ANTI-CANCER ANTI-CANCER THERAPYTHERAPY

MONOCLONAL ANTIBODIESMONOCLONAL ANTIBODIES

BY: FIROUZEH KAMALIBY: FIROUZEH KAMALI

Conventional Anti-Cancer TherapyConventional Anti-Cancer Therapy

Chemotherapy: ImperfectChemotherapy: Imperfect Systematic nature of cytoxicitySystematic nature of cytoxicity Agents lack intrinsic anti-tumor selectivityAgents lack intrinsic anti-tumor selectivity Anti-proliferative mechanism on cells in cycle, Anti-proliferative mechanism on cells in cycle,

rather than specific toxicity directed towards rather than specific toxicity directed towards particular cancer cellparticular cancer cell

Host toxicity: treatment discontinued at dose Host toxicity: treatment discontinued at dose levels well below dose required to kill all levels well below dose required to kill all viable tumor cellsviable tumor cells

HISTORYHISTORY

Emil von Behring in 1890Emil von Behring in 1890 Discovered antibodiesDiscovered antibodies

Paul Ehrlich (16 years later)Paul Ehrlich (16 years later) Coined phrase, “magic bullets and poisoned arrows”: Coined phrase, “magic bullets and poisoned arrows”:

use of antibodies to specifically target toxic use of antibodies to specifically target toxic substances in pathogenic substancessubstances in pathogenic substances

Kohler and Milstein in 1975Kohler and Milstein in 1975 Discovery of monoclonal antibodies (mAb) directed Discovery of monoclonal antibodies (mAb) directed

against well-characterized antigensagainst well-characterized antigens Use of DNA bio-engineered technologies within last Use of DNA bio-engineered technologies within last

25 years25 years

RationaleRationale

mAb as efficient carriers for delivery of anti-mAb as efficient carriers for delivery of anti-tumor agentstumor agents Enhanced vascular permeability of circulating Enhanced vascular permeability of circulating

macromolecules for tumor tissue and subsequent macromolecules for tumor tissue and subsequent accumulation in solid tumorsaccumulation in solid tumors

Normal tissue: blood vessels have intact endothelial layer that Normal tissue: blood vessels have intact endothelial layer that permits passage of small molecules but not entry of permits passage of small molecules but not entry of macromolecules (like mAb)macromolecules (like mAb)

Tumor tissue: blood vessels leaky, so small and large Tumor tissue: blood vessels leaky, so small and large molecules have access to malignant tissuemolecules have access to malignant tissue

-tumor tissue generally do not have a lymphatic drainage -tumor tissue generally do not have a lymphatic drainage system; therefore, macromolecules are retained and can system; therefore, macromolecules are retained and can accumulate in solid tumorsaccumulate in solid tumors

Patho-physiology of Tumor TissuePatho-physiology of Tumor Tissue

AngiogenesisAngiogenesis

HypervasculatureHypervasculature

Impaired lymphatic drainage Impaired lymphatic drainage

***Due to these characteristics, tumors can ***Due to these characteristics, tumors can be exploited for tumor-selective drug be exploited for tumor-selective drug delivery****delivery****

Genetic EngineeringGenetic Engineering

Remove or modify effector functions of mAb: used to Remove or modify effector functions of mAb: used to avoid unwanted side effects avoid unwanted side effects Use mAb in their natural, fragmented, chemically Use mAb in their natural, fragmented, chemically modified, or recombinant formsmodified, or recombinant formsUse of phage display antibody libraries or transgenic Use of phage display antibody libraries or transgenic animalsanimals

Identify animals that make desired antibodiesIdentify animals that make desired antibodies Animals must be immunized using the cellular antigens and Animals must be immunized using the cellular antigens and

immunization procedures used to generate conventional immunization procedures used to generate conventional antibodiesantibodies

Perform cell fusions to generate clones and isolate stable Perform cell fusions to generate clones and isolate stable clones, making mAbclones, making mAb

Most mAb used in the clinical setting were generatedMost mAb used in the clinical setting were generatedin micein mice

