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Name of the Trainee : Mary Ros Joy

Name of the Company : Biocon limited

Name of the Supervisor/Guide: Dr. Maria Melina Soares

Title of Report : Determination of the functional activity of novel bifunctional monoclonal antibodies in vitro.

Field of Training: R & D Area of the project: Immunology

ABSTRACT This work describes the screening of various novel bifunctional monoclonal antibodies which will be using for cancer immunotherapy. It involves development and standardization of different ELISAs as well as various cell based assays such as inhibition of proliferation assays, antibody dependent cell cytotoxicity assays (ADCC), Flow cytometer based cell-Receptor binding assays to find out the functionality of the bifunctional monoclonal antibodies using Epidermoid carcinoma cell line-A-431.

BACKGROUND ART

The immune system is remarkably versatile defence system that has evolved to protect animals from invading pathogenic microorganisms and cancer [1]. It is able to generate an enormous variety of cells and molecules capable of specifically recognizing and eliminating an apparently limitless variety of foreign invaders. These cells and molecules act together in a dynamic network whose complexity rivals that of the nervous system.Immunotherapy is a new class of cancer treatment that works to harness the innate powers of the immune system to fight cancer [2]. It is defined as "Treatment of disease by inducing, enhancing, or suppressing an immune response". Because of the immune system's unique properties, these therapies may hold greater potential than current treatment approaches to fight cancer more powerfully, to offer longer-term protection against the disease, to come with fewer side effects, and to benefit more patients with more cancer types. Immunotherapy designed to elicit or amplify an immune response are classified as Activation Immunotherapy. And that designed to reduce, suppress or more appropriately direct an existing immune response, as in cases of autoimmunity or allergy, are classified as Suppression Immunotherapy [3].One way the immune system normally attacks foreign substances in the body is by making large numbers of different antibodies. An antibody is a sticky protein that targets a specific antigen. Antibodies circulate in the body until they find and attach to the antigen. Once attached, they recruit other parts of the immune system to destroy the cells containing the antigen.Many copies of a specific antibody can be made in the lab. These are known as monoclonal antibodies (mAbs). These antibodies can be useful in fighting diseases because they can be designed specifically to only target a certain antigen, such as one that is found on cancer cells. Cancer treatment: One possible treatment for cancer involves monoclonal antibodies that bind only to cancer cell-specific antigens and induce an immunological response against the target cancer cell. Such mAb could also be modified for delivery of a toxin, radioisotope, cytokine or other active conjugate; it is also possible to design bi-specific antibodies that can bind with their Fab regions both to target antigen and to a conjugate or effecter cell. In fact, every intact antibody can bind to cell receptors or other proteins with its Fc region [4]. INTRODUCTION

The immune system is a network of cells, tissues, and organs that work together to defend the body against attacks by foreign invaders, such as micro-organisms and cancer. Monoclonal antibodies can be used to treat cancer.Monoclonal antibodies are now used to treat many diseases, including some types of cancer. A major advantage of these drugs is that because they are so specific, they may have only mild side effects, unlike some other cancer treatments. But researchers first have to identify the right antigen to attack. For cancer, this is not always easy, and so far mAbs have proven to be more useful against some cancers than others. Over the past 15 years or so, the US Food and Drug Administration (FDA) have approved about a dozen mAbs to treat certain cancers. As researchers have found more antigens that are linked to cancer, they have been able to make monoclonal antibodies against more and more cancers. Clinical trials of newer mAbs are now being done on many types of cancer.Types of monoclonal antibodiesTwo types of monoclonal antibodies are used in cancer treatments: Naked mAbs are antibodies that work by themselves. There is no drug or radioactive material attached to them. These are the most commonly used mAbs at this time. Conjugated mAbs are those joined to a chemotherapy drug, radioactive particle, or a toxin (a substance that poisons cells). These mAbs work, at least in part, by acting as homing devices to take these substances directly to the cancer cells [5].Squamous-cell carcinoma (SCC or SqCC) is a cancer of a kind of epithelial cell, the squamous cell. These cells are the main part of the epidermis of the skin, and this cancer is one of the major forms of skin cancer. However, squamous cells also occur in the lining of the digestive tract, lungs, and other areas of the body, and SCC occurs as a form of cancer in diverse tissues, including the lips, mouth, esophagus, urinary bladder, prostate, lung, vagina, and cervix, among others. Despite sharing the name squamous cell carcinoma, the SCCs of different body sites can show tremendous differences in their presenting symptoms, natural history, prognosis, and response to treatment. SCC is a histologically distinct form of cancer. It arises from the uncontrolled multiplication of cells of epithelium, or cells showing particular cytological or tissue architectural characteristics of squamous cell differentiation, such as the presence of keratin, tonofilament bundles, or desmosomes, structures involved in cell-to-cell adhesion.Epidermal growth factor receptor: The epidermal growth factor receptor (EGFR; ErbB-1; HER1 in humans) is the cell-surface receptor for members of the epidermal growth factor family (EGF-family) of extracellular protein ligands. EGFR exists on the cell surface and is activated by binding of its specific ligands, including epidermal growth factor and transforming growth factor (TGF). Mutations that lead to EGFR overexpression (known as upregulation) or over activity have been associated with a number of cancers, including lung cancer, anal cancers. Mutations, amplifications or misregulations of EGFR or family members are implicated in about 30% of all epithelial cancers. Mutations involving EGFR could lead to its constant activation, which could result in uncontrolled cell division a predisposition for cancer. Consequently, mutations of EGFR have been identified in several types of cancer, and it is the target of an expanding class of anticancer therapies. The identification of EGFR as an oncogene has led to the development of anticancer therapeutics directed against EGFR, including gefitinib and erlotinib for lung cancer, and cetuximab for colon cancer.

