Assignment Topic

Assignment
Topic: Recombinant anti-cancer therapeutics.

Course code: PHRM-521
Course Title: Molecular Biology and Biotechnology
Submitted To:
Dr. Abu A Sajib
Adjunct faculty
Department of Pharmacy
East West University
Submitted By :
Md. Rakayet Hossain
Id. 2017-3-79-037

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Introduction
Cancer:
Cancer is the uncontrolled development of anomalous cells anyplace in a body. Cancer advanced when the body’s ordinary control instrument stops working. Cancer starts when quality changes invigorates development of one cell or some cells and increase more than typical condition, which may comes about in arrangement of a tumor.; common categories of cancer-related or causative agents are: chemical or harmful compound exposures, ionizing radiation, a few pathogens, and human genetic mutation. (Charles Patrick Davis, 2018)
There are over 200 sorts of cancer. Cancers are the moment most common cause of passing around the world. (Chulpanova et al., 2018) .The foremost common cancer medications are confined to surgery, conventional chemotherapy and radiotherapy. In spite of the fact that these conventional anticancer therapies are compelling within the management of numerous patients, these treatments are incapable for approximately half of cancer sufferers. (Pranchevicius and Vieira, 2013)
Recombinant anti-cancer therapeutics
In spite of the fact that numerous anticancer treatments are accessible, finding a remedy for cancer proceeds to be a troublesome assignment. In this way, numerous endeavors have been made to create more successful medications, such as recombinant therapy based on a unused lesson of tumor-specific items that are delivered utilizing recombinant DNA technology. These recombinant items are utilized with the most destinations of slaughtering the tumor and stimulating safe cells to reply to the cancer cells. The vital recombinant items in anticancer treatment are vaccine, antibodies, immunotoxins, immunostimulants and combination proteins. This audit centers on the common viewpoints of these hereditarily built items, their clinical execution, current propels and future prospects for this sort of anticancer treatment. (Pranchevicius and Vieira, 2013)
Antibodies
Principle: Recombinant antibodies are monoclonal antibodies produced in vitro using manufactured qualities. (Man, 2018)
Monoclonal antibodies (mAbs) are the class of antibodies that currently contribute most to recombinant products. Initially, hybridoma technology was used to produce mAbs.
The improvement of procedures to humanize or chimarize mAbs to decrease their murine components has been an important advance within the field of counter acting agent therapeutics. These new recombinant antibodies carry on essentially to a normally occurring immunoglobulin and mirror the typical antibody-based immune response, serving as effective agents in treating patients with cancer.

The production of recombinant antibodies takes after basically similar workflow. It consists of determining the grouping of the required product taken after by refinement of the codon, at that point quality union and develop era, at that point they are transferred to a cell culture within the process called transfection and once the cell culture produces the specified recombinant antibody, it is routinely collected, filtered and analyzed or utilized for advance experimentation. For recombinant antibody production the steady cell lines such as CHO is used.

Fig: Recombinant Antibody Production.

Examples:
Drug Drug Class Therapeutic Indication Organism Class Strains Year of Approval
Trastuzumab
mAb Metastatic breast cancer, gastric
cancer
Humanized Mammalian cell (Chinese
hamster ovary) 1998
Cetuximab
mAb Metastatic colorectal cancer Chimeric
murine/human
Mammalian (murine myeloma)
cell 2004
Pertuzumab
mAb HER2-positive metastatic breast
cancer
Humanized Mammalian cell (Chinese
hamster ovary) 2012
Alemtuzumab
mAb B-cell chronic lymphocytic leukemia Humanized Mammalian cell (Chinese
hamster ovary) 2001
Vaccine
Principle: Dynamic immunotherapy against cancer is spoken to by vaccines. Most antibodies point to improve or incite a resistant response against a tumor by implies of tumor antigen-specific cytotoxic T lymphocytes (CTLs) since these cells are able to straightforwardly kill malignant cells. Research on cancer antibodies has utilized several sources of tumor antigens, such as filtered or synthesized tumor cell-surface particles (proteins, peptides or lysates) and cells or lysates of allogeneic or autologous tumor cell lines. Tumor-specific antigens (TSAs) may be a idealize target for cancer antibodies since these antigens are fundamental for tumorigenesis and cancer progression. (Pranchevicius and Vieira, 2013)
Recombinant infections can be utilized to hereditarily adjust cells, activating the expression of tumor-specific antigens. These infections can too be utilized to straightforwardly infect antigen-presenting cells to boost anti-cancer immune reactions. The upgraded introduction of the tumor antigens to immune cells leads to expanded recurrence and having expression of cytotoxic T-cells. Furthermore, transgenes expressed by the viral vector are more immunogenic than antigens managed with adjuvant. Therefore, recombinant infections show an important instrument for advancement of antitumor therapeutic vaccines.

