Monoclonal mouse antibodies against human estrogen receptor, human progesterone receptor, human ki-67, and human p53

Abstract

Breast cancer is a serious type of cancer that affects many women each year, often leading to death. To choose the right treatment for breast cancer, studying biomarkers is crucial, and one way to do this is by using the immunohistochemical technique (IHC). Biomarkers like Estrogen receptor (ER), progesterone receptor (PR), Ki67, and P53 play a crucial role in determining the prognosis, progression, and response to treatment of breast cancer. Specific monoclonal antibodies against these biomarkers were created by immunizing mice with peptides derived from these proteins. These antibodies were then tested for specificity using ELISA and IHC staining on normal and cancerous tissues, confirming their ability to recognize the receptors. Additionally, the antibodies were evaluated for isotype, affinity constant, and their ability to detect natural antigens in flow cytometry and western blot.

Description

Monoclonal Mouse Antibodies Against Human Estrogen Receptor, Human Progesterone Receptor, Human Ki-67, and Human P53

[0001] Breast cancer is one of the most significant types of cancer, particularly affecting women, and unfortunately leads to the death of many patients each year. To determine the appropriate treatment for this disease, it is essential to study the biomarkers associated with breast cancer using the immunohistochemical (IHC) technique. Key biomarkers include the estrogen receptor (ER), progesterone receptor (PR), Ki67, and P53. By examining these biomarkers through IHC, valuable insights can be gained regarding the prognosis, disease progression, and treatment response.

[0002] The investigation of these biomarkers is conducted using monoclonal antibodies. In the current patent, new specific monoclonal antibodies targeting ER, PR, Ki67, and P53 were developed by immunizing mice with peptides derived from the sequences of these proteins. Their specificity was confirmed through specific peptide enzyme-linked immunosorbent assays (ELISA) and IHC staining of various normal and cancerous tissues. The expression of these receptors was evaluated and validated.

[0003] Additionally, beyond isotype and affinity constant analysis, these antibodies were tested for their ability to recognize natural antigens using flow cytometry and western blot techniques.

[0004] C07K 16 / 28 - C12N 15 / 00

[0005] Monoclonal antibodies to human estrogen receptor

[0006] 1980 September

[0007] Extranuclear estrogen receptor protein (estrophilin) of MCF-7 human breast cancer cells was purified by passage of the cytosol fraction of a cell homogenate through an affinity column of estradiol linked to Sepharose by a substituted di-n-propyl sulfide bridge in the 17 alpha position. Elution with 50 micro M [3H]estradiol in 10% (vol / vol) dimethyl formamide / 0.5 M sodium thiocyanate gave 40% recovery of [3H]estradiol-estrophilin showing 14% of the specific radioactivity expected for the pure complex. Serum from a Lewis rat immunized with this partially purified estradiol-receptor complex contained antiestrophilin antibodies that reacted not only with nuclear and extranuclear estradiol-receptor complexes from MCF-7 cells but also with estrophilin from rat, calf, and monkey uterus, hen oviduct, and human breast cancers. Splenic lymphocytes from the immunized rat were fused with cells of two different mouse myeloma lines (P3-X63-Ag8 and Sp2 / 0-Ag14) to yield hybridoma cultures, 2% of which produced antibodies to estrophilin. After cloning by limiting dilution, three hybridoma lines secreting antiestrophilin were expanded in suspension culture and as ascites tumors in athymic mice to provide substantial quantities of monoclonal antibodies that recognize mammalian but not avian estrophilin and that show different degrees of reactivity with receptor from nonprimate sources. By growing the clone from Sp2 / 0 in the presence of [35S]methionine, radiolabeled monoclonal IgG has been prepared. These monoclonal antibodies should prove useful in the study of estrogen receptors of human reproductive tissues, in particular for the radioimmunochemical assay and immunocytochemical localization of receptors in breast cancers.

[0008] For the first time, in 1980, Greene and his colleagues produced three mAbs against ER using the hybridoma method and by fusing myeloma cells with rat spleen cells, none of which could be used in the IHC technique.

[0009] New monoclonal antibodies to oestrogen and progesterone receptors effective for paraffin section immunohistochemistry

[0010] 1997 October

[0011] Assessment of oestrogen and progesterone receptors (ER and PgR) in breast cancer is widely used for the prediction of response to endocrine therapy and as a prognostic marker. Cytosolic assays have been replaced in many centres by immunochemical techniques, which have many advantages including applicability to small samples, simplicity, and cost-effectiveness. This study describes the generation and characterisation of two novel murine monoclonal antibodies recognizing ER and PgR, designated NCL-ER-6F11 and NCL-PGR respectively, which are effective in heat-treated formalin-fixed, paraffin-embedded tissue. The antibodies have been characterized by Western blotting and by immunohistochemistry on normal and pathological breast and other tissues. NCL-ER-6F11 has been shown to compare favourably with a currently available ER antibody. These antibodies may prove of value in the assessment of hormone receptor status in human breast cancer.

