Presentation on theme: "Bispecific antibodies Touqeer Ahmed Ph.D. Atta-ur-Rahman School of Applied Bioscience, National University of Sciences and Technology 24 rd September,"— Presentation transcript:
Bispecific antibodies Touqeer Ahmed Ph.D. Atta-ur-Rahman School of Applied Bioscience, National University of Sciences and Technology 24 rd September, 2014
Bispecific antibodies Bispecific antibodies (bsAbs) contain two different binding specificities within a single molecule and these can specifically bind two different molecules together They have been used extensively for research and therapeutic approaches In principal, bsAbs could be used in any application where two molecules need to be cross-linked noncovalently within a distance of about 10 to 20 nM. Types (based on how are these produced) 1. Chemically cross-linked antibodies 2. Hybrid hybridoma antibodies
Type1-Chemically Cross-Linked Antibodies In this protocol two antibodies or their Fab fragments are cross-linked using the hetero bifunctional compound N-succinimidyl-3-(2- pyridyldithiol) propionate (SPDP) This reagent binds randomly to ε-amino groups on lysine residues and forms disulfide bonds between antibodies The resulting bispecific molecules consist of aggregates of antibodies of varying size linked together at random sites.
Epsilon Amino Group on Lysine
Type-2 Antibodies Obtained from Hybrid Hybridoma Differentially labeled hybridomas are fused and cells staining for both fluorochromes are isolated using a fluorescence-activated cell sorter (FACS) One hybridome is labeled with fluorochrome FITC (green) and the other one is labeled with TRITC (red). These are fused with poly ethylene glycol (PEG) Double labeled hybridoma are selected by FACS
Purification of Bispecific Antibodies Using affinity column chromatography and using both antigens one after the other or using two separate columns for the purification of bispecific antibodies Specificity checking in ELISA
Uses of Antibodies
Therapeutic Field Anti-tumor therapy Antibodies against tumor antigen are produced and they are converted into Immunotoxins Immunosuppression Using monoclonal antibodies against TCR, BCR, Co-receptor complex and cytokines etc Muromonab-CD3 (OKT3) Antithymocyte globulins Alemtuzumab Drug toxicity reversal Toxicity produced by drugs is treated using monoclonal antibodies against drugs, so that the functions of drugs are blocked and effect reversed. Application of Monoclonal Antibodies
Application of Monoclonal Antibodies Therapeutic Field Anti-tumor therapy Monoclonal antibodies have become an active area of drug development for anticancer therapy and other non-neoplastic diseases, because they are directed at specific targets and often have fewer adverse effects. They are created from B lymphocytes (from immunized mice or hamsters) fused with “immortal” B- lymphocyte tumor cells. Currently, several monoclonal antibodies are available in the United States for the treatment of cancer. Trastuzumab, rituximab, bevacizumab, and cetuximab are important examples. Alemtuzumab: is effective in treatment of B-cell chronic lymphocytic leukemia that no longer responds to other agents I 131 -tositumomab: is used in relapsed non-Hodgkin lymphoma. Gemtuzumab ozogamicin: which is a monoclonal antibody conjugated with a plant toxin that binds to CD33 (a cell-surface receptor that is present on the leukemia cells of 80 percent of patients with acute myelocytic leukemia)
Trastuzumab In patients with metastatic breast cancer, overexpression of transmembrane human epidermal growth factor–receptor protein 2 (HER2) is seen in 25 to 30 percent of patients. Trastuzumab [tra-STEW-zoo-mab], a recombinant DNA–produced, humanized monoclonal antibody, specifically targets the extracellular domain of the HER2 growth receptor that has intrinsic tyrosine kinase activity. The drug, usually administered with paclitaxel, can cause regression of breast cancer and metastases in a small percentage of these individuals. Trastuzumab binds to HER2 sites in breast cancer tissue and inhibits the proliferation of cells that overexpress the HER2 protein, thereby decreasing the number of cells in the S phase.
