CN118240076A - PMEPA1 specific antibody and application thereof - Google Patents

PMEPA1 specific antibody and application thereof Download PDF

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Publication number
CN118240076A
CN118240076A CN202410165870.8A CN202410165870A CN118240076A CN 118240076 A CN118240076 A CN 118240076A CN 202410165870 A CN202410165870 A CN 202410165870A CN 118240076 A CN118240076 A CN 118240076A
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pmepa1
antibody
specific antibody
binding fragment
amino acid
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靳建荣
秦龙
文飞
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Gansu Yimin Biotechnology Development Co ltd
Lanzhou University Second Hospital
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Gansu Yimin Biotechnology Development Co ltd
Lanzhou University Second Hospital
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Abstract

The invention relates to the technical field of biology and medicine, in particular to a PMEPA1 specific antibody. The PMEPA 1-specific antibody of the present invention can specifically bind to PMEPA1 and significantly inhibit proliferation of gastric cancer cells, and is therefore particularly suitable for detecting expression of PMEPA1 in a subject and for treating gastric cancer with PMEPA1 as a target.

Description

PMEPA1 specific antibody and application thereof
Technical Field
The invention relates to the technical field of biology and medicine, in particular to a PMEPA1 specific antibody and application thereof.
Background
Gastric cancer is one of the major diseases threatening the life health of humans. Surgical resection is one of the most effective modes of treatment, however most patients are diagnosed at a mid-to-late stage and are not suitable for further surgical treatment. In recent years, molecular targeted therapy and chemotherapy are widely applied to the treatment of middle and late stage gastric cancer, but the problems of large individual curative effect difference, drug resistance and the like still exist. Therefore, many researches at present focus on exploring potential molecular mechanisms of gastric cancer, and provide potential molecular markers for early diagnosis and clinical treatment of gastric cancer.
It has been found that androgen induction of prostate transmembrane protein 1 (Prostate Transmembrane Protein Androgen Induced, pmepa1) expression in gastric cancer tissue is significantly up-regulated, knockdown of PMEPA1 reduces proliferation capacity of gastric cancer cells and inhibits migration and invasion of gastric cancer cells, while overexpression of PMEPA1 produces the opposite effect. Apoptosis experiments prove that the PMEPA1 has the effect of inhibiting the apoptosis of gastric cancer cells. Indicating that PMEPA1 can be a potential molecular marker and therapeutic target of gastric cancer.
Disclosure of Invention
The present invention aims to provide a novel PMEPA1 specific antibody which is capable of binding to a PMEPA1 protein with high affinity.
The invention also aims to provide the application of the PMEPA1 specific antibody in gastric cancer treatment.
Specifically, in one aspect, the present invention provides a PMEPA1 specific antibody or antigen binding fragment thereof, comprising a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 1.2 and 3, HCDR1, HCDR2 and HCDR3, the light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 6. LCDR1, LCDR2 and LCDR3 shown in fig. 7 and 8.
In one embodiment, the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO:4, and the light chain variable region has the amino acid sequence set forth in SEQ ID NO: 9.
In one embodiment, the PMEPA1 specific antibody or antigen binding fragment thereof is a monoclonal antibody, chimeric antibody, humanized antibody, fully human antibody, fab ', fv fragment, F (ab') 2, scFv or di-scFv.
In one embodiment, the PMEPA1 specific antibody or antigen binding fragment thereof is an scFv.
In one embodiment, the PMEPA1 specific antibody or antigen binding fragment thereof specifically binds human PMEPA1.
In one embodiment, the PMEPA1 specific antibody or antigen binding fragment thereof further comprises a linker connecting the heavy chain variable region and the light chain variable region.
In one embodiment, the linker has the sequence set forth in SEQ ID NO:11, and a polypeptide comprising the amino acid sequence shown in seq id no.
In one embodiment, the PMEPA1 specific antibody or antigen binding fragment thereof has the amino acid sequence as set forth in SEQ ID NO:12, and a polypeptide having the amino acid sequence shown in FIG. 12.
In another aspect, the invention provides an isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof as described herein.
In another aspect, the invention provides an expression vector comprising a nucleic acid molecule as described herein.
