Design Problem No: Course Instructor: D.O.

A: 02/04/11 Students Roll No:

2 Deepanvita, mam RA7801A09

Course code: BTY-462 Course Tutor: D.O.S: Section No: NA 19/04/11 A7801

Declaration I declare that this homework is my individual work. I have not copied from any other students work or from any other source except where due acknowledgment is made explicitly in the text, nor has any part been written for me by another person. Students Signature: Gurbakhshish Singh Evaluators comment .................... Marks obtainedout of. Content of Design Problem should start from this page only.

Problem Statement: MABTHERA, can it be used for combating Double negative or Triple negative Breast cancer? Expectations: 1) Explanation of the terminologies involved in the above statement. 2) Explanation of the approach you propose to use for combating the disease. 3) If MABTHERA, how it can be produced at lab scale, pilot scale or large scale? 4) Mention important subtopics like why a specific antibody and how it will result in the treatment of the disease. 5) Take support of recent developments in India in the field of Immunology and Latest Antibody production Technology. 6) Use the knowledge of the course as a source for theoretical knowledge and other needed data to solve this problem.
7) Feel free to use other resources but make sure that you list the sources in your solution

as References. Use cited publications, review and research articles from reputed journals and conference proceedings of not more than 5 years old.

SOLUTION
INTRODUCTION Breast cancer is a malignant tumor that starts from cells of the breast. A malignant tumor is a group of cancer cells that may grow into (invade) surrounding tissues or spread (metastasize) to distant areas of the body. The disease occurs almost entirely in women, but men can get it, too. Double negative breast cancer refers to breast cancer that does not expres the genes for progestrone receptor (PR) and human epidermal growth receptor 2 (HER2) but expresses genes for estrogen receptor (ER). So the patient is PR-, HER2- and ER+. In some cancers it has been found that patient express genes for ER but not for HER2. So a double negative breast cancer patient is either ER-/HER2+ or ER+/HER2-. Triple-negative breast cancer refers to any breast cancer that does not express the genes for estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth receptor 2 (HER2). This subtype of breast cancer is clinically characterized as more aggressive and less responsive to standard treatment and associated poorer overall patient prognosis. It is diagnosed more frequently in younger women, women with BRCA1 mutations, and those belonging to African-American and Hispanic ethnic groups, and those having a recent birth. MabThera infusion contains the active ingredient rituximab, which is a type of medicine known as a monoclonal antibody. It works by attacking white blood cells called B cells. MabThera belongs to a class called monoclonal antibodies. Monoclonal antibodies are proteins which specifically recognise and bind to other unique proteins in the body MabThera works by binding to a protein on the surface of certain white blood cells known as B lymphocytes. This action means it can be used to treat two different diseases: cancers of the lymphatic system known as non-Hodgkins lymphomas, and the inflammatory disease of the joints known as rheumatoid arthritis Monoclonal antibody therapy is the use of monoclonal antibodies (or mAb) to specifically bind to target cells. This may then stimulate the patient's immune system to attack those cells. It is possible to create a mAb specific to almost any extracellular/ cell surface target, and thus there is a large amount of research and development currently being undergone

to create monoclonals for numerous serious diseases (such as rheumatoid arthritis, multiple sclerosis and different types of cancers BRCA1 (breast cancer 1) is a human tumor suppressor gene that produces a protein called breast cancer type 1 susceptibility protein. BRCA1 is expressed in the cells of breast and other tissue, where it helps repair damaged DNA, or destroy cells if DNA cannot be repaired. If BRCA1 itself is damaged, damaged DNA is not repaired properly and this increases risks for cancers. TRIPLE NEGATIVE BREAST CANCER CHARACTERSTICS Triple-negative breast cancers (TNBC) are themselves a subgroup of "basal-type" breast cancers. Triple-negative breast cancers (TNBC) are ER-, PR-, and HER2- i.e. they do not have genes for estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth receptor 2 (HER2) Nine out of ten women with faulty BRCA1 had triple negative breast cancer. Most samples of triple negative cancers had very low levels of 53BP1" (making them less sensitive to radiotherapy). Most TNBC cancers over-express Epidermal Growth Factor Receptor (EGFR). Some TNBC over expresses transmembrane glycoprotein NMB (GPNMB) Transmembrane glycoprotein NMB is a protein that in humans is encoded by the GPNMB gene. In melanocytic cells and osteoclasts the GPNMB gene is transcriptionally regulated by Microphthalmia-associated transcription factor. GPNMB has been identified aggressively or highly expressed in melanoma, glioma and breast cancer specimens. SYMPTOMS AND DIAGNOSIS Breast cancer is sometimes found after symptoms appear, but many women with early breast cancer have no symptoms Signs and symptoms Widespread use of screening mammograms has increased the number of breast cancers found before they cause any symptoms, but some breast cancers are not found by mammogram,