Structure of AntibodyStructure of Antibody

Presently, all intact therapeutic antibodies are murine Presently, all intact therapeutic antibodies are murine immunoglobulins of the IgG classimmunoglobulins of the IgG class

Murine immunoglobulin = glycoprotein that has a Y-shaped Murine immunoglobulin = glycoprotein that has a Y-shaped structure: 2 identical polypeptide heavy chains and 2 identical structure: 2 identical polypeptide heavy chains and 2 identical light chains linked by an S-S bondlight chains linked by an S-S bond

Chimeric antibody = genetically engineered construct containing Chimeric antibody = genetically engineered construct containing a mouse Fab portion and a human Fc portiona mouse Fab portion and a human Fc portion

3 main components3 main components Two identical Fabs (fragment-antigen binding site): the arms of Two identical Fabs (fragment-antigen binding site): the arms of

the Ythe Y An Fc (for fragment crystallizable), the stem of the YAn Fc (for fragment crystallizable), the stem of the Y

Constant region responsible for triggering effector functions that Constant region responsible for triggering effector functions that eliminate the antigen-associated cellseliminate the antigen-associated cells

Constant region must be tailored to match requirements of the antibody Constant region must be tailored to match requirements of the antibody (depending on which antigen you want it to bind to)(depending on which antigen you want it to bind to)

IgG structureIgG structure

3 MECHANISMS RESULTING IN 3 MECHANISMS RESULTING IN APOPTOSISAPOPTOSIS

Antigen cross-linkingAntigen cross-linking

Activation of death receptorsActivation of death receptors

Blockade of ligand-receptor growth or Blockade of ligand-receptor growth or survival pathwayssurvival pathways

1.1. Antigen Cross-LinkingAntigen Cross-Linking

Target growth factor receptorTarget growth factor receptor Antagonize ligand-receptor signalingAntagonize ligand-receptor signaling Growth-factor signaling mediated by the Growth-factor signaling mediated by the

receptor tyrosine kinase is inhibited receptor tyrosine kinase is inhibited EGFR (epidermal growth factor receptor)EGFR (epidermal growth factor receptor) IGF-1R (insulin-like growth factor-1 receptor)IGF-1R (insulin-like growth factor-1 receptor) FGFR (fibroblast growth factor receptor)FGFR (fibroblast growth factor receptor) PDGFR (platelet-derived growth factor receptor)PDGFR (platelet-derived growth factor receptor) VEGFR (vascular endothelial growth factor)VEGFR (vascular endothelial growth factor)

Results in arrest of tumor cell growthResults in arrest of tumor cell growth

2.2. Activation of death receptorsActivation of death receptors

Cross-link targeted surface antigens on Cross-link targeted surface antigens on tumor cells and antibody agonists that tumor cells and antibody agonists that mimic ligand-mediated activation of mimic ligand-mediated activation of specific receptorsspecific receptors Response: intracellular Ca II ions increaseResponse: intracellular Ca II ions increase Activate caspase-3 and caspase-9 (involved Activate caspase-3 and caspase-9 (involved

in cell apoptosis)in cell apoptosis)

APOPTOSIS PATHWAYAPOPTOSIS PATHWAY

3.3. Delivery of Cytotoxic AgentsDelivery of Cytotoxic Agents

Physically link antibodies to toxic Physically link antibodies to toxic substances for deliverysubstances for delivery Radio-immunoconjugates (aim of delivering Radio-immunoconjugates (aim of delivering

radiation directly to the tumor) radiation directly to the tumor) Toxin-immunoconjugates (deliver toxins Toxin-immunoconjugates (deliver toxins

intracellularly)intracellularly) Antibody-directed enzyme pro-drug therapy Antibody-directed enzyme pro-drug therapy

(ADEPT): localize enzymes to tumor cell (ADEPT): localize enzymes to tumor cell surfacessurfaces

