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Slide 1 - Monoclonal antibodies Anticancer therapy Weihua Wu
Slide 2 - What is an antibody? 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
Slide 3 - Monoclonal Antibodies 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. Given (almost) any substance, it is possible to create monoclonal antibodies that specifically bind to that substance; they can then serve to detect or purify that substance. This has become an important tool in biochemistry, molecular biology and medicine.
Slide 4 - The IgG Class of Antibodies All current therapeutic antibodies are of the IgG class. When the objective of antibody therapy is to directly kill the target cell, the isotype of choice is IgG1, since this isotype is optimal for complement fixation.
Slide 5 - The structure of antibodies http://www.path.cam.ac.uk/~mrc7/igs/mikeimages.html
Slide 6 - ppt slide no 6 content not found
Slide 7 - Definitions Fab = Fragment, antigen binding Fc = Fragment, crystalline The Fc fragment specifies other biological activities of the molecule. For example, the Fc fragment may determine whether the antibody simply prevents signaling through a receptor, or alternatively, causes the cell’s destruction through complement fixation or targeting immune effector cells.
Slide 8 - Can Antibodies Destroy Foreign ‘Objects’? The antibody alone may not be sufficient to destroy a foreign ‘object’. For example, a common test for HIV is the presence of anti-HIV antibodies in the blood. Obviously, those antibodies alone are not sufficient to protect the host from the virus.
Slide 9 - 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. 1964 Littlefield developed a way to isolate hybrid cells from 2 parent cell lines using the hypoxanthine-aminopterin-thymidine (HAT) selection media. 1975 Kohler and Milstein provided the most outstanding proof of the clonal selection theory by fusion of normal and malignant cells. This resulted in the first monoclonal antibodies, for which they received the Nobel Prize in 1984.
Slide 10 - What Diseases to Target and How? Cancer cells express a variety of antigens that are attractive targets for monoclonal antibody-based therapy. The development of monoclonal antibodies against specific targets has been largely accomplished by immunizing mice against human tumor cells and screening the hybridomas for antibodies of interest.
Slide 11 - Unfulfilled Promise? The early promise of the use of antibodies in the treatment of disease initially went unfulfilled (more than two decades) for two reasons: 1. Early antibodies displayed insufficient activation of human effector functions (i.e. the antibodies did not kill the infecting organism or cell) 2. The early antibodies were of murine (mouse) origin, and thus triggered the production of human anti-mouse antibodies (HAMA).
Slide 12 - Other obstacles to the use of monoclonal antibodies in cancer treatment Antigen distribution of malignant cells is highly heterogeneous, so some cells may express tumor antigens, while others do not. Tumor blood flow is not always optimal High interstitial pressure within the tumor can prevent the passive monoclonal antibody from binding.
Slide 13 - The types of mAb designed 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. 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) Humanized mAbs: contained only the CDRs of the rodent variable region grafted onto human variable region framework
Slide 14 - Evolution of Therapeutic Antibodies
Slide 15 - Nomenclature of Therapeutic Antibodies Terminate the name in –ximab for chimeric antibodies and –umab for humanized antibodies.
Slide 16 - Common Chemotherapy in Treatment of Cancer Shortcomings: Nature of cytotoxin Lack of in vivo selectivity The mechanism of anti-proliferation on cells cycle, rather than specific toxicity directed towards particular cancer cell Host toxicity: treatment discontinued, most of them had bad side-effects, such as no appetites, omit, lose hair
Slide 17 - Monoclonal antibodies for cancer treatment Three mechanisms that could be responsible for the cancer treatment. mAbs act directly when binding to a cancer specific antigens and induce immunological response to cancer cells. Such as inducing cancer cell apoptosis, inhibiting growth, or interfering with a key function. mAbs was modified for delivery of a toxin, radioisotope, cytokine or other active conjugates. 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 cell
Slide 18 - 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;1:118-129
Slide 19 - ‘Naked’ Monoclonal Antibodies ‘Naked’ means these antibodies are not fused to a toxin. Naked Monoclonal antibodies can kill cells via a variety of mechanisms, including: Antibody-Dependent Cellular Cytotoxicity (ADCC), Complement-Dependent Cytotoxicity (CDC), and direct induction of apoptosis. However, the precise clinical mechanisms often remain uncertain
Slide 20 - Antibody-dependent cellular cytotoxicity From Wikipedia, the free encyclopedia Antibody-Dependant Cellular Cytotoxicity ADCC is the least understood of the three mechanisms, it is mediated by either NK cells or CTL. The action of ADCC is dependant on the recognition of the objective cell by antibodies attached on the surface of the effector cell (terminally differentiated leukocyte). This process is part of the adaptive immune response due to the dependence on antibodies and therefore a former anti-body response is required for this mechanism to take effect and be effective against an invading pathogen.
Slide 21 - http://www.meds.com/immunotherapy/monoclonal_antibodies.html
Slide 22 - Rituximab (Rituxan) Rituximab is a chimeric monoclonal antibody that targets the CD20 B-cell antigen. This antigen is expressed on 90% of B-cell neoplasms The precise biological functions of CD20 are uncertain, but the antibody is believed to function by flagging the B-cells for destruction by the body’s own immune system, including ADCC, CDC, and apoptosis. This antibody thus leads to the elimination of all B-cells from the body (including cancerous ones), allowing new, healthy B-cells to be produced from lymphoid stem cells.
Slide 23 - Trastuzumab (Herceptin) Herceptin is an anti-cancer antibody that acts on HER2/neu (erbB2) receptor, which is overexpressed in breast cancer. Only cells overexpressing this receptor are susceptible. Such cells, when treated with Herceptin, undergo arrest in the G1 phase of the cell cycle and experience a reduction in proliferation. This can reduce the rate of relapse of breast cancer by 50% during the first year. The precise mechanism of action is still under investigation.
Slide 24 - Monoclonal antibodies which deliver a toxin Monoclonal antibodies can be utilized to selectively deliver a toxin to a malignant cell.
Slide 25 - Gemtuzumab ozogamicin (Mylotarg) This monoclonal antibody is conjugated to the cytotoxic agent calicheamycin It is used to treat acute myelogenous leukemia (AML), which is a cancer of the myeloid line of blood cells. This monoclonal antibody attacks the CD33 receptor, which is found in most leukemic blast cells, but not in normal hematopoietic stem cells
Slide 26 - Gemtuzumab ozogamicin (Mylotarg) Once bound to CD33, the antibody-calicheamycin complex is transported inside of the AML cells by lysosomes. To facilitate selective release inside of the cancer cells, calicheamycin is connected to gemtuzumab by a chemical linker that is stable at physiologic pH but is hydrolyzed in the acidic pH of the lysosomes that transport the antibody-calicheamycin complex into the cell.
Slide 27 - Dale L Ludwig, etal. Oncogene(2003) 22, 9097-9106 Strategy of a direct or in direct induction of apoptosis in targeted cancer cells mAbs target growth factor receptors to exert a direct effect on the growth and survival of the cancer cells by antagonizing ligand-receptor signaling. mAbs can target to cell surface antigens and directly elicit apoptotic signaling.
Slide 28 - 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
Slide 29 - mAbs development Phage display library: construction of VH and VL 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. Transgenic plants: transgenic tobacco plants to produce IgA. Transgenic animals: transgenic mouse to make humanized IgG. (Abgenix,CA)
Slide 30 - 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 Robottle cell culture process. Membrane binded cell culture process Microcarrier cell culture process Suspended cell culture process
Slide 31 - Questions?
 

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