration of cancer, said treatment and/or prophylaxis being carried out by administering a therapeutically effective amount of said medicament or formulation to said subject, thereby treating and/or preventing cancer or tumor.
Preferably, the treatment or amelioration of cancer refers to the ability to stimulate or increase immune function in a cancer patient.
Preferably, the cancer is a cancer associated with CK18 expression.
The present invention also provides a method of treating/and 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: comprising at least one of an antibody or antigen binding fragment thereof according to the invention, or a nucleic acid molecule according to the invention, or a vector according to the invention, or a host cell according to the invention.
The terms "subject" and "patient" as used herein are used interchangeably herein to refer to any animal in which an antibody-related formulation or drug, treatment as described herein may be desired. Subjects and patients thus include, but are not limited to: primates (including humans), canines, felines, rats and other mammalian subjects. Preferably, the subject is a human.
In the present invention, the term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (reduce) the progress of an undesired physiological change or disorder, such as an autoimmune disease. Beneficial or desired clinical results include, but are not limited to, results that are either detectable or undetectable, including alleviation of symptoms, diminishment of extent of disease, stabilization of disease state (i.e., not worsening), delay or slowing of disease progression, amelioration or palliation of the disease state, and palliation (whether partial or total). "treatment" also means an extended lifetime as compared to the lifetime expected when not receiving treatment. The need for treatment includes those already suffering from a condition or disorder, as well as those susceptible to a condition or disorder, or those in need of prophylaxis of such a condition or disorder.
The term "effective amount" as used herein means the amount of a drug or pharmaceutical agent that elicits the biological or pharmaceutical response in a tissue, system, animal or human that is being sought, for example, by a researcher or clinician. Furthermore, the term "therapeutically effective amount" means an amount that results in improved treatment, cure, prevention, or alleviation of a disease, disorder, or side effect, or a reduction in the rate of progression of a disease or condition, as compared to a corresponding subject that does not receive such an amount. The term also includes within its scope an amount effective to enhance normal physiological function. In general, effective amounts herein will vary depending upon factors such as the given drug or compound, pharmaceutical formulation, route of administration, type of disease or disorder, subject being treated, and the like, but can still be routinely determined by one of skill in the art. The effective amount of a compound of the present invention can be readily determined by one skilled in the art by conventional methods known in the art.
The invention also provides the use of an antibody or antigen binding fragment thereof according to the invention in combination with other medicaments. Preferably, the other drugs include diagnostic agents, prophylactic agents and/or therapeutic agents.
The antibody provided by the invention can be further applied to the preparation of immune chimeric antigen receptor and immune chimeric antigen receptor modified T cells.
Example 1
This example is the preparation of CK18 monoclonal antibodies.
1. Immunogen preparation
Sequencing correct pET28a-CK18-Rosetta (DE 3) Strain 1:100 was inoculated in 10ml LB+kan medium (plasmid as shown in FIG. 1), after overnight culture, 1:100 is inoculated in 1L LB+Kan culture medium, when the culture is carried out until the OD600 is approximately equal to 0.6, 0.5mM IPTG,220rpm is added, the culture is carried out at 20 ℃ overnight, then thalli are collected, 250ml 50mM Tris-HCl pH8.0+500mM NaCl is added into the fermentation product to be resuspended completely, the fermentation product is placed on ice for precooling for 10min and then is subjected to ultrasonic pyrolysis, the ultrasonic intensity is 300w, the ultrasonic intensity is 1s, the interval is 1s, and the total ultrasonic time is 30min. CK18 was identified as being expressed as inclusion bodies in prokaryotic expression.
The inclusion body pellet after cell disruption was resuspended in 25mM Tris-HCl pH8.0, 125mM NaCl, 0.5% Tween-20, 1mM EDTA, 1M UREA, centrifuged at vertical mix 30min,12000rpm 10min, the supernatant discarded, and washing was repeated twice. The pellet was washed with 25mM Tris-HCl pH8.0, 125mM NaCl, resuspended well and centrifuged at 12000rpm for 10min to remove residual UREA well.
After the inclusion body was washed, the inclusion body was dissolved in 25mM Tris-HCl pH8.0, 125mM NaCl, 1.5% SLS dissolution buffer, and after the dissolution was completed, the inclusion body was centrifuged at 12000rpm for 20 minutes to remove undissolved impurities.
Purifying and renaturating the dissolved supernatant by using a column Ni-NTA, and slowly reducing the SLS concentration to achieve the renaturation purpose. The final optimum washing concentration was 40mM and the optimum eluted imidazole concentration was 200mM. The target protein eluted product was dialyzed into 25mM Tris-HCl pH8.0, 125mM NaCl, 0.1% Tween-20, 50% Glycerol, and the purified recombinant CK18 protein was about 90% pure (FIG. 2).
