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

1. ANTI-CANCERANTI-CANCER
THERAPYTHERAPY
MONOCLONAL ANTIBODIESMONOCLONAL ANTIBODIES
ByBy
SATHISHKUMAR GSATHISHKUMAR G
(sathishsak111@gmail.com)(sathishsak111@gmail.com)

2. 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 towardsrather than specific toxicity directed towards
particular cancer cellparticular cancer cell

Host toxicity: treatment discontinued at doseHost toxicity: treatment discontinued at dose
levels well below dose required to kill alllevels well below dose required to kill all
viable tumor cellsviable tumor cells

3. 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 toxicuse 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) directedDiscovery of monoclonal antibodies (mAb) directed
against well-characterized antigensagainst well-characterized antigens

Use of DNA bio-engineered technologies within lastUse of DNA bio-engineered technologies within last
25 years25 years

4. RationaleRationale
mAb as efficient carriers for delivery of anti-mAb as efficient carriers for delivery of anti-
tumor agentstumor agents

Enhanced vascular permeability of circulatingEnhanced vascular permeability of circulating
macromolecules for tumor tissue and subsequentmacromolecules for tumor tissue and subsequent
accumulation in solid tumorsaccumulation in solid tumors

Normal tissue: blood vessels have intact endothelial layer thatNormal tissue: blood vessels have intact endothelial layer that
permits passage of small molecules but not entry ofpermits passage of small molecules but not entry of
macromolecules (like mAb)macromolecules (like mAb)

Tumor tissue: blood vessels leaky, so small and largeTumor 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 cansystem; therefore, macromolecules are retained and can
accumulate in solid tumorsaccumulate in solid tumors

5. Patho-physiology of Tumor TissuePatho-physiology of Tumor Tissue
AngiogenesisAngiogenesis
HypervasculatureHypervasculature
Impaired lymphatic drainageImpaired lymphatic drainage
***Due to these characteristics, tumors can***Due to these characteristics, tumors can
be exploited for tumor-selective drugbe exploited for tumor-selective drug
delivery****delivery****

6. Genetic EngineeringGenetic Engineering
Remove or modify effector functions of mAb: used toRemove or modify effector functions of mAb: used to
avoid unwanted side effectsavoid unwanted side effects
Use mAb in their natural, fragmented, chemicallyUse mAb in their natural, fragmented, chemically
modified, or recombinant formsmodified, or recombinant forms
Use of phage display antibody libraries or transgenicUse 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 andAnimals must be immunized using the cellular antigens and
immunization procedures used to generate conventionalimmunization procedures used to generate conventional
antibodiesantibodies

Perform cell fusions to generate clones and isolate stablePerform 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 generated
in micein mice

7. Structure of AntibodyStructure of Antibody
Presently, all intact therapeutic antibodies are murinePresently, all intact therapeutic antibodies are murine
immunoglobulins of the IgG classimmunoglobulins of the IgG class

Murine immunoglobulin = glycoprotein that has a Y-shapedMurine immunoglobulin = glycoprotein that has a Y-shaped
structure: 2 identical polypeptide heavy chains and 2 identicalstructure: 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 containingChimeric 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 ofTwo 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 thatConstant 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 antibodyConstant 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)

8. IgG structureIgG structure

9. 3 MECHANISMS RESULTING IN3 MECHANISMS RESULTING IN
APOPTOSISAPOPTOSIS
Antigen cross-linkingAntigen cross-linking
Activation of death receptorsActivation of death receptors
Blockade of ligand-receptor growth orBlockade of ligand-receptor growth or
survival pathwayssurvival pathways

10. 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 theGrowth-factor signaling mediated by the
receptor tyrosine kinase is inhibitedreceptor 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

11. 2.2. Activation of death receptorsActivation of death receptors
Cross-link targeted surface antigens onCross-link targeted surface antigens on
tumor cells and antibody agonists thattumor cells and antibody agonists that
mimic ligand-mediated activation ofmimic ligand-mediated activation of
specific receptorsspecific receptors

