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ANTI-CANCER THERAPY

ANTI-CANCER THERAPY. MONOCLONAL ANTIBODIES BY: FIROUZEH KAMALI. Conventional Anti-Cancer Therapy. Chemotherapy: Imperfect Systematic nature of cytoxicity Agents lack intrinsic anti-tumor selectivity

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ANTI-CANCER THERAPY

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  1. ANTI-CANCER THERAPY MONOCLONAL ANTIBODIES BY: FIROUZEH KAMALI

  2. Conventional Anti-Cancer Therapy • Chemotherapy: Imperfect • Systematic nature of cytoxicity • Agents lack intrinsic anti-tumor selectivity • Anti-proliferative mechanism on cells in cycle, rather than specific toxicity directed towards particular cancer cell • Host toxicity: treatment discontinued at dose levels well below dose required to kill all viable tumor cells

  3. HISTORY • Emil von Behring in 1890 • Discovered antibodies • Paul Ehrlich (16 years later) • Coined phrase, “magic bullets and poisoned arrows”: use of antibodies to specifically target toxic substances in pathogenic substances • Kohler and Milstein in 1975 • Discovery of monoclonal antibodies (mAb) directed against well-characterized antigens • Use of DNA bio-engineered technologies within last 25 years

  4. Rationale • mAb as efficient carriers for delivery of anti-tumor agents • Enhanced vascular permeability of circulating macromolecules for tumor tissue and subsequent accumulation in solid tumors • Normal tissue: blood vessels have intact endothelial layer that permits passage of small molecules but not entry of macromolecules (like mAb) • Tumor tissue: blood vessels leaky, so small and large molecules have access to malignant tissue -tumor tissue generally do not have a lymphatic drainage system; therefore, macromolecules are retained and can accumulate in solid tumors

  5. Patho-physiology of Tumor Tissue • Angiogenesis • Hypervasculature • Impaired lymphatic drainage ***Due to these characteristics, tumors can be exploited for tumor-selective drug delivery****

  6. Genetic Engineering • Remove or modify effector functions of mAb: used to avoid unwanted side effects • Use mAb in their natural, fragmented, chemically modified, or recombinant forms • Use of phage display antibody libraries or transgenic animals • Identify animals that make desired antibodies • Animals must be immunized using the cellular antigens and immunization procedures used to generate conventional antibodies • Perform cell fusions to generate clones and isolate stable clones, making mAb • Most mAb used in the clinical setting were generated in mice

  7. Structure of Antibody • Presently, all intact therapeutic antibodies are murine immunoglobulins of the IgG class • Murine immunoglobulin = glycoprotein that has a Y-shaped structure: 2 identical polypeptide heavy chains and 2 identical light chains linked by an S-S bond • Chimeric antibody = genetically engineered construct containing a mouse Fab portion and a human Fc portion • 3 main components • Two identical Fabs (fragment-antigen binding site): the arms of the Y • An Fc (for fragment crystallizable), the stem of the Y • Constant region responsible for triggering effector functions that eliminate the antigen-associated cells • Constant region must be tailored to match requirements of the antibody (depending on which antigen you want it to bind to)

  8. IgG structure

  9. 3 MECHANISMS RESULTING IN APOPTOSIS • Antigen cross-linking • Activation of death receptors • Blockade of ligand-receptor growth or survival pathways

  10. 1. Antigen Cross-Linking • Target growth factor receptor • Antagonize ligand-receptor signaling • Growth-factor signaling mediated by the receptor tyrosine kinase is inhibited • EGFR (epidermal growth factor receptor) • IGF-1R (insulin-like growth factor-1 receptor) • FGFR (fibroblast growth factor receptor) • PDGFR (platelet-derived growth factor receptor) • VEGFR (vascular endothelial growth factor) • Results in arrest of tumor cell growth

  11. 2. Activation of death receptors • Cross-link targeted surface antigens on tumor cells and antibody agonists that mimic ligand-mediated activation of specific receptors • Response: intracellular Ca II ions increase • Activate caspase-3 and caspase-9 (involved in cell apoptosis)

  12. APOPTOSIS PATHWAY

  13. 3. Delivery of Cytotoxic Agents • Physically link antibodies to toxic substances for delivery • Radio-immunoconjugates (aim of delivering radiation directly to the tumor) • Toxin-immunoconjugates (deliver toxins intracellularly) • Antibody-directed enzyme pro-drug therapy (ADEPT): localize enzymes to tumor cell surfaces