[0012] In another study, Bevit and his colleagues in 1997, using the hybridoma method, produced mouse monoclonal antibodies against ER that could be used in formalin-fixed paraffin-embedded tissue in the IHC method.

[0013] Development of new rabbit monoclonal antibody to estrogen receptor: immunohistochemical assessment on formalin-fixed, paraffin-embedded tissue sections

[0014] 2005 March

[0015] Evaluation of estrogen and progesterone receptors in breast cancer is widely used for the prediction of the response to endocrine therapy and as a biologic parameter closely related to disease prognosis. Immunohistochemistry is considered a specific, sensitive, and economic method for the determination of estrogen receptor / progesterone receptor status. The authors developed the first rabbit antiestrogen receptor monoclonal antibody (clone SP1) used in immunohistochemistry on formalin-fixed, paraffin-embedded tissue sections especially from breast carcinomas. This new antibody, compared with currently available antiestrogen receptor antibodies, has important advantages, including its reactivity even without heat-based antigen retrieval of fixed, embedded tissue sections in immunohistochemistry, and the predominance of nuclear immunostaining with only a very low cytoplasmic signal. A comparative study of immunohistochemistry on 61 histologic specimens from breast cancer cases showed that SP1 yields the same results as the well-known, standardized mouse monoclonal antibody to estrogen receptor (clone 1D5). Antibody affinity of SP1 is 8 times higher than that of 1D5. Thus, SP1 may prove of great value in the assessment of estrogen receptor status in human breast cancer.

[0016] In 2005, Huang and his colleagues produced a monoclonal rabbit anti-ER antibody called SP1, which was able to be used in IHC and ER detection on tissue sections fixed with formalin and paraffin.

[0017] Monoclonal and polyclonal antibodies to human progesterone receptor peptide-(533-547) recognize a specific site in unactivated (8S) and activated (4S) progesterone receptor and distinguish between intact and proteolyzed receptors

[0018] 1990 September

[0019] We have synthesized three peptides with amino acid sequences corresponding to amino acids 533-547, 597-611, and 765-779 of the human progesterone receptor (hPR). These peptides were conjugated to keyhole limpet hemocyanin and injected into mice and rabbits to develop antibodies to hPR. Antibodies to the undenatured form of PR were elicited only by the peptide with amino acid sequence 533-547. Fusion of SP2 / 0 myeloma cells with spleen cells from mice immunized with this peptide produced several active clones. Rabbit sera from immunized animals produced one antiserum that reacted with the undenatured form of PR. One monoclonal antibody (PR-AT 4.14) and one antiserum (PR-AT533) raised against peptide-(533-547) were characterized. Binding of these antibodies to the undenatured form of PR was demonstrated by analysis of the antibody-receptor complexes on sucrose density gradients and by immunoprecipitation techniques. Binding of PR to the antibodies was inhibited by excess peptide. The antibodies did not react with estrogen, glucocorticoid, or androgen receptors, but recognized PR from human breast cancer as well as calf, rabbit, mouse, and rat uteri, indicating that this epitope was conserved among these species. Based on sucrose density gradient analysis of PR prepared and labeled in the presence of proteolysis inhibitors and sodium molybdate, the antibodies bound to a site on the intact undenatured PR, but failed to bind to partially degraded steroid-binding form of the receptor, suggesting that the antibody-binding domain is at or near a site sensitive to proteolysis.

[0020] In 1990, Traish and colleagues immunized mice with PR receptor peptides conjugated to KLH and produced murine monoclonal antibodies against PR using the hybridoma method. Also, this group produced rabbit polyclonal antibodies against PR by immunizing rabbits. This group did not check their monoclonal antibodies in ELISA test and did not evaluate their ability in IHC assay.

[0021] New monoclonal antibodies to estrogen and progesterone receptors effective for paraffin section immunohistochemistry

[0022] 1997 October

[0023] Assessment of oestrogen and progesterone receptors (ER and PgR) in breast cancer is widely used for the prediction of response to endocrine therapy and as a prognostic marker. Cytosolic assays have been replaced in many centres by immunochemical techniques, which have many advantages including applicability to small samples, simplicity, and cost-effectiveness. This study describes the generation and characterisation of two novel murine monoclonal antibodies recognizing ER and PgR, designated NCL-ER-6F11 and NCL-PGR respectively, which are effective in heat-treated formalin-fixed, paraffin-embedded tissue. The antibodies have been characterized by Western blotting and by immunohistochemistry on normal and pathological breast and other tissues. NCL-ER-6F11 has been shown to compare favourably with a currently available ER antibody. These antibodies may prove of value in the assessment of hormone receptor status in human breast cancer.

