Monoclonal antibodies Anticancer therapy

THE IMMUNE SYSTEM DEFINITION: - The integrated body system of organs, tissues, cells & cell products that differentiates self from non – self & neutralizes potentially pathogenic organisms. (The American Heritage Stedman's Medical Dictionary) The Latin term “IMMUNIS” means EXEMPT, referring to protection against foreign agents. The Immune System consists of 1.Innate Immunity Primary Response 2.Acquired Immunity Secondary Response

ANATOMY OF THE IMMUNE SYSTEM

BECOMES FUNCTIONING OF THE IMMUNE SYSTEM HUMORAL (ANTIBODY MEDIATED) IMMUNE RESPONSECELL MEDIATED IMMUNE RESPONSE ANTIGEN (1 ST EXPOSURE) ANTIGENS DISPLAYED BY INFECTED CELLS ACTIVATE CYTOTOXIC T CELL GIVES RISE TO ACTIVE CYTOTOXIC T CELL ENGULFED BY STIMULATES MACROPHAGE APC HELPER T CELLS STIMULATES MEMORY HELPER T CELLS MEMORY T CELLS MEMORY B CELLS PLASMA CELLS STIMULATES B CELLS FREE ANTIGENS DIRECTLY ACTIVATE STIMULATES GIVES RISE TO SECRETE ANTIBODIES STIMULATES ANTIGEN (2 nd EXPOSURE) STIMULATES Defend against extracellular pathogens by binding to antigens and making them easier targets for phagocytes and complement Defend against intracellular pathogens and cancer by binding and lysing the infected cells or cancer cells

Lymphocytes Produce antibodies B-cells mature in bone marrow then concentrate in lymph nodes and spleen T-cells mature in thymus B and T cells mature then circulate in the blood and lymph Circulation ensures they come into contact with pathogens and each other

B -Lymphocytes There are c.10 million different B-lymphocytes, each of which make a different antibody. The huge variety is caused by genes coding for abs changing slightly during development. There are a small group of clones of each type of B- lymphocyte

B -Lymphocytes At the clone stage antibodies do not leave the B- cells. The abs are embedded in the plasma membrane of the cell and are called antibody receptors. When the receptors in the membrane recognise and antigen on the surface of the pathogen the B- cell divides rapidly. The antigens are presented to the B-cells by macrophages

B -Lymphocytes

Some activated B cells  PLASMA CELLSthese produce lots of antibodies, < 1000/sec The antibodies travel to the blood, lymph, lining of gut and lungs. The number of plasma cells goes down after a few weeks Antibodies stay in the blood longer but eventually their numbers go down too.

B -Lymphocytes Some activated B cells  MEMORY CELLS. Memory cells divide rapidly as soon as the antigen is reintroduced. There are many more memory cells than there were clone cells. When the pathogen/infection infects again it is destroyed before any symptoms show.

What are antibodies An antibody is a protein used by the immune system to identify and neutralize foreign objects like bacteria and viruses. Each antibody recognizes a specific antigen unique to its target. Monoclonal antibodies (mAb) are antibodies that are identical because they were produced by one type of immune cell, all clones of a single parent cell. Polyclonal antibodies are antibodies that are derived from different cell lines. Isotypes According to differences in their heavy chain constant domains, immunoglobulins are grouped into five classes, or isotypes: IgG, IgA, IgM, IgD, and IgE. IgG: IgG1 (66%), IgG2 (23%), IgG3 (7%) and IgG4 (4%), blood and tissue liquid. IgA:IgA1 (90%) and IgA2 (10%), stomach and intestines IgM: normally pentamer, ocassionally hexamer, multiple immunoglobins linked with disulfide bonds IgD:1% of proteins in the plasma membranes of B-lymphocytes, function unknown IgE: on the surface of plasma membrane of mast cells, play a role in immediate hypersensitive and denfensive for parasite

ANTIBODIES Derived from different B Lymphocytes cell lines POLYCLONAL.MONOCLONAL. Derived from a single B cell clone Batch to Batch variation affecting Ab reactivity & titre mAb offer Reproducible, Predictable & Potentially inexhaustible supply of Ab with exquisite specificity Enable the development of secure immunoassay systems. NOT Powerful tools for clinical diagnostic tests