AIM & OBJECTIVE

AIM:Screening of various novel bifunctional monoclonal antibodies which can be used for cancer immunotherapy.

OBJECTIVES: Check the functionality of different moieties of the construct using ELISAs. To determine the strength of binding of drug to the receptor present on the cells using Flow cytometer based binding assay. To culture and maintain A-431 cell lines of epidermoid carcinoma for conducting cell based assays. To study the Inhibition of proliferation of cancer cells in the presence of mabs. To study antibody dependent cell cytotoxicity of the mabs.

TECHNIQUES AND METHODS USED

ELISAPrinciple: Enzyme-linked immune sorbent assay is a widely-used method for measuring the concentration of a particular molecule (e.g., a hormone or drug) in a fluid such as serum or urine. It is also known as enzyme immunoassay or EIA. Amongst the available assays, ELISA test is one of the distinguished and widely used tests. It is extensively used due to the advantages like rapidity and speed in experimentation, greater sensitivity and specificity for even small amount of test samples. In ELISA test, the reaction is measurable in both qualitative and quantitative terms i.e. qualitative Elisa measures specific type of substance while qualitative Elisa measures quantity of substance in the given sample respectively [6].

Method: The protein is coated to a solid surface, such as the inner surface of the micro plate. The primary antibody dilution is added. After incubation time (time for binding to the immobilized antigen), secondary antibody or antibodies coupled to an enzyme one that produces a coloured product from a colourless substrate is added. Washing is done after each incubation periods to remove the unreacted reagents. The intensity of the colour produced is proportional to the amount of enzyme-labelled antibodies bound (and thus to the concentration of the antibodies being assayed). Here we are comparing the sample with the standard and concluding how much is comparable.

Materials: 1X Phosphate Buffered Saline (PBS) (pH 7.4) (GIBCO) ELISA plate washing buffer: 0.1% Tween - PBS solution Blocking Buffer Assay diluents Negative control Positive control Bifunctional mab. Biotin-conjugated Secondary antibodies SA-HRP TMB-substrate. CELL BASED ASSAYSCell line used is epidermoid carcinoma cell line-A-431A-431 (ATCC CRL-1555) Organism: Homo sapiens, human Tissue: skin/epidermis Product Format: frozen Morphology: epithelial Culture Properties: adherent Biosafety Level: 1 Disease: Epidermoid carcinoma Storage Conditions: liquid nitrogen vapor phase Revival of cells The vial was thawed quickly with gentle agitation in a 37C water bath. To reduce the possibility of contamination, the cap was kept out of the water. After thawing, the vial was decontaminated by spraying with 70% ethanol. The content of the vial was transferred to a centrifuge tube containing complete culture medium and centrifuged at 125 x g for 5 minutes. The process was conducted in a bio safety hood to maintain aseptic conditions. The cell pellet was resuspending with the complete medium and dispensed into a culture flask. The culture was incubated at 37C in a 5% CO2 incubator. Maintenance of the cell line The flask was visually examined for macroscopic evidence of any microbial contamination using an inverted microscope. The culture was also checked for viable cells which get attached to the bottom of the flask. Medium renewal was done twice or thrice weekly. For sub culturing the flasks, the old culture medium was removed and discarded. The cells were briefly rinsed with 0.25% (w/v) Trypsin- 0.53mM EDTA solution to remove all traces of serum which contains trypsin inhibitor. Trypsin-EDTA solution was added to the flasks and cells were observed under an inverted microscope to ensure that the cells left the flask (usually within 5 to 7 minutes). Then complete growth medium was added to the flask to neutralise trypsinisation and the cells were aspirated by gentle pipetting [7]. Materials: The base medium for this cell line is GIBCO DMEM Medium, Catalog No. To make complete growth medium, the following components were added: Fetal bovine serum to a final concentration of 10%. (FBS) 5% PenStrep 20mM HEPES

