Mechanisms of resistance to anti-EGFR monoclonal antibody treatment in metastatic colorectal cancer

Metastatic colorectal cancer (mCRC) continues to be counted as a major health problem. The introduction of newer cytotoxics, irinotecan and oxaliplatin, has achieved a significant improvement in survival rates. Novel targeted therapies (bevacizumab, and cetux-imab) in combination with most efficient chemotherapy regimens have pushed the median survival beyond the 2-year mark and increased the proportion of patients which could benefit from resection of metastatic lesions. In addition, several studies have proved that the CRC mutation profiles should influence patient selection or stratif ication in prospective trials. KRAS mutational status represents a paradigm for biomarker development in the era of molecular targeted therapies. The present article is an overview of the most important studies in the development of biomarkers for the optimization of anti-epidermal growth factor receptor (anti-EGFR) treatment in mCRC, beyond KRAS mutations, which is a work in progress. The aim will be to identify molecular markers that might be used to select patients with a higher probability of response to anti-EGFR monoclonal antibodies. Overall the accumulating evidence of the molecular biology of CRC has substantially changed the approach to mCRC treatment and has given clinicians more rational options for treating this illness.

Monoclonal antibodies as therapeutic agents in oncology and antibody gene therapy

Antibodies as therapeutic agents are mostly used in oncology, as illustrated by their applications in lymphoma, breast cancer or colorectal cancer. This review provides a brief historical sketch of the development of monoclonal antibodies for cancer treatment and summarizes the most significant clinical data for the best-established reagents to date. It also discusses strategies to improve the anti-tumor efficacy of antibody therapy, including antibody gene therapy and exploitation of bone marrow derived primary mesenchymal stem cells as the antibody gene transporter.

Molecular Markers for the Prediction of Anti-EGFR Monoclonal Antibody Treatment Efficacy in Metastatic Colorectal Cancer

The implementation of individualized targeted therapy for metastatic colorectal cancer (mCRC), in addition to standard chemotherapeutic regimens, currently is a topic under debate. Approximately 35% - 45% of mCRC patients exhibit mutated KRAS, which is considered to be an independent predictor of poor response to treatment with epidermal growth factor receptor (EGFR) monoclonal antibody. However, only about 50% of patients with wild-type KRAS respond to anti-EGFR therapy. Two major EGFR-dependent signaling pathways, RAS-RAF-MAPK and PI3K-PTEN-AKT, may be involved in the poor response to anti-EGFR. Increased EGFR gene copy number as detected by fluorescence in situ hybridization, but not increased EGFR protein expression, correlates with efficacy of anti-EGFR treatment. The identification of mutations in BRAF and PIK3CA (exon 20) and deletions in PTEN also may help clinicians screen for anti-EGFR resistance in mCRC patients with wild-type KRAS. To guide health professionals through the realm of individualized targeted therapies for mCRC, we review recent progress on identifying negative predictors and prognostic markers of anti-EGFR treatment efficacy.

Immunoscintigraphy of local recurrent rectal cancer with^(99m)Tc-labeled anti-CEA monoclonal antibody CL58

AIM: To explore a specific diagnostic method for local recurrent rectal cancer.METHODS: Immunoscintigraphy with 99mTc-labeled anti-CEA monoclonal antibody (MoAb) CL-58 was performed for patients suspected of having a postoperative local recurrent rectal cancer and the findings were compared with the results of conventional imaging and pathology.RESULTS: A total of 36 patients with a suspected local recurrent rectal cancer underwent immunoscintigraphy with 99mTc-conjugated CL58. Local recurrence of rectal cancer was identified in 31 patients and established in 30 during operation, endoscopy and pathological examination. No local recurrence was found in 5 patients without specific accumulation of 99mTc during the follow-up. Immunoscintigraphy had a positive rate of 86.11%, a specificity of 83.33%, and a sensitivity of 100%.CONCLUSION: Immunoscintigraphy has a highly specific and predictive value for detecting local recurrent rectal cancer, especially after abdominal perineal resection (APR).