In another aspect, the invention provides a host cell comprising an expression vector as described herein.
In another aspect, the invention provides a conjugate comprising a PMEPA1 specific antibody or antigen binding fragment thereof as described herein and a coupling moiety.
In one embodiment, the coupling moiety is selected from the group consisting of protein tags, such as His, flag, GST, MBP, HA, myc, GFP; a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin; therapeutic agents, such as anti-inflammatory drugs or immunosuppressants; or, an additional biologically active polypeptide.
In another aspect, the invention provides a pharmaceutical composition comprising a PMEPA1 specific antibody or antigen binding fragment thereof, a nucleic acid molecule, an expression vector, a host cell or conjugate as described herein, and a pharmaceutically acceptable carrier and/or excipient.
In another aspect, the invention provides the use of a PMEPA1 specific antibody or antigen binding fragment thereof, a nucleic acid molecule, an expression vector, a host cell or a conjugate as described herein in the manufacture of a medicament for detecting expression of PMEPA1 in a sample or for preventing and/or treating a cancer associated with PMEPA 1.
In one embodiment, the cancer associated with PMEPA1 is selected from gastric cancer.
Definition of the definition
As used herein, the term "antibody" refers to an immunoglobulin molecule capable of specifically binding a target (e.g., a carbohydrate, polynucleotide, lipid, polypeptide, etc.) through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term includes not only whole polyclonal or monoclonal antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, but also antigen binding fragments thereof (e.g., fab ', F (ab') 2, fv fragments, single chain (e.g., scFv, di-scFv, (scFv) 2) and domain antibodies (including, e.g., shark and camelbody), as well as fusion proteins comprising antibodies, and any other modified configuration of immunoglobulin molecules comprising an antigen recognition site.
As used herein, the term "antigen binding fragment" includes antigen compound binding fragments of such antibodies, including Fab, F (ab') 2, fd, fv, scFv, antibody minimal recognition units, and single chain derivatives of such antibodies and fragments, e.g., scFv-Fc, and the like, preferably scFv.
The term "scFv" means a molecule comprising an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker, which retains the ability to bind an antigen. Such scFv molecules may have the general structure: NH 2-VL-linker-VH-COOH or NH 2-VH-linker-VL-COOH. As used herein, the linker used in the scFv is not limited and may be any linker known in the art for linking VH and VL in scFv. As an example, the transmembrane domain may have a sequence as set forth in SEQ ID NO:11, and a polypeptide comprising the amino acid sequence shown in seq id no.
As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in an antibody variable region that are responsible for antigen binding. Three CDRs are contained in the antibody and are designated CDR1, CDR2 and CDR3. The exact boundaries of these CDRs may be defined according to various numbering systems known in the art, e.g. as in the Kabat numbering system, chothia numbering system or IMGT numbering system. For a given antibody, one skilled in the art will readily identify the CDRs defined by each numbering system. And, correspondence between different numbering systems is well known to those skilled in the art.
As used herein, the term "framework region" or "FR" residues refer to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.
As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed. The strength or affinity of a specific binding interaction may be represented by the equilibrium dissociation constant (K D) of the interaction. In the present invention, the term "K D" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and antigen.
The specific binding properties between two molecules can be determined using methods well known in the art. One method involves measuring the rate of antigen binding site/antigen complex formation and dissociation. Both the "binding rate constant" (k a or k on) and the "dissociation rate constant" (k dis or k off) can be calculated from the concentration and the actual rate of association and dissociation (see MALMQVIST M, nature,1993, 361:186-187). The ratio of K dis/kon is equal to the dissociation constant K D (see Davies et al Annual Rev Biochem,1990; 59:439-473). The K D、kon and K dis values may be measured by any effective method. In certain embodiments, the dissociation constant may be measured in Biacore using Surface Plasmon Resonance (SPR). In addition to this, bioluminescence interferometry or Kinexa can be used to measure the dissociation constant.
In some embodiments, the invention also provides variants of a PMEPA1 specific antibody as described herein that have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence of a PMEPA1 specific antibody as described herein and substantially retain the biological function of the antibody from which it is derived (e.g., the biological activity of specifically binding to PMEPA 1).