[0024] In another study, Bevit et al., 1997, using the hybridoma method, produced murine monoclonal antibodies against the progesterone receptor, which were used in IHC using formalin-fixed paraffin-embedded tissue samples.

[0025] Development of new rabbit monoclonal antibody to progesterone receptor (Clone SP2): no heat pretreatment but effective for paraffin section immunohistochemistry

[0026] 2006 June

[0027] Evaluation of estrogen and progesterone receptor (ER, PgR) status in breast cancer is widely used for the prediction of the response to endocrine therapy and as a biologic parameter closely related to disease prognosis. The IHC method is considered to be a specific, sensitive, and economical method for determining ER and PgR status. The authors developed the first rabbit anti-PgR mAb (clone SP2) used in IHC on formalin-fixed, paraffin-embedded tissue sections from breast carcinomas. This new antibody, compared with currently available anti-PgR antibodies, has important advantages, including its reactivity even without heat-based antigen retrieval of fixed-embedded tissue sections in IHC and the predominance of nuclear immunostaining with only very low cytoplasmic signal. A comparative study of IHC on 107 histologic specimens from breast cancer cases showed that SP2 yields the same results as the wellknown mouse mAb to PgR (clone 1A6). The antibody affinity of SP2 is 12 times higher than that of 1A6. Thus, SP2 may prove of great value in the assessment of PgR status in human breast cancer.

[0028] In 2006, Huang and colleagues produced the first anti-PR rabbit monoclonal antibody, called SP2, and this antibody was able to bind to the PR protein in breast cancer tissues. with formalin to detect in the IHC test.

[0029] New Ki-67-equivalent murine monoclonal antibodies (MIB 1-3) generated against bacterially expressed parts of the Ki-67 cDNA containing three 62 base pair repetitive elements encoding for the Ki-67 epitope

[0030] 1993 June

[0031] In a study in 1933, Key and his colleagues produced murine monoclonal antibodies against Ki67 with the names MIB1, MIB2 and MIB3 by the hybridoma method. Among these three types of antibodies, MIB1 and MIB3 were able to detect Ki67 antigen in paraffinized tissue slides containing this antigen after being fixed with formalin in IHC test.

[0032] Monoclonal Antibodies against Specific p53 Hotspot Mutants as Potential Tools for Precision Medicine

[0033] 2021 August

[0034] The large number of mutations identified across all cancers represents an untapped reservoir of targets that can be useful for therapeutic targeting if highly selective, mutation-specific reagents are available. We report here our attempt to generate such reagents: monoclonal antibodies against the most common R175H, R248Q, and R273H hotspot mutants of the tumor suppressor p53. These antibodies recognize their intended specific alterations without any cross-reactivity against wild-type (WT) p53 or other p53 mutants, including at the same position (as exemplified by anti-R248Q antibody, which does not recognize the R248W mutation), evaluated by direct immunoblotting, immunoprecipitation, and immunofluorescence methods on transfected and endogenous proteins. Moreover, their clinical utility to diagnose the presence of specific p53 mutants in human tumor microarrays by immunohistochemistry is also shown. Together, the data demonstrate that antibodies against specific single-amino-acid alterations can be generated reproducibly and highlight their utility, which could potentially be extended to therapeutic settings.

[0035] In a 2018 study conducted by Hwang and his colleagues, mouse monoclonal antibodies against different P53 antigen mutations were produced, and according to the results of this study, these antibodies can be used to detect P53 mutations using the IHC method.

[0036] WO2004024747

[0037] HIGH AFFINITY MONOCLONAL ANTIBODY FOR RECOGNIZING THE ESTROGEN RECEPTOR (ER) AND METHOD FOR CREATING THE ANTIBODY

[0038] High affinity monoclonal antibodies for recognizing estrogen receptor (clone SP1) with immunohistochemistry and methods for creating such an antibody are disclosed. The lagomorph derived ER antibody provides a significant advantage over the currently available mouse ER antibodies in that there is no need for target retrieval when performing immunohistochemistry. Furthermore, the very low background when the lagomorph derived ER antibody is used in immunohistochemistry is also impressive. The immunohistochemistry comparative study with about fifty clinical specimens showed that the new ER (clone SP1) antibody and favorable results when compared to mouse monoclonal ER antibodies (clone 1D5). The lagomorph derived ER antibody may prove of great value in the assessment of ER status in human breast cancer. Humanized versions of the ER antibody may also provide therapeutic benefits.

[0039] The mentioned patent is similar to our claimed one but their methods are different and also there is no mention of Human Ki-67 and Human P53 as antigens.