2. Immunization of mice
Pure BALB/C mice are selected, 50 mug CK18 antigen is immunized for the first time, freund's complete adjuvant is added, and injection is carried out by mutual pushing method for emulsification, and then subcutaneous multipoint injection is carried out.
After ∈3 weeks
The second immunization dose is the same as the first immunization dose, and the incomplete Freund's adjuvant is added for subcutaneous injection;
after ∈3 weeks
The third immunization dose is the same as the third immunization dose, no adjuvant is added, the intraperitoneal injection is carried out, and the titer is measured after 5-7 days of blood sampling;
∈2-3 weeks
Boosting, 100 μg CK18 antigen, intraperitoneal injection,
after ∈3 days
Spleen-taking fusion
3. Cell fusion
Myeloma cells SP2/0 were combined with spleen cells at 1:10, washing with serum-free incomplete culture solution 1 time in a 50ml centrifuge tube, centrifuging at 1200rpm for 8min; the supernatant was discarded and the residual liquid was pipetted. The bottom of the centrifugal tube is lightly flicked to loosen the cell sediment slightly. 1ml of 45% PEG (molecular weight 3250) solution pre-warmed at 37℃was added over 90s with gentle shaking. The mixture was subjected to a water bath at 37℃for 90s.
The incomplete culture solution was added at 37℃to terminate the PEG effect, and 1ml, 2ml, 3ml, 4ml, 5ml and 6ml were added every 2min, respectively. Centrifuge at 800rpm for 6min. The supernatant was supplemented and resuspended in HAT selection medium containing 20% calf serum.
The cells were added to the 96-well plate of the existing feeder cell layer, and 100. Mu.l each well was added. Placing the culture plate at 37deg.C and 5% CO 2 Culturing in an incubator.
4. Cloning and screening of hybridoma cells
Monoclonal hybridoma cells were assayed for reactivity of the cell supernatants of each clone with CK18 recombinant antigen using limiting dilution, and cells from positive wells were transferred to 24-well plates for expansion culture.
5. Epitope screening of CK18 antibodies
0.5ml liquid paraffin was injected intraperitoneally into BALB/C mice 1X 10 weeks after 1-2 weeks 6 And inoculating the positive hybridoma cells for 7-10 days, collecting ascites, and purifying to obtain the antibody.
Antibody epitope screening was performed using CK18A247-A299 and A323-368 polypeptides to screen antibodies to the epitope of interest. Wherein the CK18A 247-299 amino acid sequence is as follows: IMADIRAQYDELARKNREELDKYWSQQIEESTTVVTTQSAEVGAAETTLTELR (SEQ ID NO.: 7); the CK18A 323-368 amino acid sequence is: LREVEARYALQMEQLNGILLHLESELAQTRAEGQRQAQEYEALLNI (SEQ ID NO.: 8).
Example 2
The present example is for antibody activity detection, coating conditions: cold Bao Refeng, CK 18-full length, ED, coating concentration: 1ug/ml 10 ug/plate, coating solution: 0.05M CB 100 ul/well, blocking solution: CEA blocking solution 130 ul/well, reactant concentration: the enzyme concentration is shown in the following table: 1:1000, reaction mode: adding the secondary antibody for 30min and developing for 10min and stopping adding the sample for 30min and the secondary antibody for 30min.
The detection result shows that the 18# -26# antibody has activity.
FIG. 3 shows the results of a CK18-25# antibody activity detection Logistic curve fit, where the equation y= (A-D)/(1+ (x/C)/(B))/(n+D, A=2.91389, B= -8.32167, C=3.60835, D=0.07198, n=0.88425, r2= 0.99918).
Example 3
The present example is a CK18 full length laboratory mouse titer assay, detecting laboratory mouse serum, wherein the coating conditions: cold Bao Refeng, CK18 full length-Ag, coating concentration: 1ug/ml 10 ug/plate, coating solution: 0.05mcb 100 ul/well, blocking solution: CEA blocking solution 130 ul/well, reactant concentration: the enzyme concentration is shown in the following table: 1:1000, reaction mode: adding the secondary antibody for 30min and developing for 10min and stopping adding the sample for 30min and the secondary antibody for 30min.
The experimental result shows that the titer of the 1-4# mice is about 80 ten thousand on average, and the high titer of the 5# mice is 163 ten thousand compared with other mice.
Fig. 4 shows exemplary post-tail blood titer detection results of immunized mice, wherein the Logistic curve fit equation is y= (a-D)/([ (1+ (x/C)/(B) ]ζ+d, where a=2.38848, b= -22.05521, c=5.93496, d=0.03741, n=0.56833, r+2= 0.99183.