More specifically, the variants differ from the PMEPA1 specific antibodies as described herein only in conservative substitutions of one or more (e.g., conservative substitutions of up to 20, up to 15, up to 10, up to 5, or up to 1) amino acid residues.
As used herein, the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matched positions shared by the two sequences divided by the number of positions to be compared x 100. For example, if 6 out of 10 positions of two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions in total are matched). Typically, the comparison is made when two sequences are aligned to produce maximum identity. Such alignment may be conveniently performed using, for example, a computer program such as the Align program (DNAstar, inc.) Needleman et al (1970) j.mol.biol.48: 443-453. The percent identity between two amino acid sequences can also be determined using the algorithm of E.Meyers and W.Miller (Comput. ApplBiosci.,4:11-17 (1988)) which has been integrated into the ALIGN program (version 2.0), using the PAM120 weight residue table (weight residue table), the gap length penalty of 12 and the gap penalty of 4. Furthermore, percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J mobiol. 48:444-453 (1970)) algorithm that has been incorporated into the GAP program of the GCG software package (available on www.gcg.com) using the Blossum 62 matrix or PAM250 matrix and the GAP weights (GAP WEIGHT) of 16, 14, 12, 10, 8, 6 or 4 and the length weights of 1,2,3, 4,5 or 6.
As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the desired properties of a protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions may be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions that replace an amino acid residue with an amino acid residue having a similar side chain, such as substitutions with residues that are physically or functionally similar (e.g., of similar size, shape, charge, chemical nature, including the ability to form covalent or hydrogen bonds, etc.) to the corresponding amino acid residue. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative substitutions of amino acids are well known in the art (see, e.g., brummell et al, biochem.32:1180-1187 (1993); kobayashi et al Protein Eng.12 (10): 879-884 (1999); and Burks et al Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).
As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes, such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC), or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, papilloma vacuolation virus (e.g., SV 40). A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication origin.
As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, a prokaryotic cell such as e.g. escherichia coli or bacillus subtilis, a fungal cell such as e.g. yeast cells or aspergillus, an insect cell such as e.g. S2 drosophila cells or Sf9, or an animal cell such as e.g. fibroblasts, CHO cells, COS cells, NSO cells, heLa cells, BHK cells, HEK 293 cells or other human cells. Host cells may include single cells or cell populations. Host cells may include immune cells. Immune cells include T cells, B cells, NK cells, monocytes, macrophages or dendritic cells or any combination thereof.
The vector may be introduced into the host cell by conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E.coli, competent cells, which are capable of absorbing DNA, can be obtained after an exponential growth phase and treated by the CaCl 2 method using procedures well known in the art. Another approach is to use MgCl 2. Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods may be used: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.
As used herein, the term "pharmaceutically acceptable carrier and/or excipient" refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and active ingredient, which is well known in the art and includes, but is not limited to: pH modifiers, surfactants, adjuvants, ionic strength enhancers, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives.
As used herein, the term "preventing" refers to a method performed in order to prevent or delay the occurrence of a disease or disorder or symptom (e.g., a disease associated with high expression of PMEPA 16) in a subject. As used herein, the term "treatment" refers to a method that is performed in order to obtain beneficial or desired clinical results. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., no longer worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and diminishment of symptoms (whether partial or total), whether detectable or undetectable. Furthermore, "treatment" may also refer to an extension of survival compared to the expected survival (if not treated).
As used herein, the term "subject" refers to a mammal, such as a primate mammal, e.g., a human.
As used herein, the term "sample" may be any suitable sample from a subject to be tested for expression of PMEPA1, e.g. the sample may be a tissue from the subject, a cell isolated from a tissue, a blood or serum sample, etc.
As used herein, "a cancer associated with PMEPA 1" refers to any cancer associated with PMEPA1 and/or PMEPA1 expressing cells.
As used herein, "cancer" refers to any medical condition characterized by malignant cell growth or tumor, abnormal proliferation, infiltration, or metastasis, may be benign or malignant, and includes both solid tumors and non-solid cancers (e.g., hematological malignancies), such as leukemia. As used herein, "solid tumor" refers to a solid mass of neoplastic and/or malignant cells.