either because the test was not done or because, even under ideal conditions, mammograms do not find every breast cancer. The most common signs of breast cancer are: A new lump or mass
A painless, hard mass that has irregular edges

But, breast cancers can be tender, soft, or rounded. For this reason, it is important that any new breast mass or lump be checked by a health care professional experienced in diagnosing breast diseases. Other possible signs of breast cancer include:

Swelling of all or part of a breast (even if no distinct lump is felt) Skin irritation or dimpling Breast or nipple pain Nipple retraction (turning inward) Redness, scaliness, or thickening of the nipple or breast skin Nipple discharge (other than breast milk) Sometimes a breast cancer can spread to underarm lymph nodes and cause a lump or

swelling there, even before the original tumor in the breast tissue is large enough to be felt. Proceedings to Diagnose Breast Cancer (Triple Negative) Physical Examination Breasts will be thoroughly examined for any lumps or suspicious areas and to feel their texture, size, and relationship to the skin and chest muscles. Any changes in the nipples or the skin of breasts will be noted. The lymph nodes in the armpit and above the collarbones may be palpated (felt), because enlargement or firmness of these lymph nodes might indicate spread of breast cancer. We may also probably do a complete physical exam to judge general health of the patient and whether there is any evidence of cancer that may have spread. If breast symptoms and/or the results of physical exam suggest breast cancer might be present, more tests will likely be done. These might include imaging tests, looking at samples of nipple discharge, or doing biopsies of suspicious areas.

Imaging Tests Used to Evaluate Breast Disease Imaging tests use x-rays, magnetic fields, sound waves, or radioactive substances to create pictures of the inside of your body. Imaging tests may be done for a number of reasons, including to help find out whether a suspicious area might be cancerous, to learn how far cancer may have spread, and to help determine if treatment is working. Diagnostic Mammograms Mammograms are mostly used for screening, but they can also be used to examine the breast of a woman who has a breast problem. This can be a breast mass, nipple discharge, or an abnormality that was found on a screening mammogram. In some cases, special images known as cone views with magnification are used to make a small area of abnormal breast tissue easier to evaluate. A diagnostic mammogram can show:

That the abnormality is not worrisome at all. In these cases the woman can usually return to having routine yearly mammograms. That a lesion (area of abnormal tissue) has a high likelihood of being benign (not cancer). In these cases, it is common to ask the woman to come back sooner than usual for her next mammogram, usually in 4 to 6 months.

That the lesion is more suspicious, and a biopsy is needed to tell if it is cancer

BIOPSY During a biopsy, the doctor removes a sample of the suspicious area to be looked at under a microscope. A biopsy is done when mammograms, other imaging tests, or the physical exam finds a breast change (or abnormality) that is possibly cancer. A biopsy is the only way to tell if cancer is really present. There are several types of biopsies, such as Fine needle aspiration biopsy, Core (large needle) biopsy Surgical biopsy. We can use any one of the above mentioned biopsy technique depending on specific situation.