While the invention has been described with reference to exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. Various modifications or changes may be made to the exemplary embodiments of the present disclosure without departing from the scope or spirit of the invention. The scope of the claims is to be accorded the broadest interpretation so as to encompass all modifications and equivalent structures and functions.
Claims (12)
1. An antibody or antigen-binding fragment thereof that targets cytokeratin 18, the antibody or antigen-binding fragment thereof comprising:
1, wherein the heavy chain comprises the variable regions of the antigen complementarity determining regions CDR1, CDR2, and CDR 3; and/or
2, wherein the light chain comprises the variable regions of the antigen complementarity determining regions CDR1, CDR2, and CDR 3.
2. An antibody or antigen-binding fragment thereof that targets cytokeratin 18, the antibody or antigen-binding fragment thereof comprising:
3, wherein the heavy chain comprises the variable regions of the antigen complementarity determining regions CDR1, CDR2, and CDR 3; and/or
4, wherein the light chain comprises the variable regions of the antigen complementarity determining regions CDR1, CDR2, and CDR 3.
3. An antibody or antigen-binding fragment thereof that targets cytokeratin 18, the antibody or antigen-binding fragment thereof comprising:
a heavy chain amino acid sequence set forth in SEQ ID No. 5, wherein said heavy chain comprises the variable regions of the antigen complementarity determining regions CDR1, CDR2, and CDR 3; and/or
The light chain amino acid sequence set forth in SEQ ID NO. 6 wherein the light chain comprises the variable regions of the antigen complementarity determining regions CDR1, CDR2 and CDR 3.
4. The antibody or antigen-binding fragment thereof according to any one of claims 1-3, wherein the antibody has any one of the amino acid sequences shown in (I) or (II):
(I) An amino acid sequence having at least 90%, preferably at least 95%, still preferably at least 98%, most preferably at least 99% homology to the amino acid sequences shown in SEQ ID No. 1-6;
(II) an amino acid sequence obtained by modifying, substituting, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO. 1-6;
wherein the variant having the amino acid sequence shown in (I) or (II) still retains the activity of targeting cytokeratin 18.
5. The antibody or antigen-binding fragment thereof of any one of claims 1-4, wherein the antibody comprises at least one of a monoclonal antibody, a chimeric antibody, a humanized antibody, a bispecific antibody, or a multispecific antibody; the antigen binding fragments include Fab fragments, fab ', F (ab') 2 At least one of a fragment, a single chain variable fragment scFv, a scFv-Fc fragment or a single chain antibody ScAb.
6. An isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof according to any one of claims 1-5.
7. A vector comprising the nucleic acid molecule of claim 6.
8. A host cell comprising the vector of claim 7.
9. A method of producing an anti-cytokeratin 18 antibody, comprising the steps of introducing a polynucleotide encoding the antibody or antigen-binding fragment thereof into the host cell of claim 8, and culturing the host cell under conditions suitable for expression of the polynucleotide, thereby producing the antibody; or alternatively
The method comprises the following steps:
(1) Preparing recombinant cytokeratin 18 antigen;
(2) Inoculating the antigen obtained in the step (1) into a mouse to prepare a monoclonal antibody;
(3) Positive hybridoma cells are screened using an antigen comprising the amino acid sequence set forth in SEQ ID No. 7-8.
10. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-5, or a nucleic acid molecule according to claim 6, or a vector according to claim 7, or a host cell according to claim 8, and optionally a pharmaceutically acceptable carrier.
11. Use of an agent in the manufacture of a medicament or formulation for the treatment and/or prophylaxis of cancer or a tumor in a subject, wherein the treatment and/or prophylaxis treats and/or prevents cancer or tumor by administering to the subject a therapeutically effective amount of the medicament or formulation, wherein the agent comprises an antibody or antigen-binding fragment thereof according to any one of claims 1-4, or a nucleic acid molecule according to claim 5, or a vector according to claim 6, or a host cell according to claim 7;
the cancer or tumor comprises: acute Myelogenous Leukemia (AML), multiple Myeloma (MM), chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), hodgkin's lymphoma, gastric cancer peritoneal metastasis, liver cancer, leukemia, renal tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal cancer, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), brain glioma, endometrial cancer, or a combination thereof.
12. Kit for the detection of CK18, characterized in that it comprises an antibody or an antigen-binding fragment thereof and/or a recombinant cytokeratin 18 antigen according to any one of claims 1 to 5,
wherein the antigen comprises the amino acid sequence set forth in SEQ ID No. 7-8.
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