1. MONOCLONAL ANTIBODIES BY: FIROUZEH KAMALI
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 Paul Ehrlich (16 years later)
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

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

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) 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

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