Laboratory Examination of Breast Cancer Tissue 1. The biopsy samples of breast tissue are looked at in the lab to determine whether breast cancer is present and if so, what type it is. 2. The lab may also perform certain tests that can help determine how quickly a cancer is likely to grow and (to some extent) what treatments are likely to be effective. Sometimes these tests aren't done on the biopsy sample, but instead they are performed on the whole cancer specimen when it is removed by either lumpectomy or mastectomy. 3. If a benign condition is diagnosed, no further treatment would be required. 4. An important step in evaluating a breast cancer is to test a portion of the cancer removed during the biopsy (or surgery) to see if they have estrogen and progesterone receptors. Cancer cells may contain neither, one, or both of these receptors. Breast cancers that contain estrogen receptors are often referred to as ER-positive (or ER+) cancers, while those containing progesterone receptors are called PR-positive (or PR+) cancers. 5. Women with hormone receptorpositive cancers tend to have a better prognosis and are much more likely to respond to hormone therapy than women with cancers without these receptors. 6. If the woman breast cells do not contain any of the receptors i.e ER, PR and HER2, she is lacking genes coding for these receptors and hence would be suffering from triple negative breast cancer. 7. All breast cancers, with the exception of lobular carcinoma in situ (LCIS), should be tested for these hormone receptors when they have the breast biopsy or surgery. About 2 of 3 breast cancers contain at least one of these receptors. This percentage is higher in older women than in younger ones. Receptors are proteins on the outside surfaces of cells that can attach to certain substances, such as hormones, that circulate in the blood. Normal breast cells and some breast cancer cells have receptors that attach to estrogen and progesterone. These 2 hormones often fuel the growth of breast cancer cells. This is not the case with triple negative breast cancer.

TREATMENT Breast cancers are classified by whether or not they express the genes for estrogen receptor, progesterone receptor or Her2/neu. These three receptors are known to help the cancer develop, and the most successful breast cancer treatments are hormone-based drugs that directly target these receptors. It is important to know what subtype the cancer is before commencing treatment as different drugs target different receptors. "Triple-negative" breast cancer cells do not express any of these receptors. This means they are generally unresponsive to such standard receptor-mediated treatments. However, other forms of chemotherapy can still generate positive outcomes. Some reports even suggest they are more susceptible to non-receptor mediated therapies than other tumours. Triple-negative tumors do not respond to endocrine agents or trastuzumab and can only be treated with chemotherapy. Although triple-negative breast cancer can be treated with chemotherapy, early relapse and metastasis (spread to other tissues) is common. So in this solution we would try to find new approach to target breast cancer i.e. by using monoclonal antibody along with chemotherapy. This therapy would be more specific and would involve monoclonal antibodies targeting Epidermal Growth Factor Receptor (EGFR) and transmembrane glycoprotein NMB (GPNMB) which are over-expressed during triple negative breast cancer. MabThera (Rituximab) MabThera consists of a human IgG1 kappa constant region, with a variable region isolated from a murine anti-CD20 antibody. The variable murine regions are specific for the CD20 antigen, while the human component allows effective use of complement and cellmediated lysis mechanisms in vivo; this means that MabThera can effectively eradicate lymphoma cells in a variety of ways. MabThera has low potential for immunogenicity and resultant production of antiMabThera antibodies, because the majority of the molecule is of human origin.

Fig: MabThera (Rituximab) combines murine anti-CD20 variable regions with a human constant region

MabThera (Rituximab) effectively eradicates CD20+ cells in lymphoma patients by several distinct mechanisms, including complement-dependent cytotoxicity (CDC), antibodydependent cellular cytotoxicity (ADCC) and apoptosis But treatment of triple negative breast cancer involves targeting over expressed Epidermal growth Factor receptors and do not involve targeting B cells. So Rituximab cannot be used for treating triple negative breast cancer but other monoclonal antibodies can be used in addition to chemotherapy to enhance the suppressing of cancer few times. Most BRCA1-associated breast cancers are triple-negative and basal-like and express basal markers such as CK-5 and EGFR. Efforts to link sporadic BBCs with BRCA1pathway dysfunction are ongoing. One possible link may involve ID4, a down regulator of BRCA1, being over expressed in BBCs (Basal Breast Cancers). BRCA1 (Breast Cancer 1) protein mediates the response to DNA-damaging agents and the repair of double-stranded DNA breaks; therefore, BRCA1 dysfunction causes deficient double-stranded DNA break repair and allows affected tumors to have enhanced apoptotic responses to platinum agents. Treatments such as chemotherapy and radiation cant tell the difference between fastgrowing healthy cells and cancer cells, so targeted therapies are been applied. These targeted therapies work by shutting down a specific process the cancer cells use to grow and thrive.

