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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.  
  • 22. 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
  • 23. RITUXIMAB (Rituxan)
    • 1 st 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
  • 24. Toxic effects of Rituximab
    • Short-lived mild reactions to infusion after first treatment: fever, chills, rigors, rash, and nausea
  • 25. 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
  • 26. 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