THE SPECIFIC PHOTODYNAMIC EFFECTS OF MONOCLONAL ANTIBODIES CONJUGATED WITH HEMATOPORPHYRIN DERIVATIVE ON GASTRIC CANCER IN VITRO AND IN VIVO

Murine monoclonal antibody (MoAb) BB4.3, raised against the human gastric cancer cell line BGC823, was puriffied with Protein A-Sepharose CL-4B affinity chromatography and identified as IgG2a. It was then conjugated with a hematoporphyrin derivative (HPD) by using carbodiimide. The qualitative analysis of this conjugate showed that the amount of free HPD was negligible and there were no IgG aggregates among the conjugates. The conjugate retained both the antibody and photochemical activity of HPD.In vitro, the phototoxic effect of this HPD-BB4.3 conjugate on target cells was about 15 times higher than that of free HPD. The quality of selective photocytotoxicity was proven by the greater cytotoxi-city this conjugate showed than that of corresponding normal mouse IgG (NIgG) conjugated with HPD. It showed less cytotoxicity to colon cancer cell line B-80 (negative reaction to MoAb BB4.3) than to BGC825. Moreover, its cytotoxicity to BGC823 cells could be blocked specifically by excess BB4.3 antibody, but not by another MoAb 3G9, which combines with BGC823 at different binding sites from MoAb BB4.3.Nude mice inoculated with 2 × 10- BGC823 cells were given HPD-BB4.3, HPD, HPD-NIgG, HPD plus BB4.3 and PBS, respectively then exposed to light. Four out of six animals treated with the HPD-BB4.3 conjugate remained tumor-free for a long period. Although two developed tumors, there was a significant difference between the HPD-BB4.3-treated group and all the control groups in tumor induction time, tumor growth rate, and survival time (p<0.001). The HPD-BB4.3 conjugate inhibited the growth of established tumors by more than 40% in comparison with control groups (p<0.05).

MONOCLONAL ANTIBODIES AGAINST HUMAN GASTRIC CANCER

Spleen cells from Balb/c mice immunized with five human gastric cancer cell lines in sequence were fused with murine myeloma cell line SP2/0, and hybridomas 3F4, 3G9 and 3H11, secreting monoclonal antibodiees (MoAbs) against gastric cancer, were obtained through selective culture and screening. These MoAbs have both good selectivity and a high positive rate of reaction for gastric cancer, reaching 5/5 and 84.8% to 93.5% for gastric cancer cells and tissues respectively. The reaction of MoAbs with normal cells and tissues was neglible.The corresponding antigens of the MoAbs were sensitive to digestion by trypsin and pronase and resistant to treatment with sodium periodate, indicating their nature as proteins. The antigen 3G9 could be visualized with Western blotting as two bands with molecular weights of 100KD and 70KD, however no band was found for antigens 3F4 and 3H11. There was a high expression of antigens in the majority of gastric cancer cells and tissues independent of his-topathological type of gastric cancer. A low expression of antigens was seen with other tumors and fetal gastrointestinal tissues. These could be considered as gastric cancer-associated antigens or on-cofetal antigens with a quite extensive distribution.

Monoclonal antibodies that target the immunogenic proteins expressed in colorectal cancer

In an attempt to improve upon the end results obtained in treating colorectal cancer it was apparent that the earlier the diagnosis that could be obtained, the better the chance for obtaining desired results. In the case of more advanced tumors typified by later stage colorectal cancer, surgical debulking is an important part of the treatment strategy. Here the use of additional therapeu-tic modalities including chemotherapy and present day immunotherapy has failed to accomplish the desired im-provements that have been sought after. Adjuvant ther-apy, has offered little to the overall survival. The concept of early detection is now recognized as the initial step in reaching proper end results and can readily be demon-strated from colorectal cancer studies. Here survival has been found to be a reflection of the stage at which the tumor is first identified and treated. When specific mono-clonals targeting colorectal cancer are employed diagnos-tically, we have been able to demonstrate detection of colorectal cancer at its inception as a premalignant lesion, such that genotypic features can be identified before the phenotypic appearance of cancer can be noted.