  14. General Drug Delivery System • Drug molecules bound to macromolecule through spacer molecule • Drug released from macromolecule after cellular uptake of the conjugate • Targeting moiety = monoclonal antibody

  15. TOXIN IMMUNOCONJUGATES • Cell surface antigen must internalize upon mAb binding • When drug is released, it interferes with protein synthesis to induce apoptosis • 3 methods to attach cytotoxic drug to variable regions of mAb • a. Couple drug to lysine moieties in the mAb • b. Generation of aldehyde groups by oxidizing the carbohydrate region and subsequent reaction with amino-containing drugs or drug derivatives • c. Couple drugs to sulfhydryl groups by selectively reducing the interchain disulfides near the Fc region of the mAb

  16. Direct attachment of mAb to drug by S-S bonding

  17. Immunoconjugate • BR96-doxorubicin conjugate (BR96-DOX) • Promising toxin-immunoconjugate • mouse/human chimeric mAb • Targets antigen over-expressed on surface of human carcinoma cells of breast, colon, lung, and ovary • Disulfide reduction attaches mAb to drug, BR96 • Dose that can be safely administered every 3 weeks is insufficient

  18. Other examples of toxin-immunoconjugates • KS1/4-MTX • Conjugate of methotrexate (MTX) • Coupling of MTX to the lysine moieties of the mAb • No significant clinical response • KS1/4-DAVLB • Conjugate of vinca alkaloid derivatives • Vinca alkaloid derivatives attached to amino groups of lysine residues on KS1/4 mAb • No significant clinical response

  19. Why are these toxin-immunoconjugates unsuccessful? • Cause gastrointestinal toxicity • Inner regions of solid tumors poorly vascularized and have low blood flow (reduce amount of immunoconjugate reaching these parts of the tumor) • Antigen expression is heterogenous on tumor cells • Restricts the amount of cells that can be effectively targeted by antibody conjugates

  20. ADEPT ENZYMES (Antibody-directed enzyme pro-drug therapy) • Chemically link the mAb to the enzyme of interest; can also be a fusion protein produced recombinantly with the antibody variable region genes and the gene coding the enzyme • Convert subsequently administered anti-cancer pro-drugs into active anti-tumor agents • Upon binding to targeted enzymes, it is converted into active drug

  21. Anti-growth factor mAb Therapy • Angiogenesis • Formation of nascent blood vessels • VEGF • One of the most upregulated antigens in cancer • Protect endothelial cells from apoptosis via activation of PKC pathways and upregulation of anti-apoptotic proteins such as Bcl-2 • Activity mediated by tyrosine kinase receptors, VEGFR 1 and VEGFR 2 • Functions indirectly as survival factor for tumor cells • Inhibit VEGF signaling • Block the receptor • Inhibits tumor growth and metastasis • Deprives tumors of nutrient-providing blood vessels

  22. RITUXIMAB (Rituxan) • 1st therapeutic mAb approved by FDA in 1997 • High-level expression of the gene encoding Rituximab was found • a mouse-chimeric mAb • Contains the human IgG1 and murine variable regions that target CD20 B-cell antigen • CD20 antigen function: cell cycle progression • Binding Rituximab to CD-20 causes: autophosphorylation, activation of serine/tyrosine protein kinases, and induction of oncogene expression --- induces apoptosis • Response rates of 50% to 70% in follicular lymphomas • Response rates of 90% to 100% when used in combination with various chemotherpay procedures • Concluded that the dose of 4, once-weekly 375 mg/m squared IV infusions of Rituximab was safe and effective in patients with relapse or refractory B non-Hodgkin’s lymphoma

  23. Toxic effects of Rituximab • Short-lived mild reactions to infusion after first treatment: fever, chills, rigors, rash, and nausea

  24. Factors affecting pharmacokinetic parameters • Circulating target antigens (which can lead to rapid clearance) • Antigen-antibody internalization in cells (which affect serum clearance and half-life) • Antibody size and domains with the Fc region • Fragments have shorter half-lives and more rapid clearance rates than their full-sized immunoglobulins

  25. FUTURE • Researchers hope to define the optimal combinations of the use of mAb with conventional chemotherapeutic agents and with radiation therapy • Determine best therapy candidates and expand clinical trials to other tumor types

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