In particular, the cancer associated with PMEPA1 may be selected from gastric cancer.
Advantageous effects of the invention
The PMEPA1 specific antibody provided by the invention can be specifically combined with PMEPA1 and can obviously inhibit the proliferation of gastric cancer cells, so that the PMEPA1 specific antibody is particularly suitable for detecting the expression of PMEPA1 in a subject and treating gastric cancer by taking the PMEPA1 as a target point.
Drawings
FIG. 1 shows the results of flow cytometry detection of PMEPA1 specific antibodies selected in example 1.
FIG. 2 shows the ELISA detection results of the PMEPA1 specific antibodies selected in example 1.
FIG. 3 shows the effect of the PMEPA 1-specific antibodies selected in example 1 on gastric cancer cell proliferation.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Example 1: screening of PMEPA 1-specific antibodies
Expression and purification of PMEPA1 recombinant protein: full length PMEPA1 gene CDS sequence was amplified from a human PMEPA1 gene CDS sequence template and cloned into pET-28a vector (supplied by the university of Lanzhou second Hospital Sun Hui) to obtain recombinant plasmid pET-28a-PMEPA1 (full length). The recombinant plasmid is transformed into BL21 competent cells, and protein expression is induced by IPTG after culturing until OD600 apprxeq 0.6-0.8. And ultrasonically crushing the thalli, collecting inclusion bodies, washing, dissolving, dialyzing and renaturating the inclusion bodies, and purifying Ni-NTA protein to obtain the PMEPA1 recombinant protein.
Immunization of mice: 200 μg of purified BCA-quantified PMEPA1 recombinant protein (with the addition of an appropriate amount of natural immunoadjuvant, final concentration of 1 mg/mL) was taken and the first mouse immunization was performed by intraperitoneal injection. After 14 days, a second immunization with 100. Mu.g of the PMEPA1 full-length recombinant protein was performed, and after 28 days, a third immunization with 50. Mu.g of the PMEPA1 full-length recombinant protein was performed for a total of 42 days, and the immunization was performed in the same manner as described above. Mice were blood samples immunized for 30 days for ELISA assays to determine serum antibody titers.
Microfluidic sorting constructs a positive B cell pool: after the mice after the immunization are killed, spleens of the mice are taken, B lymphocytes are enriched and separated by utilizing a magnetic bead sorting technology, and positive B cells expressing high-affinity antibodies are enriched and separated based on a microfluidics technology.
Screening for high affinity and high specificity PMEPA1 scFv: and (3) reverse transcription is carried out on mRNA of the enriched positive B cells based on an autonomous optimized single-cell reverse transcription technology, antibody variable region sequences of the B cells are amplified by combining nested PCR, temperature gradient PCR and touchdown PCR technologies, and the antibody sequences are cloned to a Lenti-cmv-DP3 display vector to complete the preparation of the scFv library. And packaging the PMEPA1 antibody sequence plasmid into 293T cells by adopting a lentiviral packaging system, thereby obtaining the PMEPA1 single-chain antibody cell display library. Based on a flow cytometry detection means and an enzyme-linked immunosorbent assay, a high-affinity and high-specificity antibody aiming at PMEPA1 is screened, and the amino acid sequence of the antibody is shown as SEQ ID NO:12, the nucleotide sequence is shown as SEQ ID NO: shown at 13.