The structure of antibodies

Antibodies Also known as immunoglobulins Globular glycoproteins The heavy and light chains are polypeptides The chains are held together by disulphide bridges Each ab has 2 identical ag binding sites – variable regions. The order of amino acids in the variable region determines the shape of the binding site

ENGINEERED ANTIBODIES

How Abs work Some act as labels to identify antigens for phagocytes Some work as antitoxins i.e. they block toxins for e.g. those causing diphtheria and tetanus Some attach to bacterial flagella making them less active and easier for phagocytes to engulf Some cause agglutination (clumping together) of bacteria making them less likely to spread

Different Immunoglobulins

TypeNumber of ag binding sites Site of actionFunctions IgG2Blood Tissue fluid CAN CROSS PLACENTA Increase macrophage activity Antitoxins Agglutination IgM10Blood Tissue fluid Agglutination IgA2 or 4Secretions (saliva, tears, small intestine, vaginal, prostate, nasal, breast milk) Stop bacteria adhering to host cells Prevents bacteria forming colonies on mucous membranes IgE2TissuesActivate mast cells  HISTAMINE Worm response

History of Mab development 1890 Von Behring and kitasato discovered the serum of vaccinated persons contained certain substances, termed antibodies 1900 Ehrlich proposed the “ side-chain theory” 1955 Jerne postulated natural selection theory. Frank Macfarlane Burnet expended. Almost the same time, Porter isolated fragment of antigen binding (Fab) and fragment crystalline (Fc) from rabbit y-globulin Littlefield developed a way to isolate hybrid cells from 2 parent cell lines using the hypoxanthine-aminopterin-thymidine (HAT) selection media Kohler and Milstein provided the most outstanding proof of the clonal selection theory by fusion of normal and malignant cells 1990 Milstein produced the first monoclonal antibodies.

The types of mAb designed A.Murine source mAbs: rodent mAbs with excellent affinities and specificities, generated using conventional hydrioma technology. Clinical efficacy compromised by HAMA(human anti murine antibody) response, which lead to allergic or immune complex herpersensitivities. B.Chimeric mAbs: chimers combine the human constant regions with the intact rodent variable regions. Affinity and specificity unchanged. Also cause human antichimeric antibody response (30% murine resource) C.Humanized mAbs: contained only the CDRs of the rodent variable region grafted onto human variable region framework

Chemotherapy Shortcomings: A. Nature of cytotoxin B. Lack of in vivo selectivity C. The mechanism of anti-proliferation on cells cycle, rather than specific toxicity directed towards particular cancer cell D. Host toxixity: treatment discontinued, most of them had bad side-effects, such as no appetites, omit, lose hair

Monoclonal antibodies for cancer treatment Three mechanisms that could be responsible for the cancer treatment. A. mAbs act directly when binding to a cancer specific antigen and induce immunological response to cancer cells. Such as inducing cancer cell apoptosis, inhibiting growth, or interfering with a key function. B. mAbs was modified for delivery of a toxin, radioisotope, cytokine or other active conjugates.toxinradioisotopecytokine C. it is also possible to design bispecific antibodies that can bind with their Fab regions both to target antigen and to a conjugate or effector cellbispecific

mAbs treatment for cancer cells ADEPT, antibody directed enzyme prodrug therapy; ADCC, antibody dependent cell-mediated cytotoxicity; CDC, complement dependent cytotoxicity; MAb, monoclonal antibody; scFv, single-chain Fv fragment. Carter P: Improving the efficacy of antibody-based cancer therapies. Nat Rev Cancer 2001;1:

Dale L Ludwig, etal. Oncogene(2003) 22, Strategy of a direct or in direct induction of apoptosis in targeted cancer cells 1.mAbs target growth factor receptors to exert a direct effect on the growth and survival of the cancer cells by antagonizing ligand-receptor signaling. 2.mAbs can target to cell surface antigens and directly elicit apoptotic signaling.