General Drug Delivery SystemGeneral Drug Delivery System

Drug molecules Drug molecules bound to bound to macromolecule macromolecule through spacer through spacer moleculemolecule Drug released from Drug released from

macromolecule after macromolecule after cellular uptake of the cellular uptake of the conjugateconjugate

Targeting moiety = Targeting moiety = monoclonal antibodymonoclonal antibody

TOXIN IMMUNOCONJUGATESTOXIN IMMUNOCONJUGATES

Cell surface antigen must internalize upon mAb bindingCell surface antigen must internalize upon mAb bindingWhen drug is released, it interferes with protein When drug is released, it interferes with protein synthesis to induce apoptosissynthesis to induce apoptosis3 methods to attach cytotoxic drug to variable regions of 3 methods to attach cytotoxic drug to variable regions of mAbmAb

a. Couple drug to lysine moieties in the mAba. Couple drug to lysine moieties in the mAb b. Generation of aldehyde groups by oxidizing the carbohydrate b. Generation of aldehyde groups by oxidizing the carbohydrate

region and subsequent reaction with amino-containing drugs or region and subsequent reaction with amino-containing drugs or drug derivativesdrug derivatives

c. Couple drugs to sulfhydryl groups by selectively reducing the c. Couple drugs to sulfhydryl groups by selectively reducing the interchain disulfides near the Fc region of the mAbinterchain disulfides near the Fc region of the mAb

Direct attachment of mAb to drug Direct attachment of mAb to drug by S-S bondingby S-S bonding

ImmunoconjugateImmunoconjugate

BR96-doxorubicin conjugate BR96-doxorubicin conjugate (BR96-DOX)(BR96-DOX)

Promising toxin-Promising toxin-immunoconjugateimmunoconjugate

mouse/human chimeric mAbmouse/human chimeric mAb Targets antigen over-Targets antigen over-

expressed on surface of expressed on surface of human carcinoma cells of human carcinoma cells of breast, colon, lung, and ovarybreast, colon, lung, and ovary

Disulfide reduction attaches Disulfide reduction attaches mAb to drug, BR96mAb to drug, BR96

Dose that can be safely Dose that can be safely administered every 3 weeks is administered every 3 weeks is insufficientinsufficient

Other examples of toxin-Other examples of toxin-immunoconjugatesimmunoconjugates

KS1/4-MTXKS1/4-MTX Conjugate of methotrexate (MTX)Conjugate of methotrexate (MTX) Coupling of MTX to the lysine moieties of the mAbCoupling of MTX to the lysine moieties of the mAb No significant clinical responseNo significant clinical response

KS1/4-DAVLBKS1/4-DAVLB Conjugate of vinca alkaloid derivativesConjugate of vinca alkaloid derivatives Vinca alkaloid derivatives attached to amino groups of Vinca alkaloid derivatives attached to amino groups of

lysine residues on KS1/4 mAblysine residues on KS1/4 mAb No significant clinical responseNo significant clinical response

Why are these toxin-Why are these toxin-immunoconjugates unsuccessful?immunoconjugates unsuccessful?Cause gastrointestinal toxicityCause gastrointestinal toxicityInner regions of solid tumors poorly Inner regions of solid tumors poorly vascularized and have low blood flow vascularized and have low blood flow (reduce amount of immunoconjugate (reduce amount of immunoconjugate reaching these parts of the tumor)reaching these parts of the tumor)Antigen expression is heterogenous on Antigen expression is heterogenous on tumor cellstumor cells Restricts the amount of cells that can be Restricts the amount of cells that can be

effectively targeted by antibody conjugateseffectively targeted by antibody conjugates

ADEPT ENZYMES (Antibody-ADEPT ENZYMES (Antibody-directed enzyme pro-drug therapy)directed enzyme pro-drug therapy)

Chemically link the mAb to the enzyme of Chemically link the mAb to the enzyme of interest; can also be a fusion protein produced interest; can also be a fusion protein produced recombinantly with the antibody variable region recombinantly with the antibody variable region genes and the gene coding the enzymegenes and the gene coding the enzyme