We know that targeting estrogen and progesterone receptors and HER2 isnt helpful for triplenegative breast cancer as they are not present in this case.

Using Cetuximab
Women with metastatic triple-negative breast cancer respond twice as well to chemotherapy if Cetuximab is added. Targeting the epidermal growth factor receptor (EGFR) can double the response rate compared to only cisplatin chemotherapy. A platinum agent is used because preclinical data suggested that triple-negative tumors may have defects in BRCA1-mediated DNA repair and thus may be sensitive to DNAdamaging agents such as platinum. Patients with triple negative tumors usually have a poor prognosis because the tumor tends to grow and spread rapidly and also because they generally do not respond well to several therapies. MECHANISM Cetuximab binds specifically to the epidermal growth factor receptor (EGFr, HER1, cErbB-1) on both normal and tumor cells. EGFr is over-expressed in triple negative breast cancers. Cetuximab competitively inhibits the binding of epidermal growth factor (EGF) and other ligands, such as transforming growth factor alpha.

Fig: Mechanism of Action of Cituximab drug

 Binding of cetuximab to the EGFr blocks phosphorylation and activation of receptorassociated kinases (Tyrosine Kinase), resulting in inhibition of cell growth, induction of apoptosis, decreased matrix metalloproteinase secretion and reduced vascular endothelial growth factor production. ADMINISTRATION There are various ways applied by different researchers and doctors to administer Cetuximab drug along with cisplatin Dozes given: 400 mg/m2 initial dose of Cituximab drug and then 250 mg/m2 weekly plus up to six 3weekly cycles of cisplatin given by intravenous infusion. Cisplatin can be given 75 mg/m intravenously on Day 1 for every 3 weeks, with a maximum of 6 cycles. Administration of the Investigational Medicinal Product (IMP) will be stopped upon the first occurrence of disease progression and the dozes can be changed according to the circumstances.

Using Glembatumumab vedotin

Glembatumumab vedotin (also known as CDX-011 and CR011-vcMMAE) is an antibody-drug conjugate (ADC) that targets cancer cells expressing transmembrane glycoprotein NMB (GPNMB) MECHANISM The fully-human monoclonal antibody glembatumumab (CR011) is linked to monomethyl auristatin E (MMAE). The linkage is stable in the bloodstream. The antibody binds to GPNMB on the cancer cells, the antibody-drug conjugate (ADC) is internalised, the linkage is broken and MMAE i.e. Glembatumumab vedotin is released to kill the cell. In preclinical studies glembatumumab vedotin was capable of killing GPNMB expressing melanoma and breast cancer cells in vitro and inducing partial or complete regression of GPNMB-expressing tumors in mouse models.

Using Bevacizumab
To get the oxygen and nutrients to grow and spread, tumors create new blood vessels through a process called angiogenesis. Avastin (chemical name: bevacizumab) is a medicine that interferes with the activity of the VEGF (Vascular Endothelial Growth Factor) protein, which stimulates this process. MECHANISM Avastin attaches itself to VEGF, preventing VEGF from interacting with receptors on the blood vessels. By blocking this interaction, Avastin keeps VEGF from stimulating angiogenesis.

Monoclonal Antibodies are used with Chemotherapy

Monoclonal antibody therapy can suppress a specific process the cancer cells use to grow and thrive but this therapy has not been found successful to remove or eliminate cancer from the body tissues. So monoclonal antibodies are used along with traditional chemotherapy process so that they can help to suppress cancer at faster rate as compared to chemotherapy alone. CISPLATIN Cisplatin, cisplatinum, or cis-diamminedichloroplatinum(II) (CDDP) (trade names Platinol and Platinol-AQ) is a chemotherapy drug used to treat various types of cancers, including sarcomas, some carcinomas (e.g. small cell lung cancer, and ovarian cancer), lymphomas, breast cancers and germ cell tumors. It is the platinum-based anti-cancer drug. These platinum complexes react in vivo, binding to and causing crosslinking of DNA, which ultimately triggers apoptosis (programmed cell death).