Example:
Drug Drug Class Therapeutic Indication Organism Class Strains Year of Approval
Quadrivalent HPV Vaccine Cervical, vulvar, vaginal and anal
cancer caused by HPV 16 and 18 Viral VLPs of the major capsid
(L1) protein of HPV 6, 11,16
and 18 2006
Sipuleucel-T Vaccine Prostate cancer Human Patient’s peripheral blood
mononuclear cells 2010
Immunostimulants
Principal: Immunostimulants known as immunostimulators are attractive substances that activate the immune system of humans and animals for avoidance of diseases and improvement of the body’s common resistance to various viral and bacterial diseases. e.g. Cancer. (JMMID)
The advancement of products that stimulate immune cells of either the adaptive or the natural resistant response gives evidence for the capabilities of immunotherapy after a tumor has developed to the point of causing clinical infection. In addition, interferon’s, a course of cytokines with multifunctional properties, can induce pro-apoptotic quality expression, causing direct impacts on cancer cells and inhibiting angiogenesis. Of the three interferon sorts found, as it were sort I interferon’s, and particularly interferon-? (IFN-?), have applications in anticancer treatment. (Pranchevicius and Vieira, 2013)
IL-2 is a T cell development factor that promotes T cell expansion and survival. It stimulates the cytotoxic action of CD8 and normal killer (NK) cells and balances the differentiation of T cells in reaction to antigen. In addition, IL-2 induces the generation and multiplication of antibodies by B cells, stimulates the development of administrative T cells, and is fundamental for activation-induced cell death. (Shahbazi and Bolhassani, 2018)

Fig: INF? Mechanism. (Solis et al., 2006)
Example:
Drug Drug Class Therapeutic Indication Organism Class Strains Year of Approval
IFN-?2a Immunostimulant interferon
Hairy cell leukemia, Kaposi sarcoma Human Escherichia coli 1986
Aldesleukin Immunostimulant interleukin
Metastatic renal cell carcinoma,
metastatic melanoma
Human Escherichia coli 1992
Immunotoxin
Principle: Recombinant immunotoxins (ITs) are bifunctional chimeric molecules composed of an antibody part connected to a toxin component. Immunotoxins get their toxic potency from the toxin and their specificity from the antibody. (Allahyari et al., 2017)
For this purpose, various bacterial toxins have been used in RITs targeting cancer cells, and among these toxins, Pseudomonas exotoxin (PE) and Diphtheria toxin (DT) are the most commonly used bacterial toxins.

Once antibody–drug conjugates (ADCs) and immunotoxins are internalized into an endocytic compartment after binding on the cell surface. The ADCs travel to lysosomes, where the drug is released from the antibody, inducing drug penetration in the cytosol, disruption of microtubule dynamics, and cell death. Modified Pseudomonas exotoxin (PE) toxin is cleaved from immunotoxin by the furin protease and transported to the endoplasmic reticulum (ER) through the Golgi. The toxin catalyzes ADP ribosylation of eukaryotic elongation factor 2 (eEF2), inducing inhibition of protein synthesis and cell death. The T domain of Diphtheria toxin (DT) forms a pore in the membrane of the endosome, allowing transit of DT in the cytosome. DT also catalyzes inhibitory modification of eEF2. (Li et al., 2017)

Fig: Mechanism of Action of Immunotoxins. (Li et al., 2017)
Since of this phenomenon, immunotoxins are very effective agents. Thus, immunotoxins can be used against solid and non-solid tumors but have an improved reaction against the second since immunotoxins are large enough to pass through the tumor tissue. (Pranchevicius and Vieira, 2013)
Examples:
Drug Drug Class Therapeutic Indication Organism Class Strains Year of Approval
Gemtuzumab
ozogamicin
Antibody-conjugated CD33-positive acute myeloid
leukemia
Humanized Mammalian cell 2000
Brentuximab
vedotin
Antibody-conjugated Hodgkin lymphoma, systemic
anaplastic large-cell lymphoma Chimeric
murine/human
Mammalian cell (Chinese
hamster ovary) Yttrium-90
Ibritumomab
Tiuxetan
Antibody-conjugated Non-Hodgkin lymphoma Murine Mammalian cell (Chinese
hamster ovary) 2002
Conclusion
Advancement in anticancer treatment has been surprising and has represented new hopes for patients. These progresses would not be possible without biotechnology support.

Reference
Allahyari, H., Heidari, S., Ghamgosha, M., Saffarian, P. and Amani, J. (2017). Immunotoxin: A new tool for cancer therapy. Tumor Biology, 39(2), p.101042831769222.