[0040] IN201711016149

[0041] GENERATION AND CHARACTERIZATION OF HYBRIDOMA CLONES PRODUCING MONOCLONAL ANTIBODIES AGAINST SPECIFIC DOMAINS OF HUMAN PREGNANE AND XENOBIOTIC RECEPTOR (PXR)

[0042] The present invention relates to the generation and characterization of hybridoma clones producing monoclonal antibodies against specific domains of human Pregnane and Xenobiotic Receptor (PXR, NR1I2) utilizing the purified full-length protein as an antigen. It also relates to a method of generation of monoclonal antibody using the full-length human PXR protein as antigen. Further, it relates to the usefulness of antibodies for various immunological tools like ELISA, immunoblotting, immunoprecipitation, immunofluorescence, immunohistochemistry, flowcytometry etc. In addition, the antibodies may identify expression of PXR protein in different diseased states and provide important prognostic or predictive information in clinical samples in different cancerous states like endometrial cancer, breast cancer, prostate cancer and epithelial ovarian carcinoma. The present invention demonstrates the functionality and usefulness of mouse anti-human PXR monoclonal antibodies to elucidate the role of PXR in various disease outcomes. The monoclonal antibodies raised herein against human PXR are consistently produced from a replenishing cellular source implying a reliable and economical source of antibodies as a useful immunological tool in biological research and immunodiagnostics.

[0043] The said invention is close to our claimed one because of their common purpose but its receptor is Xenebiotic while ours is the human estrogen Receptor and human progesterone.

[0044] United States Patent Application 20110150935

[0045] TARGETED TREATMENT FOR PATIENTS WITH ESTROGEN RECEPTOR NEGATIVE AND PROGESTERONE RECEPTOR NEGATIVE BREAST CANCERS

[0046] Treatments for estrogen receptor and progesterone receptor negative breast cancer or estrogen receptor, progesterone receptor and c-erbB2 negative (triple negative) breast cancer are provided.

[0047] The mentioned method has similarities with our claimed one and the important similarity between them is their receptors but on the other hand, the method’s treatment is for a specific state of the cancer and its process and proteins are far from ours.

[0048] Monoclonal antibodies 2A5, 2H1, and 2A6 were created. The 2A5 antibody targets a specific amino acid sequence in the human progesterone receptor and has an affinity constant of 2. 69 nM. The 2H1 antibody binds to a specific amino acid sequence in the human Ki67 protein with an affinity constant of 2. 3 nM. The 2A6 antibody targets a particular amino acid sequence in the human P53 protein with an affinity constant of 3. 24 nM.

[0049] Cancer is one of the most common diseases in the world, which, despite significant advances in diagnosis and treatment, still accounts for a high percentage of related deaths. After diagnosing cancer in a patient, doctors examine biological molecules known as cancer biomarkers in cancer samples to choose the type of treatment and also to receive information about the prognosis and course of the disease. One of the most important cancers is breast cancer, which is the second leading cause of cancer death in women. Important biomarkers of breast cancer include estrogen receptor (ER), progesterone receptor (PR), Ki67, and P53. Examining the level of expression of these receptors in breast cancer provides doctors with useful information in the field of choosing or not choosing the type of treatment for patients, as well as the prognosis of the disease. The gold standard method for investigating these biomarkers in cancer tissues is immunohistochemistry (IHC). The study of these tumor markers requires specific antibodies especially monoclonal antibodies. After the cancer samples are prepared for longer storage, they are fixed using formalin, and as a result, the proteins in them lose their natural structure. For this reason, most of the available monoclonal antibodies cannot identify their target proteins in formalin-fixed tissues in the IHC technique. The subject of this patent is the production of new monoclonal antibodies usable in the IHC technique using formalin-fixed tissues against human ER, PR, Ki67, and P53. Antibodies produced in this study are new in the world and Iran and are able to identify their target molecules in normal and cancerous tissues that express them.Solution of Problem

[0050] A- Production of new monoclonal antibodies against ER, PR, Ki67, and P53 with the ability to identify these markers in the IHC technique in cancer patients with breast cancer.

[0051] Diagnostic monoclonal antibodies against cancer markers have been produced by various researchers and commercial companies, and they differ from each other depending on the type of antibody and the epitope they identify on the target molecule. Therefore, although all these antibodies identify the same target, depending on the technique used (for example, IHC) or the method used (manually or with an automation device), they may have different functions and therefore have different sensitivity and specificity. They show themselves. Therefore, the production of new antibodies against these cancer markers is still being done all over the world so that the best antibodies in terms of performance, sensitivity, and specificity can enter the market of laboratory and diagnostic products. This also applies to therapeutic antibodies, so that different types of antibodies against a specific tumor marker (for example, HER2 or CD20) have been produced, each of which has biological effects based on the type of epitope they identify on the target molecule so they are different and unique in this sense.

[0052] To make the said monoclonal antibody, mice were immunized with the designed peptide, and their spleen cells were fused with myeloma cells and monoclonal antibody-producing hybrid cells were produced. After checking the cells and selecting cells producing anti-ER, PR, Ki67, and P53 antibodies that can be used in the IHC test, the selected cells were subcloned in several steps to obtain pure monoclonal antibody-producing cells. Then the cells were multiplied and ascites were collected after injection into mice. The obtained ascites were purified with Protein G column and pure antibody was obtained. Finally, the purified antibody was used to perform specificity tests. Also, the produced antibody was compared with a commercial antibody to detect the expression of ER, PR, Ki67, and P53 in the tissue samples of breast cancer patients, and its sensitivity, specificity, and accuracy were calculated.