Convert subsequently administered anti-cancer Convert subsequently administered anti-cancer pro-drugs into active anti-tumor agentspro-drugs into active anti-tumor agents Upon binding to targeted enzymes, it is converted into Upon binding to targeted enzymes, it is converted into

active drugactive drug

Anti-growth factor mAb TherapyAnti-growth factor mAb Therapy

AngiogenesisAngiogenesis Formation of nascent blood vesselsFormation of nascent blood vessels

VEGFVEGF One of the most upregulated antigens in cancerOne of the most upregulated antigens in cancer Protect endothelial cells from apoptosis via activation of PKC Protect endothelial cells from apoptosis via activation of PKC

pathways and upregulation of anti-apoptotic proteins such as pathways and upregulation of anti-apoptotic proteins such as Bcl-2Bcl-2

Activity mediated by tyrosine kinase receptors, VEGFR 1 and Activity mediated by tyrosine kinase receptors, VEGFR 1 and VEGFR 2VEGFR 2

Functions indirectly as survival factor for tumor cellsFunctions indirectly as survival factor for tumor cells

Inhibit VEGF signalingInhibit VEGF signaling Block the receptorBlock the receptor Inhibits tumor growth and metastasisInhibits tumor growth and metastasis Deprives tumors of nutrient-providing blood vesselsDeprives tumors of nutrient-providing blood vessels

RITUXIMAB (Rituxan)RITUXIMAB (Rituxan)

11stst therapeutic mAb approved by FDA in 1997 therapeutic mAb approved by FDA in 1997 High-level expression of the gene encoding Rituximab was foundHigh-level expression of the gene encoding Rituximab was found a mouse-chimeric mAba mouse-chimeric mAb Contains the human IgG1 and murine variable regions that target CD20 Contains the human IgG1 and murine variable regions that target CD20

B-cell antigenB-cell antigenCD20 antigen function: cell cycle progressionCD20 antigen function: cell cycle progressionBinding Rituximab to CD-20 causes: autophosphorylation, activation of Binding Rituximab to CD-20 causes: autophosphorylation, activation of serine/tyrosine protein kinases, and induction of oncogene expression --- serine/tyrosine protein kinases, and induction of oncogene expression --- induces apoptosisinduces apoptosis

Response rates of 50% to 70% in follicular lymphomas Response rates of 50% to 70% in follicular lymphomas Response rates of 90% to 100% when used in combination with Response rates of 90% to 100% when used in combination with various chemotherpay proceduresvarious chemotherpay proceduresConcluded that the dose of 4, once-weekly 375 mg/m squared IV Concluded that the dose of 4, once-weekly 375 mg/m squared IV infusions of Rituximab was safe and effective in patients with infusions of Rituximab was safe and effective in patients with relapse or refractory B non-Hodgkin’s lymphomarelapse or refractory B non-Hodgkin’s lymphoma

Toxic effects of RituximabToxic effects of Rituximab

Short-lived mild Short-lived mild reactions to infusion reactions to infusion after first treatment: after first treatment: fever, chills, rigors, fever, chills, rigors, rash, and nausearash, and nausea

Factors affecting pharmacokinetic Factors affecting pharmacokinetic parametersparameters

Circulating target antigens (which can lead to Circulating target antigens (which can lead to rapid clearance)rapid clearance)

Antigen-antibody internalization in cells (which Antigen-antibody internalization in cells (which affect serum clearance and half-life)affect serum clearance and half-life)

Antibody size and domains with the Fc regionAntibody size and domains with the Fc region Fragments have shorter half-lives and more rapid Fragments have shorter half-lives and more rapid

clearance rates than their full-sized immunoglobulinsclearance rates than their full-sized immunoglobulins

FUTUREFUTURE

Researchers hope to define the optimal Researchers hope to define the optimal combinations of the use of mAb with combinations of the use of mAb with conventional chemotherapeutic agents conventional chemotherapeutic agents and with radiation therapyand with radiation therapy

Determine best therapy candidates and Determine best therapy candidates and expand clinical trials to other tumor typesexpand clinical trials to other tumor types