Binding Assay

Principle: Flow cytometry is alaser based, biophysical technology employed incell counting, sorting, biomarker detection andprotein engineering, by suspending cells in a stream of fluid and passing them by an electronic detection apparatus. When sample solution is injected into a flow cytometer, the particles are randomly distributed. The sample is ordered into asingle particlestream then can be interrogated by the machines detection system [8].

Method: Primary dilutions are prepared in tubes using filtered PBS. Trypsinised cells that resuspended in PBS is added to the tubes and incubated. After incubation washed with PBS and Secondary is added and incubated. After washing samples are given for flow cytometer acquisition. Materials: A 431 cell line Medium: DMEM High Glucose Penicillin streptomycin Fetal Bovine Serum (FBS) 0.25% Trypsin EDTA Negative and Positive control Bifunctional mab. Dulbecco Phosphate Buffered Saline (DPBS) Phosphate Buffered Saline

Proliferation Assay

Principle: The Proliferation Assay allows determining the number of cells that are growing in the absence or presence of certain proliferation affecting agents, e.g. Drug A certain number of cells are seeded in the wells of a 96 well plate. At the same time proliferation affecting agent is added. The cells are incubated for a certain time at 37C.After incubation Alamar Blue is added which monitors the reducing environment of the proliferating cell. Alamar Blue is soluble, stable in culture medium and is non-toxic. The continuous monitoring of cells in culture is therefore permitted. Proliferation may be monitored with Alamar Blue using a standard spectrophotometer, a standard spectrofluorometer, a spectrophotometric microtiter well plate reader, or spectrofluorometric microtiter well plate reader.

Method: Drug dilution is prepared and according to the template added to the assay plate. To this trypsinised cells are added. Incubated at 37 C After incubation add Alamar Blue Again incubate and take out fluorescence reading.

Materials: A 431 cell line Medium: DMEM High Glucose Penicillin streptomycin Fetal Bovine Serum (FBS) 0.25% Trypsin EDTA Negative and Positive control Bifunctional mab. Dulbecco Phosphate Buffered Saline (DPBS) Alamar blue

Antibody-Dependent Cellular Cytotoxicity (ADCC)

Principle: Antibody-dependent cellular cytotoxicity (ADCC) is one of the most potent molecular mechanisms used by the immune system to eliminate tumor cells. It is a mechanism of cell-mediated immune defense whereby an effecter cell of the immune system actively lyses a target cell, whose membrane-surface antigens have been bound by specific antibodies. It is one of the mechanisms through which antibodies, as part of the humoral immune response. Classical ADCC is mediated by natural killer (NK) cells; macrophages, neutrophils and eosinophils can also mediate ADCC.

Method: Cells are seeded on 96 well plate one day before and incubated at 37 C On the day of experiment blood is collected from the donor and PBMCs are separated. From the previously seeded plate media is removed and prepared drug dilutions are added. Plate is incubated for some time then separated PBMCs are added. Again incubated at 37 C. Add CytoTox-Glo to the wells Incubate and take out the luminescence reading.Materials: A 431 cell line Medium: DMEM High Glucose Penicillin streptomycin Fetal Bovine Serum (FBS) 0.25% Trypsin EDTA Medium: RPMI 1640 Negative and Positive control Bifunctional mab. Dulbecco Phosphate Buffered Saline (DPBS) CytoTox-Glo Cytotoxicity Assay Kit Ficoll Paque Plus

EQUIPMENTS HANDLED

Flow cytometerIt is a laser based, biophysical technology employed in cell counting, sorting, biomarker detection and protein engineering, by suspending cells in a stream of fluid and passing them by an electronic detection apparatus. It allows simultaneous multi parametric analysis of the physical and/or chemical characteristics of up to thousands of particles per second.