[0053] A) design, synthesis, and conjugation of ER, PR, Ki67, and P53 peptides to the carrier molecule

[0054] A peptide derived from the sequence of ERα proteins with the sequence KIPLERPLGEVYLDSSKPAVYNYPEGAAYEFNAAAAAANAQVYGQTGLPYG, PR with sequence PLYSDFQPPALKIKEEEEEGAEASARSPRSYLVAGANPAAFPDFPLGPPPP, Ki67 with sequence AGTLPGSKRQLQTPKEKAQALEDLAGFKELFQTPGHTEELVAAGKTTKIP and P53 with sequence LNEALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKKLMFKTEGPDSD were designed and commercially produced. The produced peptides were conjugated with Keyhole limpet hemocyanin (KLH) carrier by m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) cross-linker to increase immunogenicity.

[0055] B) the method of producing hybridomas producing specific monoclonal antibodies against ER, PR, Ki67, and P53

[0056] Monoclonal antibody-producing clones against human ER, PR, Ki67, and P53 were produced by the hybridoma method. In short, Balb / C mice were hyperimmunized with conjugated peptides for 6 to 8 weeks. Then their spleen cells were fused with SP2 / 0 myeloma cells in the presence of Polyethylene glycol solution, and then using a selective medium containing HAT (Hypoxanthine, Aminopterin, Thymidine), monoclonal antibody-producing clones were identified. Supernatant media of antibody-producing hybridomas were analyzed using ELISA and IHC tests, and target antibody-producing clones were identified. Finally, after four times of single-cell cloning, these hybridomas were injected into the peritoneum of mice to prepare ascites. Then the resulting ascites were purified using protein G column affinity chromatography.Advantage Effects of the Invention

[0057] The prepared antibodies act against the following cancer markers. Each of these monoclonal antibodies has unique characteristics in terms of specificity, sensitivity, affinity, and epitope identified in the target protein.

[0058] Monoclonal antibody 1B9 against ER

[0059] Monoclonal antibody 2A5 against PR

[0060] 2H1 monoclonal antibody against Ki67

[0061] Monoclonal antibody 2A6 against P53

[0062] Shows a flowchart of clone recognition.

[0063] Presents a flowchart of how the clones get examined.

[0064] Shows a flow chart in which Balb / C mice were hyperimmunized with conjugated peptides for 6 to 8 weeks and then their spleen cells were fused with SP2 / 0 myeloma cells in the presence of Polyethylene glycol solution and then using a selective medium containing HAT (Hypoxanthine, Aminopterin, Thymidine), monoclonal antibody-producing clones were identified.

[0065] is a continuation flowchart of the previous one because it shows Supernatant media of antibody-producing hybridomas were analyzed using ELISA and IHC tests, and target antibody-producing clones were acknowledged.Examples

[0066] A- The method of using the invention:

[0067] 1B9 (anti-ER), 2A5 (anti-PR), 2H1 (anti-Ki67), and 2A6 (anti-P53) antibodies produced in this study can be used to detect the presence of ER, PR, Ki67, and P53 molecules in cancer tissues. Patients, especially those with breast cancer, used the IHC test and thereby identified the subtype of breast cancer tissue and adopted the appropriate treatment method for those patients.

[0068] B- How to consume:

[0069] The produced antibodies will be prepared and sold as a solution in a formulation that can be stored at a temperature of 4 degrees Celsius. Antibodies should be diluted to a final 1 μg / ml concentration using the formulation solution when administered. These antibodies are added to the tissues as the primary antibody in the immunohistochemical method to cover the entire tissue and must be incubated for 1 hour at room temperature so that the monoclonal antibodies bind to the target antigens.

[0070] The examination of the binding of produced antibodies to target antigens in the IHC test:

[0071] The best clones in terms of sensitivity for use in IHC tests for each of the target antigens were identified and selected by the investigations and used for further tests. The selected anti-ERα monoclonal antibody-producing clone was named 1B9. To confirm the specificity of clone 1B9 produced, it was used to stain cancerous and normal tissues that express ERα. In this study, clone 1B9 was able to successfully identify ERα antigens like the commercial clone in normal endometrial, normal cervix, normal vagina, normal breast and breast, endometrial and lung cancers. The results of these staining are shown in picture 1.

[0072] The selected anti-PR monoclonal antibody-producing clone was named 2A5. To confirm the specificity of clone 2A5 produced, it was used to stain cancerous and normal tissues expressing PR. In this study, clone 2A5 successfully identified PR antigens like the commercial clone in normal endometrial, normal cervix, normal breast and breast, and ovarian and endometrial cancers. The results of these staining are shown in picture 2.