Example 2: preparation of PMEPA 1-specific antibodies
(1) Cell culture: 293T cells in the logarithmic growth phase were diluted to a density of 4X 10 5/mL after digestion with Trypsin and seeded in 2L roller bottles; adding 200ml of DMEM high-sugar culture medium (containing 3% of fetal calf serum) into each roller bottle, regulating the rotating speed of the roller bottle to 20 revolutions per hour, enabling cells to be uniformly attached to the surface of the roller bottle for about 24 hours, and carrying out transfection when the cell attachment surfaces are converged to 90%;
(2) Transient transfection: cloning the nucleotide sequence of the PMEPA1 specific antibody identified in example 1 into a pcDNA3 vector, transiently transfecting into 293T cells;
(3) Harvesting the supernatant: collecting cell expression supernatant on the 5 th day after transfection, centrifuging the supernatant for 15min at 4500rmp, filtering with 0.45 μm filter membrane, preserving the filtrate at 4deg.C, and preparing for purification;
(4) Antibody purification:
Connect 1mL Protein AHP column to purification pump and set flow rate about 1mL/min;
Washing the purification column with 20mL of elution buffer, discarding the washing solution;
washing the purification column with 20mL of binding buffer, discarding the washing solution;
Placing a liquid inlet pipe in the lower filtrate collected in the step (3), collecting filtrate after flowing through the purification column, repeating the step, and passing the collected filtrate through the purification column for the second time;
Washing the purification column with 20mL of binding buffer and detecting the presence of the hybrid protein in the wash with coomassie brilliant blue (ensuring complete elution of the non-specifically bound protein in this step), discarding the wash;
Slowly flowing the elution buffer through the purification column at a flow rate of 1mL/min, collecting the elution, monitoring the protein concentration in the elution by using a Coomassie brilliant blue reagent in the elution process, ensuring complete elution of target antibody protein, adding a neutralization buffer into all the collected elution according to a ratio of 10:1, performing dialysis and ultrafiltration concentration, measuring the concentration of the concentrated solution, marking, taking part of the concentrated solution of the antibody for verification, and preserving in a refrigerator at the temperature of the rest of minus 20 ℃.
Example 3: detection of binding of PMEPA1 specific antibodies to PMEPA1 recombinant proteins by flow cytometry
The 293T cells screened in example 1, which displayed the PMEPA1-specific antibodies, were cultured for 24 hours, and then the cells were harvested, 1ug of PMEPA1 antigen was incubated, and the binding of the PMEPA1-specific antibodies to the antigen was confirmed by flow cytometry, and the secondary antibody used was APC-anti-human IgG, and the results are shown in FIG. 1.
Example 4: ELISA detection of binding of PMEPA1 specific antibodies to PMEPA1 recombinant proteins
(1) Antigen coating: diluting the antigen (PMEPA 1 recombinant protein prepared in example 1) to 10. Mu.g/mL with a1 Xcarbonate coating buffer (Na 2CO3(1.59g)+NaHCO3 (2.93 g), adjusting pH, and fixing the volume to 50 mL), and coating the diluted antigen in a 96-well plate (setting a positive control group, a negative control group, and an experimental group, each group having 3 multiplex wells) at 0.5. Mu.g/200. Mu.L;
(2) Closing: the wells were pipetted and washed 3 times with wash buffer (0.05% solution of Tween-20 in1 XPBS) and 100. Mu.L of blocking solution (skimmed milk powder (1 g) +1XTBST (20 mL)) was added to each well and incubated for 1h at 37 ℃;
(3) Incubation of antibody to be tested: washing the 96-well plate in a plate washer for 5 times, adding antibodies to be detected according to different groups, standing for 1h at 37 ℃, discarding liquid in the holes, washing for 3min by using a washing buffer solution table, and repeating the washing for three times;
(6) And (3) incubation of enzyme-labeled secondary antibodies: 100 mu L of diluted enzyme-labeled secondary antibody (diluted 1:5000) is added into each well of a 96-well plate, and the mixture is incubated for 30min at 37 ℃ in a dark place;
(7) Color development: washing 96-well plate in a plate washer for 6-7 times, adding 100TMB color development liquid into each well, and developing color at 37deg.C for 5-15min in dark place;
(8) Detecting the absorbance: 50. Mu.L of stop solution (the liquid in the well turns yellow) was added to each well, and the absorbance was measured at 450nm by a microplate reader.
The results are shown in FIG. 2, which shows that the binding activity of the PMEPA 1-specific antibodies selected in example 1 to the PMEPA1 protein is substantially equivalent to that of the commercial PMEPA1 monoclonal antibodies. The ELISA also detects whether the antibody can recognize other antigens, and the result shows that the PMEPA1 specific antibody can not recognize other antigens and has good specificity.