PURIFICATION AND IDENTIFICATION OF THE ANTIGEN ASSOCIATED WITH MONOCLONAL ANTIBODY 3H_(11) AGAINST GASTRIC CANCER CELL

The object of this study is to investigate the properties of gastric cancer assoiciated antigen by McAb3H11 against gastric cancer cell line. Antigen purified by affinity chromatography and further charac- terized by biochemical and immunological techiques. Results showed that assoiciated antigen of McAb 3H11 was mainly located on the membrane of target cells. It was sensitive to heat and digestion of proteases,but resistant to treatment with sodium periodate. Schiff’s reagent staining was negative. SDS-PAGE and IEF- analysis showed that the antigen was a 210 kD molecular weight with pl of 8. 5 protein. The molecular weight of antigen extracted from target cell with tunicamycin was not changed. We concluded that the cor- responding antigen of McAb 3H11 might be an alkaline protein.

CYTOTOXICITY OF INDIRECT IMMUNOTOXIN MEDIATED BY ANTI-GASTRIC CANCER MONOCLONAL ANTIBODIES ON TUMOR CELLS

In the present study, an indirect assay was employed to investigate 5 anti-gastric cancer monoclonal antibodies for their cytotoxic potential as ricin A chain-containing immunotoxins. The tumor cell, were treated with dilutions of tested antibody followed by ricin A chain coupled to goat anti-mouse immunoglobulin. The cytotoxic effect was determined with tetrazolium colorimetric assay. The results showed that among the 5 antibodies chosen, MGb2 and MG7 could be well used for preparation of effective A chain immunotoxins.

New era in cancer immunotherapy: Twenty years to the discovery of monoclonal antibodies harnessing the immune system to eradicate tumors

The better understanding of the mechanism in which the immune system responds to the developing cancer provided the outcome in a new era in cancer immunotherapy. The tumor suppressive effect on the immune system is caused by negative T cell receptor signaling that abrogate immunity against the cancer cells. Novel monoclonal antibodies that target co-inhibitory receptors on T cells block the tumor induced inhibition of the immune system and enable the immune system to eradicate the tumors. The development of such antibodies started twenty years ago by the preparation of a monoclonal antibody termed BAT. A single administration of the antibody to tumor bearing mice resulted in striking anti tumor activity that was mediated by the lymphocytes. These studies provided a basis for the new era of cancer immunotherapy. The present review summarizes twenty years to the discovery of monoclonal antibodies harnessing the immune system to eradicate tumors.

IMMUNOELECTRON MICROSCOPIC STUDY ON THE LOCALIZATION OF MONOCLONAL ANTIBODIES ON GASTRIC CANCER CELLS

The localization of three monoclonal antibodies (MAb) against gastric cancer was studied on two human gastric cancer cell lines by immunoelectron microscopic technique. It has shown that the corresponding antigens of MAb 3G9 and 3H11 were distributed on the microvilli (M) and non-microvillus (NM) plasma membrane of target cells, with various M to NM ratios depending on the MAbs and target cells used. However, the corresponding antigens of MAb PD4 was only localized on the surface of round or finger-like bulges of target cells and never on the microvilli and non-microvillous plasma membrane. Since the nature and function of these tumor antigens have not been identified yet, the implication of the different distributions of these antigens remians to be clarifated.

THE ALTERNATIVE PATHWAY OF HUMAN T CELL ACTIVATION BY MONOCLONAL ANTIBODIES(A COMPARATIVE STUDY BETWEEN NORMAL INDIVIDUALS AND CANCER PATIENTS)

This paper described T cell proliferative response by an alternative pathway in normal subjects and In patients with malignant diseases. Two McAbs, Anti-CCTl and Lo-CD2-act recognizing two distinct epitopes on E-receptor (CD2) were used to costimulate PBMC. Proliferative responsiveness was measured by 3H-thymidine incorporation. It was found that 82% of 72 nonnal subjects gave proliferative response whereas only 23% of the 93 patients did. The average cpm±SD in patients with bladder cancer (118±2314), kidney cancer (1619±2719) or lymphoma (2518±4057) was significantly lower than that in normal subjects (4935±2314), (P<0.001). These results indicate that T cell proliferation through the alternative pathway was significantly depressed in patients with cancer, and this can be used as a new parameter to monitor the immune status of cancer patients.