[0073] The clone producing the selected anti-Ki67 monoclonal antibody was named 2H1. To confirm the specificity of clone 2H1 produced, it was used to stain cancerous and normal tissues that express Ki67. In this study, the 2H1 clone was able to successfully identify Ki67 antigens like the commercial clone in the tissues of the normal tonsil, normal testis, and breast, stomach, prostate, colorectal, ovarian, brain, and pancreatic cancers. The results of these staining are shown in picture 3.

[0074] The clone producing the selected anti-P53 monoclonal antibody was named 2A6. To confirm the specificity of clone 2A6 produced, it was used to stain cancerous and normal tissues that express P53. In this study, clone 2A6 was able to successfully identify P53 antigens in stomach, bladder, oral, brain, and breast cancer tissues like the commercial clone. The results of these staining are shown in picture 4.

[0075] [Pic. 1] As seen in part A of picture 1, clone 9B1 was able to identify this antigen in normal tissues of the endometrium, cervix, and vaginal wall, which are natural expressions of ER. In the normal state, stromal and ductal cells of endometrial and cervical tissues and some of the smooth muscles in these tissues naturally express the ER marker. Also, cells of the basal layer of the vaginal wall express this marker. Antibody 9B1 was able to detect ER with minimal non-specific staining and good reactivity and identify it in the target cells. In parts B to E of normal breast tissue and various cancers expressing ER antigens using positive control antibodies (SP1), negative control Ig mouse Normal, and clone 9B1 were stained. The hybridization of normal breast tissue, in which some cells express a small amount of ER, showed that the 9B1 clone was the same as the commercial clone. It can detect a small number of cells expressing ER. Staining of breast, endometrial, and lung cancers using clones 9B1 showed that this clone can identify ER-positive cells in these tissues like the commercial clone (parts C to E).

[0076]

[0077] [Pic. 2] The staining results show that clone 5A2 can express PR antigen in the nucleus of ductal cells, stroma, and some smooth muscles. Also, this antibody reacts with normal vaginal tissue that does not express the PR biomarker. Clone 5A2 was able to identify cells expressing PR in normal cancer tissue, just like IHC clone 651 to diagnose breast cancer. This shows that this clone can detect the low expression of PR. In this coloring, some ductal cells were stained by two antibodies and stromal cells were negative (picture 2, part B). Parts C to G show staining results of two different breast cancers, two different ovarian cancers, and one endometrial cancer expressing PR with the use of IHC 651 and 5A2 clones showing that in these cancers, the 5A2 clone is even more reactive than the control antibody can identify the PR molecule in these tissues. Non-specific staining in these tissues in tissues dyed with clone 5A2 was not observed.

[0078]

[0079] [Pic. 3] The results of the IHC test showed that clone 1H2 can detect 67Ki in the follicular and basal cells of the surface epithelium of the normal tonsil (part H). Also, the basal cells of the normal testis, which naturally express 67Ki, were stained by clone 1H2. Staining of normal pancreatic and breast tissues showed that the 1H2 clone did not react non-specifically with the cells of these tissues (Part I). In the next stage, 67Ki-positive ovarian, breast, brain, pancreas, colorectal and prostate cancer tissues were stained with IHC clones 1H2 and 067 (parts A to G). The results indicated that clone 1H2 was able to detect positive cells with much better reactivity than commercial clone 067 IHC. As it is evident in these images, the number of positive cells identified by clone 1H2 was more than that of the commercial clone, and also the non-specific background staining produced by the clone was much less than that of clone 067IHC. Even in one of the investigated prostate cancer tissues, the commercial clone, unlike the 1H2 clone, could not identify a few positive cells (Part G).

[0080]

[0081] [Pic. 4] Among the few parts of the body that express 53P naturally, the basal cells of the oral tissue are. For this purpose, the tissue of the oral mass was examined by IHC clones 6A2 and 053 (Part E) and the results showed that both clones can identify the wild form 53P. Staining of various cancer tissues expressing mutated 53P including stomach, bladder, brain, and breast cancers showed that both commercial antibody and 6A2 can detect 53P in the nucleus of cancer cells expressing it with the least amount of non-specific staining. (Parts A to G). To obtain the amount of non-specific staining, two normal tissues of the tonsil and breast were stained using clone 6A2, and no non-specific staining was observed in these tissues (part H).