Fig: Cisplatin

MECHANISM

Following administration, one of the chloride ligands is slowly displaced by water (an aqua ligand), in a process termed aquation. The aqua ligand in the resulting [PtCl(H2O)(NH3)2]+ is itself easily displaced, allowing the platinum atom to bind to bases. Of the bases on DNA, guanine is preferred. Subsequent to formation of [PtCl(guanine-DNA)(NH3)2]+, crosslinking can occur via displacement of the other chloride ligand, typically by another guanine.

Cisplatin crosslinks DNA in several different ways, interfering with cell division by mitosis. The damaged DNA elicits DNA repair mechanisms, which in turn activate apoptosis when repair proves impossible.

Production of Monoclonal Antibodies

Antibodies or immunoglobulins are a crucial component of the immune system, circulating in the blood and lymphatic system, and binding to foreign antigens expressed on cells. Once bound, the foreign cells are marked for destruction by macrophages and complement. In the context of cancer immunotherapy, monoclonal antibodies have brought to light a wide array of human tumor antigens. In addition to target cancer cells, antibodies can be designed to act on other cell types and molecules necessary for tumor growth. For example, antibodies can neutralize growth factors and thereby inhibit tumor expansion. In the case of triple negative breast cancer, we are going to produce antibody against Epidermal Growth Factors Receptor which will specifically bind to them and suppresses the growth of tumor. Monoclonal antibodies are made by injecting human cancer cells or proteins from cancer cells, into mice so that their immune systems create antibodies against foreign antigens. The murine cells producing the antibodies are then removed and fused with laboratory-grown cells to create hybrid cells called hybridomas. Hybridomas can indefinitely produce large quantities of these pure antibodies.
1. A sample of B cells is extracted from the spleen of the mouse and added to a culture of

myeloma cells (cancer cells). The intended result is the formation of hybridomas, cells

formed by the fusion of a B cell and a myeloma cell. The fusion is done by using polyethylene glycol, a virus or by electroporation (Campbell MA, pers. comm.)
2. The next step is to selct for the hybridomas. The myeloma cells are HGPRT- and the B

cells are HGPRT+. HGPRT is hypoxanthine-guanine phosphoribosyl transferase, an enzyme involved in the synthesis of nucleotides from hypoxanthine, an amino acid. The culture is grown in HAT (hypoxanthine-aminopterin-thymine) medium, which can sustain only HGPRT+ cells. The myeloma cells that fuse with another myeloma cell or do not fuse at all die in the HAT medium since they are HGPRT-. The B cells that fuse with another B cell or do not fuse at all die because they do not have the capacity to divide indefinitely. Only hybridomas between B cells and myeloma cells survive, being both HGPRT+ and cancerous.

Fig: Summarised production of Monoclonal Antibody

3. Each hybridoma is cultured and screened after doing SDS-PAGE (sodium dodecyl

sulfate - polyacrylamide gel electrophoresis) and Western blots to confirm that hybridoma cells are producing desired specific antibody. The probe used is the epitope of the antibody that is desired, which may be labeled by radioactivity or immunofluorescence. Once we are sure that a certain hybridoma is producing the right antibody, we can culture that hybridoma population indefinitely using air lift fermenters and culturing these cells in a suspension culture and harvest monoclonal antibodies from it. Large scale production of Monoclonal Antibodies The success of in vitro techniques for growing hybridoma cells with high MAb yields is based primarily on two factors: (i) Use of chemostats to feed the growing cells continuously with medium maintained with optimum pH, 02 content, and nutrients and (ii) Development of several different types of culture vessels and systems for high-density cell growth. Serum-free medium or reduced serum concentrations are used to culture Monoclonal Antibodies. Problems using large culture vessels and large volumes of medium have been reduced by application of a microencapsulation technique. With this method, seed cultures of hybridoma cells are encapsulated in semi-permeable alginate membranes which permit cell growth by perfusion of nutrients and oxygen. Cultures with densities of greater than 108 cells per ml of packed microcapsule beads are obtained, a significant increase over standard cell culture, and thereby an increased MAb yield, is achieved