Schematic diagram of a flow cytometer, showing focusing of the fluid sheath, laser, optics (in simplified form, omitting focusing), and photo multiplier tubes (PMTs), analogue-to-digital converter, and analysis workstationA beam of light (usually laser light) of a single wavelength is directed onto a hydro dynamically focused stream of liquid. A number of detectors are aimed at the point where the stream passes through the light beam: one in line with the light beam (Forward Scatter or FSC) and several perpendicular to it (Side Scatter or SSC) and one or more fluorescence detectors. Each suspended particle from 0.2 to 150 micrometers passing through the beam scatters the ray, and fluorescent chemicals found in the particle or attached to the particle may be excited into emitting light at a longer wavelength than the light source. This combination of scattered and fluorescent light is picked up by the detectors, and, by analyzing fluctuations in brightness at each detector, it is then possible to derive various types of information about the physical and chemical structure of each individual particle. FSC correlates with the cell volume and SSC depends on the inner complexity of the particle (i.e., shape of the nucleus, the amount and type of cytoplasmic granules or the membrane roughness). This is because the light is scattered off of the internal components of the cell.

HemocytometerThe hemocytometer is a device used to count cells.It consists of a thick glass microscope slide with a rectangular indentation that creates a chamber. This chamber is engraved with a laser-etched grid of perpendicular lines. The device is carefully crafted so that the area bounded by the lines is known, and the depth of the chamber is also known. It is therefore possible to count the number of cells or particles in a specific volume of fluid, and thereby calculate the concentration of cells in the fluid overallTo use the hemocytometer, first make sure that the special coverslip provided with the counting chamber is properly positioned on the surface of the counting chamber. When the two glass surfaces are in proper contact Newton's rings can be observed. If so, the cell suspension is applied to the edge of the cover slip to be sucked into the void by capillary action which completely fills the chamber with the sample. The number of cells in the chamber can be determined by direct counting using a microscope, and visually distinguishable cells can be differentially counted. The number of cells in the chamber is used to calculate the concentration or density of the cells in the mixture the sample comes from. It is the number of cells in the chamber divided by the chamber's volume, which is known from the start, taking account of any dilutions and counting shortcuts:[9]

Hemocytometer grid:red square = 1mm2, 100nlgreen square = 0.0625mm2, 6.25nlyellow square = 0.04mm2, 4nlblue square = 0.0025mm2, 0.25nlat a depth of 0.1mm.

Micro plate Readers (also known as plate readers) These are laboratory instruments designed to detect biological, chemical or physical events of samples in micro titer plates. They are widely used in research, drug discovery, bioassay validation and quality control in the pharmaceutical and biotechnological industry. Common detection modes for micro plate assays are absorbance, fluorescence intensity, luminescence, time-resolved fluorescence, and fluorescence polarization [10].

Other equipment used: Inverted microscope Biosafety cabinet Multi-channel pipette 96 well plate washer Automated cell counter

RESULT

Due to proprietary nature of this work, the results cannot be included in this report.

CONCLUSION

The in-vitro ELISAs have confirmed that the two domains of the bifunctional antibody are functional. The ADCC assay demonstrates that the Fc portion of the antibody moiety is also intact, accessible & functional. The bifunctional antibody will be tested for therapeutic efficacy in preclinical animal tumor models. From the assays done for finding out the functionality of the bifunctional monoclonal Antibodies its concluded that they are comparable with the standards. And they are responding nicely to each assay. So they can be used for further developmental studies.Further development in bifunctional monoclonal antibodies will be a milestone in cancer therapy.

REFERENCE

1. http://en.wikipedia.org/wiki/Immune_system2. Ault KA, Future II study group. Effect of prophylactic human papillomavirus L1 virus like particle vaccine on risk of cervical intraepithelial neoplasia grade 2, grade 3, and adenocarcinoma in situ: A combined analysis of four randomised clinical trials. Lancet. 2007; 369:186118683. Morgan RA, Dudley ME, Wunderlich JR, et al. Cancer regression in patients after transfer of genetically engineered lymphocytes. Science. 2006; 314:126129.4 . http://en.wikipedia.org/wiki/Monoclonal_antibodies5. http://www.cancer.org/treatment/treatmentsandsideeffects/treatmenttypes/immunotherapy/immunotherapy-monoclonal-antibodies6. http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/E/Elisa.html7. http://www.atcc.org/products/all/CRL-1555.aspx8. http://en.wikipedia.org/wiki/Flow_cytometry9. https://en.wikipedia.org/wiki/Hemocytometer10. http://en.wikipedia.org/wiki/Plate_reader

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