Charles Patrick Davis, P. (2018). Cancer Causes, Types, Treatment, Symptoms ; Signs. online MedicineNet. Available at: https://www.medicinenet.com/cancer/article.htm#what_is_cancer Accessed 30 Oct. 2018.

 Chulpanova, D., Solovyeva, V., Kitaeva, K., Dunham, S., Khaiboullina, S. and Rizvanov, A. (2018). Recombinant Viruses for Cancer Therapy. Biomedicines, 6(4), p.94.

Li, M., Liu, Z., Liu, X., Hui, Q., Lu, S., Qu, L., Li, Y., Zhou, Y., Ren, H. and Hu, P. (2017). Clinical targeting recombinant immunotoxins for cancer therapy. OncoTargets and Therapy, Volume 10, pp.3645-3665.

Man, T. (2018). Recombinant Antibodies: An Overview. online Info.gbiosciences.com. Available at: https://info.gbiosciences.com/blog/recombinant-antibodies-an-overview Accessed 30 Oct. 2018.

Pranchevicius, M. and Vieira, T. (2013). Production of recombinant immunotherapeutics for anticancer treatment. Bioengineered, 4(5), pp.305-312.

Shahbazi, S. and Bolhassani, A. (2018). Immunostimulants: Types and Functions. online Jommid.pasteur.ac.ir. Available at: http://jommid.pasteur.ac.ir/article-1-131-en.html Accessed 30 Oct. 2018.

Solis, M., Goubau, D., Romieu-Mourez, R., Genin, P., Civas, A. and Hiscott, J. (2006). Distinct functions of IRF-3 and IRF-7 in IFN-alpha gene regulation and control of anti-tumor activity in primary macrophages. Biochemical Pharmacology, 72(11), pp.1469-1476.

Assignment Topic

Assignment
Topic: Production of Monoclonal Antibody using Hybridoma Technology.

Course code: PHRM-407
Course Title: Pharmaceutical Biotechnology.

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Section: 01.

Submitted To:
Najneen Ahmed
Senior Lecturer
Department of Pharmacy
East West University
Submitted By :
Shanzida Hossain Anonna
Id. 2016-1-70-069
Submission date: 5-11-2018
1. Introduction:
Monoclonal antibodies are laboratory-produced molecules designed to serve as substitute antibodies that can restore, improve or mimic the immune system’s attack on cancer cells. They are planned to tie to antigens that are generally more various on the surface of cancer cells than healthy cells. (Mayo Clinic, 2018)
A hybridoma is a biologically constructed hybrid of a mortal, antibody- producing, lymphoid cell, and a malignant, or immortal myeloma cell. Hybridomas cells are been designed to produce a desired antibody in large amounts, to produce monoclonal antibodies and now this procedure is popularly known as hybridoma technology. (Pandey, 2010) The production of monoclonal antibodies was invented by César Milstein and Georges J. F. Köhler in 1975. (Wakchaure and Ganguly, 2015)
2. Production of Monoclonal Antibodies:
2.1 Hybridoma technology procedure: There are basically two stages within the production of monoclonal antibodies (mAbs) –
a) The acceptance of antibody producing lymphoid cells in vivo and the selection of antibody-producing hybridoma cells in vitro; and
b) The in vitro/in vivo proliferation of selected hybridoma clones. (Marx et al., 1997)
The generation of mAbs producing cells requires the use of animals, usually mice; and is carried out in the following way:
Step 1: Immunization
Mice are immunized with an antigen that’s prepared for infusion either by emulsifying the antigen with Freund’s adjuvant or other adjuvant. Intact cells, entire membranes, and microorganisms are sometimes utilized as immunogens. In nearly all laboratories, mice are used to deliver the specified antibodies. In common, mice are immunized every 2-3 weeks. When an adequate antibody concentration is come to in serum, immunized mice are put to dead and the spleen cut out to use as a source of cells for combination with myeloma cells. (Institute for Laboratory Animal Research National Research Council, 1999)
Step 2: Preparation of Myeloma Cells
Myeloma cells are immortalized cells that are refined with 8 azaguanine to ensure their sensitivity to the hypoxanthine aminopterin-thymidine (HAT) choice medium utilized after cell combination. A week before cell fusion, myeloma cells are developed in 8-azaguanine. to testing the growth ability. (Institute for Laboratory Animal Research National Research Council, 1999)

Fig 1: Stages of hybridoma production. (Greenfield, 2014)
Step 3: Fusion of Myeloma Cells with Immune Spleen Cells
The altogether washed lymphocytes (? cells) are mixed with Hypoxanthine-guanine phosphoribosyl transferase  (HGPRT)  imperfect myeloma cells. The mixture of cells is exposed to polyethylene glycol (PEG) for a short period, since it is harmful. PEG is expelled by washing and the cells are kept in a new medium. These cells are composed of a blend of hybridomas, free myeloma cells and free lymphocytes.  (Jha, 2018)
Step 4: Selection of Hybridomas
The cells are then cultured in (HAT) the hypoxanthine aminopterin-thymidine medium, only the hybridoma cells develop, whereas the rest will gradually die.