Air lift fermenters are ideal to grow hybridoma cells at large scale. Cultures in such vessels are both aerated and agitated by air bubbles introduced at the bottom of vessels. The vessel has an inner draft tube through which the air bubbles and the aerated medium rise since aerated medium is lighter than non aerated one; this results in mixing of the culture as well as aeration.

The air bubbles lift to the top of the medium and the air passes out through an outlet. The cells and the medium that lift out of the draft tube move down outside the tube and are recirculated. O2 supply is quite efficient but scaling up presents certain problems. Fermenters of 2-90 I are commercially available, but 2000 I fermenters are being used for the production of monoclonal antibodies. Alternatively hollow fibre chambers may also be used for culturing hybridoma cells at large scale to produce large amount of monoclonal antibodies.

CONCLUSION
Triple-negative breast cancer (TNBC) refers to any breast cancer that does not express the genes for estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth receptor 2 (HER2). They are ER-, PR-, and HER2-. Most TNBC cancers over-express Epidermal Growth Factor Receptor (EGFR). Triple-negative tumors do not respond to endocrine agents or trastuzumab and can only be treated with chemotherapy. Although triple-negative breast cancer can be treated with chemotherapy, use of monoclonal antibodies to target over expressing receptors like Epidermal Growth Factor Receptor (EGFR) and Vascular endothelial growth Receptor can enhance the suppressing of cancer Treatments such as chemotherapy and radiation cant tell the difference between fastgrowing healthy cells and cancer cells, so targeted therapies are been applied. These targeted therapies work by shutting down a specific process the cancer cells use to grow and thrive. We know that targeting estrogen and progesterone receptors and HER2 isnt helpful for triplenegative breast cancer as they are not present in this case. MabThera drugs are used. Cetuximab drug used to target Epidermal Growth Factor Receptor (EGFR); Bevacizumab drug used to attach to vascular endothelial growth receptor (VEGR) and Glembatumumab vedotin (also known as CDX-011 and CR011-vcMMAE) is an antibody-drug conjugate (ADC) used to target cancer cells expressing transmembrane glycoprotein NMB (GPNMB) BRCA1 (Breast Cancer 1) protein mediates the response to DNA-damaging agents and the repair of double-stranded DNA breaks; therefore, BRCA1 dysfunction causes deficient double-stranded DNA break repair and allows affected tumors to have enhanced apoptotic responses to platinum agents. Cisplatin drug is used in chemotherapy as it contains platinum. We conclude that triple negative breast cancer can be best treated and cured with the help of chemotherapy using cisplatin drug along with monoclonal antibody therapy involving Cetuximab, Bevacizumab and Glembatumumab vedotin drugs

REFERENCES
http://www.breastcancer.org/symptoms/diagnosis/trip_neg/ http://news.drugs-expert.com/cancer-news/triple-negative-breast-cancer-responds-well-tocetuximab-addition-to-chemotherapy/ http://clinicaltrials.gov/ct2/show/NCT00463788 http://www.nature.com/nrd/journal/v3/n7/fig_tab/nrd1445_F1.html http://www.pharmgkb.org/do/serve?objId=PA10040#tabview=tab1 http://www.cancer.org/Cancer/BreastCancer/DetailedGuide/breast-cancer-diagnosis http://www.bio.davidson.edu/Courses/molbio/MolStudents/01rakarnik/mab.html http://www.meds.com/immunotherapy/monoclonal_antibodies.html http://www.molecular-plant-biotechnology.info/animal-tissue-culture-and-hybridomatechnology/airlift-fermenters.htm
http://cmr.asm.org/cgi/reprint/1/3/313.pdf