SELECTIVE KILLING OF GASTRIC CANCER CELLS BY MONOCLONAL ANTIBODY CONJUGATED WITH A CHAIN OF RICIN

A specific cytotoxic agent against gastric cancer was constructed by covalently coupling the ricin A chain to monoclonal artibody, MGb2. MGb2 was modified by SPDP to introduce the 3-(2-pyridylthio) propionyl radical and then treated with a reduced A chain to give a disulfide linked conjugate that retained the original binding specificity of the antibody moiety. The conjugate obtained retained the activity of the antibody and the biological activity of the A chain well.

Monoclonal Antibodies: An Emerging Class of Therapeutics in Non Small Cell Lung Cancer

Lung cancer is the leading cause of cancer-related deaths in industrialized countries and non small cell lung cancer (NSCLC) accounts for 85% of all lung cancers. Cisplatin doublet based chemotherapy, which is the recommended regimen in first line therapy in advanced or metastatic NSCLC, improves survival but in low proportion. Monoclonal antibodies (mAbs) are a novel promising therapeutic class used with great results in inflammatory diseases such as rheumatoid arthritis. Antibodies are natural proteins with modular structure, specific pharmacodynamics and pharmacokinetics and possibly produced against any antigens, thus giving them several advantages over small drug therapeutics. In solid tumors, therapeutic mAbs improved progression free survival (PFS) and overall survival (OS) of patients with breast and colon cancers and had considerably changed the treatment in clinical practice. In NSCLC, bevacizumab, an anti-VEGF mAb, and cetuximab, an anti-EGFR mAb, are the most studied antibodies. Bevacizumab acts on angiognenesis and improved PFS of non squamous NSCLC but in low proportion as shown in two large phase III trials. It was approved by European Medicines Agency (EMEA) and Food and Drug administration (FDA) as a first line therapy in combination with cisplatin doublet chemotherapy. Cetuximab slightly enhanced OS but did not improve PFS in two large phase III trials. These results added to high adverse effect lead to cetuximab refusal by EMEA and FDA in NSCLC. At first glance, the results of mAbs in NSCLC are somewhat disappointing, in contrast to the benefits obtained with mAb treatments in other solid tumors. However, many other mAbs directed against novel targets, such as IGF1-R or CTLA-4, and new mAbs targeting VEGFR and EGFR pathways with different pharmacodymamical and pharmacokinetic properties are under evaluation and may change our vision of taking care of patients with NSCLC. In conclusion, it seems that mAbs therapy in NSCLC clearly marks the start of a new era in NSCLC treatment, with promises in improving patient survival and quality of life.

Development and Characterization of Monoclonal Antibody Specifically Against TSP50

Testis-specific protease 50（TSP50） has been identified as a testis-specific protein that is expressed abnormally in most human breast cancer samples,which makes it an attractive molecular marker and a potential target for diagnosis and therapy.In the present study,we prepared a panel of monoclonal antibodies（mAbs） with high specificity and sensitivity against TSP50 by hybridoma method and characterized them by ELISA,Western blot,immunofluroescence and immunohistochemical analyses.The results show that all of the 9 different clones can specifically bind to TSP50.The mAbs against TSP50 we generated could be good tools for both basic and clinical studies.

Inhibiting effects of ^(131)I-labeled anti-CEA monoclonal antibodies injected intrasplenically on liver metastasis from human colonic adenocarcinoma

I-labeled anti-CEA monoclonal antibody injected intrasplenically has better effects on inhibiting liver metastasis from human colonic adenocarcinoma than that through tail vein in nu/nu mice model.