(Jha, 2018)
Step 5: Screening the Products
The culture medium from each hybridoma culture is occasionally tested for the specified antibody specificity. The two procedures specifically ELISA and RIA are commonly used for this purpose. In both the tests, the antibody binds to the particular antigen and the unbound antibody and other components of the medium can be washed off. Thus, the hybridoma cells creating the specified antibody can be identified by screening. The antibody emitted by the crossover cells is referred to as monoclonal antibody. (Jha, 2018)
Step 6: Cloning and Propagation:
The single hybrid cells producing the desired antibody are isolated and cloned. (Jha, 2018)
Step 7: Characterization and Storage:
The monoclonal antibody should be subjected to biochemical and biophysical characterization for the required specificity. The mAbs must be characterized for their capacity to resist freezing, and defrosting. (Jha, 2018)
3. Application of Monoclonal antibody
In cancer treatment monoclonal antibody used as chemotherapy drug.

Radioimmunoassay is a alternative cancer therapy, where monoclonal antibody is coupled with radioisotope such that the cancer cell is killed by irradiation.
In organ transplantation monoclonal antibody is used because they help to better cross matching.

Humanized monoclonal antibody is used to detect the infant with bronchopulmonary dysplasia.

For the detection of bacterial and viral disease monoclonal antibody are applied.

Various disease like renal cancer, lymphoma, myeloma cancer, rheumatoid arthritis,
Metastatic breast cancer etc. where monoclonal antibody treatment is approved. (Kumar, et al., 2012)
4. Conclusion
The use of monoclonal antibodies is various and incorporates the prevention, determination, and treatment of disease. Also monoclonal antibodies are important for the investigation of parasites antigen. This hybridoma technology creates great opportunity for treatment and curing the disease by producing monoclonal antibody, which is more specific and more potent drug. So this hybridoma technology brought a revolutionary in life science.

5. Reference
Greenfield, E. (2014). Antibodies. 2nd ed. New york: Cold Spring Harbor Laboratory Press, pp.208-209.

Institute for Laboratory Animal Research National Research Council (1999). Monoclonal Antibody Production. A Report of the Committee on Methods of Producing Monoclonal Antibodies. Washington, DC: NATIONAL ACADEMY PRESS, pp.6-8.

Jha, N. (2018). Monoclonal Antibodies: Production, Advantages and Limitations. online Biology Discussion. Available at: http://www.biologydiscussion.com/antibodies/monoclonal-antibodies-production-advantages-and-limitations/10068 Accessed 4 Nov. 2018.

Kumar, A. Singh, M. ; Gupta, SM. (2012) “Hybridoma Technology”. In: Biotechnology in medicine and agriculture: principles and practices. (eds. Kumar A, Pareek A ; Gupta SM) I. K. International publishing house Pvt. Ltd., New Delhi, India, pp. 338-367.

Marx, U., Embleton, M., Fischer, R., Gruber, F., Hansson, U., Heuer, J., de Leeuw, W., Logtenberg, T., Merz, W., Portetelle, D., Romette, J. and Straughan, D. (1997). Monoclonal Antibody Production. The Report and Recommendations of ECVAM Workshop 231,2. Angera, Italy: The European Centre for the Validation of Alternative Methods, pp.121-137.

Marx, U., Embleton, M., Fischer, R., Gruber, F., Hansson, U., Heuer, J., de Leeuw, W., Logtenberg, T., Merz, W., Portetelle, D., Romette, J. and Straughan, D. (1997). Monoclonal Antibody Production. The Report and Recommendations of ECVAM Workshop 231,2. Angera, Italy: The European Centre for the Validation of Alternative Methods, pp.121-137.

Mayo Clinic. (2018). Monoclonal antibody drugs for cancer: How they work. online Available at: https://www.mayoclinic.org/diseases-conditions/cancer/in-depth/monoclonal-antibody/art-20047808 Accessed 4 Nov. 2018.

Pandey, S. (2010). HYBRIDOMA TECHNOLOGY FOR PRODUCTION OF MONOCLONAL ANTIBODIES. International Journal of Pharmaceutical Sciences Review and Research, 1(2), pp.88-94.

Wakchaure, R. and Ganguly, S. (2015). Importance of Transgenic Fish to Global Aquaculture: A Review. Fisheries and Aquaculture Journal, 06(04).

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