Heterodimer formation of members of the HER family and downstream signaling Signaling downstream of HER family activation is dependent on heterodimerization of the HER family member triggered by ligand binding to the extracellular ligand-binding domain (with the exception of HER2, which has no identified ligand and is always in an open conformation that allows dimerization). Phosphorylation of the HER kinase domains (with the exception of HER3, which does not have a kinase domain) initiates a downstream cascade resulting in VEGF transcription and other physiological responses required for carcinogenesis. Abbreviations: AR, amphiregulin; BTC, betacellulin; EPG, epigen; EPR, epiregulin; HB-EFG, heparin-binding EGF-like ligand; NRG, neuregulin. Nature Reviews Clinical Oncology 9, (January 2012)
Cell Division in Eukaryotes In eukaryotic cells, cell division starts with the most important process of DNA replication The cell cycle consists of four distinct phases: – G 1 phase – S phase (synthesis) – G 2 phase – M phase (mitosis) M phase is composed of two tightly coupled processes: Mitosis, in which the cell's chromosomes are divided between the two daughter cells Cytokinesis, in which the cell's cytoplasm divides in half forming two distinct cells G 1, S and G 2 phases are collectively known as interphase
Trastuzumab: Mechanism of action: How the antibody causes its anticancer effect remains to be elucidated. Several mechanisms have been proposed: – for example, down-regulation of HER2-receptor expression – an induction of antibody-dependent cytotoxicity, – or a decrease in angiogenesis due to an effect on vascular endothelial growth factor. Efforts are being directed toward identifying those patients with tumors that are sensitive to the drug.
Trastuzumab Pharmacokinetics: Trastuzumab is administered IV. Trastuzumab does not penetrate the blood-brain barrier. Adverse effects: – The most serious toxicity associated with the use of trastuzumab is congestive heart failure. The toxicity is worsened if given in combination with anthracycline. – Extreme caution should be exercised when giving the drugs to patients with preexisting cardiac dysfunction. – Other adverse effects include infusion-related fever and chills, headache, dizziness, nausea, vomiting, abdominal pain, and back pain, but these effects are well tolerated. – Cautious use of the drug is recommended in patients who are hypersensitive to the Chinese hamster ovary cell components of the proteins or to benzyl alcohol (in which case sterile water can be used in place of the bacteriostatic solution provided for preparation of the injection).
Rituximab Rituximab (ri-TUCKS-ih-mab) was the first monoclonal antibody to be approved for the treatment of cancer. It is a genetically engineered, chimeric monoclonal antibody directed against the CD20 antigen that is found on the surfaces of normal and malignant B lymphocytes. CD20 plays a role in the activation process for cell-cycle initiation and differentiation. The CD20 antigen is expressed on nearly all B-cell non-Hodgkin lymphomas but not in other bone marrow cells. Rituximab has proven to be effective in the treatment of posttransplant lymphoma and in chronic lymphocytic leukemia.
Rituximab Mechanism of action: The Fab domain of rituximab binds to the CD20 antigen on the B lymphocytes, and its Fc domain recruits immune effector functions, inducing complement and antibody dependent, cell-mediated cytotoxicity of the B cells. The antibody is commonly used with other combinations of anticancer agents, such as cyclophosphamide, doxorubicin, vincristine (Oncovin), and prednisone (CHOP).
Rituximab Pharmacokinetics: Rituximab is infused IV and causes a rapid depletion of B cells (both normal and malignant). The fate of the antibody has not been described.
Rituximab Adverse effects: Severe adverse reactions have been fatal. It is important to infuse rituximab slowly. Hypotension and bronchospasm may occur. Chills and fever commonly accompany the first infusion, especially in patients with high circulating levels of neoplastic cells, because of rapid activation of complement, which results in the release of tumor necrosis factor α and interleukins. Pretreatment with diphenhydramine, acetaminophen, and bronchodilators can ameliorate these problems. Cardiac arrhythmias can also occur.