[0082]

[0083] Examining the specificity of monoclonal antibodies produced in the western blot test:

[0084] To check the capability of the produced monoclonal antibodies in identifying the natural form of the target molecules, first, lysates were prepared from HeLa (expressing ER, PR, and Ki67) and MDA-MB-231 (expressing P53) cell lines. The concentration of the prepared lysates was calculated using the Enhanced BCA method. 40 μg of each of the lysates was used in each well of SDS-PAGE electrophoresis gel, and for each antibody, two wells were considered as reduced and non-reduced. After performing electrophoresis and transfer of proteins to a nitrocellulose membrane, 1B9, 2A5, 2H1, and 2A6 antibodies were used as primary antibodies and HRP-conjugated sheep anti-mouse Ig as secondary antibodies. The final bands were recorded by ECL substrate in the darkroom and on photographic film.

[0085] [Pic. 5] shows the immunoblot pattern of an anti-ER antibody named 9B1 in reaction with ER molecule present in HeLa cancer cell lysates in resuscitation (1) and non-resuscitation (2) states:

[0086]

[0087] As can be seen in picture 5, clone 9B1 can recognize the band with a molecular weight of 65 kilodaltons related to ERα in two states of regeneration and non-regeneration.

[0088] [Pic. 6] shows the immunoblot pattern of an anti-PR antibody named 5A2 in the reaction with the PR molecule present in HeLa cancer cell lysates in regeneration (1) and non-regeneration (2) states:

[0089]

[0090] As can be seen in picture 6, clone 5A2 is weakly capable of recognizing two isoforms, PRA and PRB, with molecular weights of 94 and 120 kilodaltons in the recovery state.

[0091] [Pic. 7] shows an immunoblot pattern of anti-67Ki antibody named 1H2 in the reaction with the 67Ki molecule present in HeLa cancer cell lysates in recovery (1) and non-regeneration (2) states:

[0092]

[0093] As can be seen in picture 7, clone 1H2 can recognize the 67Ki protein close to 319 kilodaltons in the regeneration and non-regeneration states.

[0094] [Pic. 8] shows the immunoblot pattern of the anti-53P antibody named 6A2 in the reaction with the 53P molecule present in 231MB-MDA cancer cell lysates in regeneration (1) and non-regeneration (2) states:

[0095]

[0096] As can be seen in picture 8, clone 6A2 can recognize the band with a molecular weight of 53 kilodaltons, corresponding to 53P, in two states of regeneration and non-regeneration.

[0097] Examining the binding of produced antibodies to natural target molecules by indirect flow cytometry method:

[0098] Indirect flow cytometry was used to check and confirm the ability of monoclonal antibodies to bind to the natural form of their target antigens in cells. In these experiments, 1F9G5 antibody was used as a control isotype along with the produced antibodies as the primary antibody, and FITC-conjugated sheep anti-mouse Ig antibody was used as the secondary antibody. According to the results of this experiment shown in pictures 9 to 12, clones 1B9, 2A5, 2H1, and 2A6 were able to successfully identify their target antigens inside the tested cells. It should be noted that the HeLa cell line (expressing ER, PR, and Ki67) was used to investigate clones 1B9, 2A5, and 2H1, and the MDA-MB-231 cell line (expressing P53) was used to investigate clone 2A6.

[0099] [Chart. 1] shows the results of flow cytometry show intracellular staining of the HeLa cell line using monoclonal antibody 9B1. In this picture, the graph related to the isotype antibody is shown in gray color and the graph related to the 9B1 antibody is marked in white color:

[0100]

[0101] [Chart. 2] shows the results of flow cytometry show intracellular staining of the HeLa cell line using 5A2 monoclonal antibody. In this picture, the diagram related to the isotype antibody is shown in gray color and the diagram related to the 5A2 antibody is marked in white color:

[0102]

[0103] [Chart. 3] displays the results of flow cytometry showing intracellular staining of the HeLa cell line using monoclonal antibody 1H2. In this picture, the graph related to the isotype antibody is shown in gray color and the graph related to the 1H2 antibody is marked in white color:

[0104]

[0105] [Chart. 4] The results of flow cytometry show intracellular staining of MDA-231 cell line using 6A2 monoclonal antibody. In this picture, the graph related to isotype antibody is shown in gray color and the graph related to 6A2 antibody is marked in white color:

[0106]

[0107] Determination of the affinity constant of produced antibodies:

[0108] ELISA method was used to check the antibody affinity constant, during which successive concentrations of antibodies were added to successive concentrations of target antigens (ER, PR, Ki67, and P53 peptides). Then a graph was drawn, where the logarithm of the antibody concentration was placed on the X axis and the amount of light absorption on the Y axis. Then 3 sigmoid curves were used and the affinity constant was calculated according to the following formula:

[0109] Ka= n-1 / 2(n[Ab′]-[Ab])

[0110] n= ([Ag] / [Ag′])

[0111] [Ab]: antibody concentration at OD50 (half maximum OD value) at [Ag] concentration

[0112] [Ab′]: antibody concentration at OD50 (half maximum OD value) at [Ag′] concentration

[0113] According to the obtained results, the affinity constant for antibody 1B9 is equal to 0.565 nmol, for antibody 2A5 is equal to 2.69 nmol, for clone 2H1 is equal to 2.3 nmol and for clone 2A6 is equal to 3.24 nmol.