Example 5: PMEPA1 specific antibodies useful for the treatment of gastric cancer
SNU-216 gastric cancer cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum, after subculturing, the cells were inoculated in 96-well plates at 1×10 4 cells/mL, treated gastric cancer cells with 100 μg/mL of the PMEPA 1-specific antibody prepared in example 2 after 24 hours, and after 6 days of drug action, cell proliferation was detected using CCK-8 detection kit, and as a result, as shown in FIG. 3A, it was shown that proliferation of SNU-216 gastric cancer cells was significantly inhibited after PMEPA 1-specific antibody treatment.
In addition, cloning experiments of SNU-216 gastric cancer cells under treatment of PMEPA1 specific antibodies were also performed, and the results are shown in FIGS. 3B-C, which show that the cloning of SNU-216 gastric cancer cells is significantly inhibited after treatment with PMEPA1 specific antibodies.
The experimental data show that the PMEPA1 specific antibody has a good gastric cancer cell inhibition effect, and can be used as a therapeutic drug for treating gastric cancer.
In the description of the specification, reference to the term "one embodiment," "a particular embodiment," "an example," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The foregoing is merely illustrative and explanatory of the invention, as various modifications and additions may be made to the particular embodiments described, or by similar arrangements, by those skilled in the art, without departing from the scope of the invention or beyond the scope of the appended claims.

Claims (10)

1. A PMEPA1 specific antibody or antigen binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1.2 and 3, HCDR1, HCDR2 and HCDR3, the light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 6. LCDR1, LCDR2 and LCDR3 shown in fig. 7 and 8.
2. The PMEPA1 specific antibody or antigen binding fragment thereof according to claim 1, wherein the heavy chain variable region has the amino acid sequence as set forth in SEQ ID NO:4, and the light chain variable region has the amino acid sequence set forth in SEQ ID NO: 9.
3. The PMEPA 1-specific antibody or an antigen binding fragment thereof according to claim 1 or 2, which is a monoclonal antibody, a chimeric antibody, a humanized antibody, a fully human antibody, a Fab ', an Fv fragment, F (ab') 2, a scFv or a di-scFv,
Preferably, the PMEPA1 specific antibody or antigen binding fragment thereof is an scFv.
4. A PMEPA 1-specific antibody or an antigen binding fragment thereof according to claim 3, further comprising a linker connecting the heavy chain variable region and the light chain variable region,
Preferably, the linker has the sequence as set forth in SEQ ID NO:11, and a sequence of the amino acids shown in the formula (I),
Preferably, the PMEPA1 specific antibody or antigen binding fragment thereof has the amino acid sequence as set forth in SEQ ID NO:12, and a polypeptide having the amino acid sequence shown in FIG. 12.
5. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-4.
6. An expression vector comprising the nucleic acid molecule of claim 5.
7. A host cell comprising the expression vector of claim 6.
8. A conjugate comprising the PMEPA 1-specific antibody or an antigen binding fragment thereof according to any one of claims 1 to 4, and a coupling moiety,
Preferably, the coupling moiety is selected from the group consisting of protein tags, such as His, flag, GST, MBP, HA, myc, GFP; a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin; therapeutic agents, such as anti-inflammatory drugs or immunosuppressants; or, an additional biologically active polypeptide.
9. A pharmaceutical composition comprising the PMEPA1 specific antibody or antigen-binding fragment thereof according to any one of claims 1-4, a nucleic acid molecule according to claim 5, an expression vector according to claim 6, a host cell according to claim 7 or a conjugate according to claim 8, and a pharmaceutically acceptable carrier and/or excipient.
10. Use of a PMEPA1 specific antibody or antigen binding fragment thereof according to any one of claims 1 to 4, a nucleic acid molecule according to claim 5, an expression vector according to claim 6, a host cell according to claim 7 or a conjugate according to claim 8 in the manufacture of a medicament for detecting expression of PMEPA1 in a sample or for preventing and/or treating a cancer associated with PMEPA1,
Preferably, the cancer associated with PMEPA1 is gastric cancer.
CN202410165870.8A 2024-02-05 2024-02-05 PMEPA1 specific antibody and application thereof Pending CN118240076A (en)

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