GENERATION OF MONOCLONAL ANTIBODY AGAINST HUMAN ANDROGEN RECEPTOR WITH SYNTHETIC PEPTIDE

Objective: Preparation of anti-human androgen receptor(hAR) monoclonal antibody (McAb). Methods: Four cells lines of hybridoma secreting specific monoclonal antibodies against AR were first established by fusion SP2/0 cell with spleen cell from BALB/c mice immunized with the coupling complex of hAR-KLH. Results: Paraffin-embedded sections of 45 prostate cancers were detected. There was an overall concordance of 91% using Immunohistochemistry between AR polyclonal antibody from Zymed and hAR-N McAb selfmade. Conclusion: The results show that the McAb obtained in this study would be a useful tool to detect the AR status in prostate cancer.

Therapeutic Implications of Monoclonal Antibody

Background: The coronavirus disease 2019 (COVID-19) pandemic is a distinct public health issue that calls for the quick development of novel treatments and viral detection. Due to their high specificity and reliability, monoclonal antibodies (mAbs) have emerged as useful diagnostic and therapeutic tools for a variety of diseases. As a result, several scientists have jumped right into developing Ab-based assays for the identification of SARS-CoV-2 and Ab drugs for use as COVID-19 therapy agents. Since the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is essential for viral infection and has a known precise structure, it has become a key target for the creation of therapeutic antibodies. The use of Ab cocktails is anticipated to be a key component of an efficient COVID-19 treatment plan since SARS-CoV-2 is an RNA virus with a high mutation rate, particularly when subjected to the selection pressure of aggressively applied preventive vaccinations and neutralizing Abs. Furthermore, SARS-CoV-2 infection could provoke an overzealous immune response, leading to a cytokine storm that accelerates the onset of a severe disease. Abs to counteract cytokine storms are also actively being researched as COVID-19 therapies. Abs are now used in SARS-CoV-2 detection assays, including immunoglobulin and antigen tests, in addition to their use as medicines. In order to stop the spread of COVID-19, such Ab-based detection tests are essential surveillance tools. In this article, we’ll go over several important ideas related to mAb-based COVID-19 pandemic detection tests and treatments. Objective: To understand the role of hybridoma technology in therapeutic implications. 1) To study the basic concepts and options in hybridoma technology;2) To study the applications of hybridoma technology;3) To explore how hybridoma technology is applied in diagnostic histopathology. Method: For this method generally there is use of mouse or mammals are transfect with the Ags to find out the formation of antibody afterwards isolate the antibody which has been formed after injecting the antigens for a number of weeks. Following are the steps for mAbs: Step 1: In this step immunization of mouse is done;Step 2: Spleen is used for the isolation of B cells;Step 3: Cultivation of cancerous cells;Step 4: Merging of B cells with Myeloma cells;Step 5: This step cell lines are separated;Step 6: in the next step screening the suitable cell lines;Step 7: observation of multiplication in vitro as well as in vivo;Step 8: Harvesting. Discussion: Now a day there are many diseases which has been cured easily at the mean time it’s very difficult to diagnose and get the treatment. Due to advancement of monoclonal antibodies are used in the diagnosis and treatments such as COVID-19, SARS and SARS COV-2. Therefore important part of the monoclonal antibodies are its used in the diagnosis as well as in the treatment tools.

Translational research case studies:Development of antibodies for cancer diagnosis and therapy

Antibodies are primary tools in several areas of biomedical sciences, including basic research, diagnostics, and molecular therapeutics. Antibodies are widely used in diagnostic applications for clinical medicine. Analysis of cells and tissues in pathology laboratories includes the use of antibodies on tissue sections. Further, antibodies are making rapid inroads into medical therapeutics, driven by technological evolution from chimeric and humanized to fully human antibodies. The therapeutic antibody market has the potential to reach $30 billion by 2010. Our lab has developed a monoclonal antibody, named Met4 that was raised against the extracellular domain of Met specifically with the goal of measuring Met in FFPE tissues. The Met receptor kinase is expressed on the cell surface of a significant number and variety of human primary solid tumors and in their metastases. The characterization of the Met4 antibody suggests it should possess adequate performance for quantification of Met expression in clinical specimens. We have also generated a fully human Fab fragment against EGFR; conjugated it to taxol as an immuno-chemotherapy agent; and investigated its in vitro antitumor efficacy on EGFR positive A431 epidermoid carcinoma cells.