[0114] Isotype determination of produced monoclonal antibodies:

[0115] The indirect ELISA method was used to determine the isotype of monoclonal antibodies produced in this research. Briefly, 1B9 antibody and control antibodies were coated in an ELISA plate. After the blocking step, isotype-determination antibodies were added to them. Then the HRP-conjugated rabbit anti-sheep antibody was added to the wells and after adding the TMB substrate, the optical absorption was read at a wavelength of 450 nm. The results showed that the isotype of clones 1B9, 2A5, 2H1, and 2A6 is of IgG1 type.

[0116] Comparison of the ability to detect target antigens in samples collected from breast cancer patients using produced antibodies and commercial antibodies:

[0117] To check the quality of produced antibodies, it was necessary to compare the results of staining breast cancer tissues using these antibodies with the results of commercial antibodies. The data obtained from the samples were calculated as Hscore. Hscore is a number between 0 and 300, which is the result of the product of the number of positive cells in each microscope (a number between 0 and 100%), and the staining intensity of positive cells (weak 1, medium 2, or strong 3) is obtained.

[0118] To check the quality of produced antibodies, it was necessary to compare the results of staining breast cancer tissues using these antibodies with the results of commercial antibodies. The data obtained from the samples were calculated as Hscore. Hscore is a number between 0 and 300, which is the result of the product of the number of positive cells in each microscope (a number between 0 and 100%) and the staining intensity of positive cells (weak (1), medium (2) or strong (3)) is obtained. Examining the Hscore obtained from the staining of breast cancer tissue samples using produced and commercial clones showed that the average Hscore data obtained from the antibodies produced in the examined samples are almost similar to the data obtained from the clones It is commercial and the correlation between their results is significant (P < 0.001). The results of this study are presented in Table 1.

[0119] [Table. 1] Shows the evaluation of the ability to detect target antigens in samples collected from breast cancer patients using produced antibodies and commercial antibodies:

[0120] Calculation of sensitivity, specificity, and accuracy values of produced antibodies:

[0121] To obtain the level of specificity, accuracy, and specificity of antibodies produced after staining breast cancer samples using commercially produced clones, scoring was done by a pathologist, and the number of positive and negative samples of each antibody was calculated with SPSS software. The obtained numbers were included in the formulas for calculating the sensitivity, specificity, and accuracy of antibodies and these values were calculated. The formulas used are:

[0122] [Formula. 1]

[0123]

[0124] [Formula. 2]

[0125]

[0126] [Formula. 3]

[0127]

[0128] The results of this study showed that the sensitivity, specificity, and accuracy of the produced antibody is acceptable and the produced antibody is almost of the same accuracy. Commercial antibodies can identify the target antigen. This shows that the antibodies produced in this study can be a good substitute for commercial antibodies for use in diagnostic and research tests.

[0129] [Table. 2] Shows the calculation of the values of sensitivity, specificity, and accuracy of produced antibodies:

[0130] A- Industrial application of the invention

[0131] Monoclonal antibodies against ER, PR, Ki67, and P53 antigens produced in this study can be used to diagnose and choose the type of treatment for breast cancer patients. To produce these antibodies in the industrial phase, the cells producing these antibodies can be injected into mice and ascites taken from them. Then he purified and isolated the antibodies. Finally, the optimal concentration of antibodies was added to the antibody storage formulation and used in IHC tests.

[0132] B- How to produce in the industrial phase

[0133] To produce monoclonal antibodies produced in the industrial phase, the cells that produce these antibodies were melted and after their multiplication and their number reached 5 million cells, each of them was given to several BALB / C mice that were previously given Pristane. It has been injected, we inject it intraperitoneally. After one to two weeks, we separate the ascites fluid formed in the mice's stomach and purify them using the G protein chromatography column. We aliquot the purified antibodies and store them at minus 20 degrees Celsius until use.

Claims

The Monoclonal antibodies, 2A5 anti-human progesterone receptor, 2H1 anti-human Ki67 protein, and 2A6 anti-human P53 protein are produced.According to claim 1, the 2A5 antibody specifically binds to the amino acid sequence PLYSDFQPPALKIKEEEGAEASARSPRSYLVAGANPAAFPDFPLGPPPP of the human progesterone receptor, has an affinity constant of 2.69 nM and isotype IgG1.According to claim 1, the 2H1 antibody specifically binds to the amino acid sequence AGTLPGSKRQLQTPKEKAQALEDLAGFKELFQTPGHTEELVAAGKTTKI of human Ki67 protein, has an affinity constant of 2.3 nM and isotype IgG1.According to claim 1, the 2A6 antibody specifically binds to the amino acid sequence LNEALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKKLMFKTEGPDSD human P53 protein, has an affinity constant of 3.24 nM and isotype IgG1.

Images