Until Feb 28, 2005, 18 mAbs were approved by FDA, which were applied in the treatment of organ transplant, Cancer, Asthma, Hematopoietic malignancies and psoriasis. The first approved mAbs was OKT-3, which is a murine IgGa2 protein to deplete T cells in patients with acute rejection of renal allotransplant. HAMA response Jancie, M Recheit, etal. Nature biotechnology, 2005, Sep,Vol. 23, No.9 Stamatis-Nick C. J Allergy Clin. Immunol, Oct. 2005

mAbs development 1. Phage display library: construction of V H and V L gene libaries and expression of them on a filamentous bacterophage. The phage expressing an antigen-bonding domain specific for a particular antigen to screen the mAbs. 2. Transgenic plants: transgenic tobacco plants to produce IgA. 3. Transgenic animals: transgenic mouse to make humanized IgG. (Abgenix,CA)

EVOLUTION OF MONOCLONAL ANTIBODY 1. TRANSGENIC DNA SPLICING / GENE KNOCK OUT 2. LIBRARIES a.BACTERIOPHAGE b. mRNA c. Cell Surface

Conventional production of mAbs The hybridoma technology: spleen cells from immunized mice are fused with the murine myeloma cells. The several process had been developed at large scale. According to the different cell culture methods, it can calisifed into four fields 1. Robottle cell culture process. 2. Membrane binded cell culture process 3. Microcarrier cell culture process 4. Suspended cell culture process

FDA APPROVED MONOCLONAL ANTIBODIES H2003PsoriasisRaptiva ™ Genentech/Xoma C1997Non-Hodgkin’s LymphomaRituxan®BiogenIdec/Genentech/Roc he M2003Non-Hodgkin’s LymphomaBexxar®Corixa/GlaxoSmithKline C2004Colorectal CancerErbitux ™ BMS/ImClone Systems H2003AsthmaXolair®Novartis/Genentech/Tanox PD2002Rheumatoid ArthritisHumira ™ Abbott/CAT H2001Chronic Lymphocytic Leukemia Campath®Schering /ILEX Oncology H2000Acute Myleoid LeukemiaMylotarg ™ Wyeth C1998Acute Transplant RejectionSimulect®Novartis C1998Crohn’s, Rheumatoid Arthritis Remicade®J & J HHHH Breast Cancer Colorectal Cancer Herceptin® Avastin ® Genentech/Roche H1998Viral Respiratory DiseaseSynagis®MedImmune/Abbott H1997Acute Transplant RejectionZenapax®PDLI M2002Non-Hodgkin’s LymphomaZevalin ™ BiogenIdec C1994Acute Cardiac ConditionsReoPro®J&J/Eli Lilly M1986Organ Transplant RejectionOrthoclone-OKT®Ortho Biotech Antibody Type (2) Date of FDA ApprovalIndications Name of Product (1) Company Name

Applications of Monoclonal Antibodies Diagnostic Applications Biosensors & Microarrays Therapeutic Applications Transplant rejection Muronomab-CD3 Cardiovascular disease Abciximab Cancer Rituximab Infectious Diseases Palivizumab Inflammatory disease Infliximab Clinical Applications Purification of drugs, Imaging the target Future Applications Fight against Bioterrorism

REPORT HIGHLIGHTS The global market for therapeutic monoclonal antibodies (mAbs) was estimated at $44.6 billion in With the rollout of at least eight new therapeutic mAb products and expanded indications for existing products expected during the forecast period, the global mAb market is expected to rise at a compound annual growth rate (CAGR) of 5.3% to nearly $58 billion in The U.S. is projected to be the largest single market for therapeutic mAbs from 2011 to This particular market was nearly $19.8 billion in 2010 and reached $20.1 billion by BCC projects this market will grow to $27.4 billion by 2016, a CAGR of 6.4%. Sales of mAbs in the rest of the world will remain higher than in the U.S.— despite a higher CAGR in the U.S. versus the rest of the world. This market is expected to grow from $24.6 billion in 2011 to $30.3 billion in 2016 at a CAGR of 4.3%.

Why should we be interested ? mAbs drive the development of multibillion dollar biotechnology industry. Many of the leading pharmaceutical companies have entered the mAb sector, attracted by quicker and less costly development, higher success rates, premium pricing, and a potentially reduced threat from generics The outlook for monoclonal antibody therapeutics is healthy. The ongoing success of existing products, combined with a bulging pipeline of new products awaiting approval and limited generic erosion, point towards robust growth in this segment