Response: intracellular Ca II ions increaseResponse: intracellular Ca II ions increase

Activate caspase-3 and caspase-9 (involvedActivate caspase-3 and caspase-9 (involved
in cell apoptosis)in cell apoptosis)

12. APOPTOSIS PATHWAYAPOPTOSIS PATHWAY

13. 3.3. Delivery of Cytotoxic AgentsDelivery of Cytotoxic Agents
Physically link antibodies to toxicPhysically link antibodies to toxic
substances for deliverysubstances for delivery

Radio-immunoconjugates (aim of deliveringRadio-immunoconjugates (aim of delivering
radiation directly to the tumor)radiation directly to the tumor)

Toxin-immunoconjugates (deliver toxinsToxin-immunoconjugates (deliver toxins
intracellularly)intracellularly)

Antibody-directed enzyme pro-drug therapyAntibody-directed enzyme pro-drug therapy
(ADEPT): localize enzymes to tumor cell(ADEPT): localize enzymes to tumor cell
surfacessurfaces

14. General Drug Delivery SystemGeneral Drug Delivery System
Drug moleculesDrug molecules
bound tobound to
macromoleculemacromolecule
through spacerthrough spacer
moleculemolecule

Drug released fromDrug released from
macromolecule aftermacromolecule after
cellular uptake of thecellular uptake of the
conjugateconjugate

Targeting moiety =Targeting moiety =
monoclonal antibodymonoclonal antibody

15. TOXIN IMMUNOCONJUGATESTOXIN IMMUNOCONJUGATES
Cell surface antigen must internalize upon mAb bindingCell surface antigen must internalize upon mAb binding
When drug is released, it interferes with proteinWhen drug is released, it interferes with protein
synthesis to induce apoptosissynthesis to induce apoptosis
3 methods to attach cytotoxic drug to variable regions of3 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 carbohydrateb. Generation of aldehyde groups by oxidizing the carbohydrate
region and subsequent reaction with amino-containing drugs orregion and subsequent reaction with amino-containing drugs or
drug derivativesdrug derivatives

c. Couple drugs to sulfhydryl groups by selectively reducing thec. 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

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

17. ImmunoconjugateImmunoconjugate
BR96-doxorubicin conjugateBR96-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 ofexpressed on surface of
human carcinoma cells ofhuman carcinoma cells of
breast, colon, lung, and ovarybreast, colon, lung, and ovary

Disulfide reduction attachesDisulfide reduction attaches
mAb to drug, BR96mAb to drug, BR96

Dose that can be safelyDose that can be safely
administered every 3 weeks isadministered every 3 weeks is
insufficientinsufficient

18. 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 ofVinca 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

19. Why are these toxin-Why are these toxin-
immunoconjugates unsuccessful?immunoconjugates unsuccessful?
Cause gastrointestinal toxicityCause gastrointestinal toxicity
Inner regions of solid tumors poorlyInner regions of solid tumors poorly
vascularized and have low blood flowvascularized 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 onAntigen expression is heterogenous on
tumor cellstumor cells

Restricts the amount of cells that can beRestricts the amount of cells that can be
effectively targeted by antibody conjugateseffectively targeted by antibody conjugates

20. ADEPT ENZYMES (Antibody-ADEPT ENZYMES (Antibody-
directed enzyme pro-drug therapy)directed enzyme pro-drug therapy)
Chemically link the mAb to the enzyme ofChemically link the mAb to the enzyme of
interest; can also be a fusion protein producedinterest; can also be a fusion protein produced
recombinantly with the antibody variable regionrecombinantly with the antibody variable region
genes and the gene coding the enzymegenes and the gene coding the enzyme
Convert subsequently administered anti-cancerConvert 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 intoUpon binding to targeted enzymes, it is converted into
active drugactive drug

22. 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 PKCProtect endothelial cells from apoptosis via activation of PKC
pathways and upregulation of anti-apoptotic proteins such aspathways and upregulation of anti-apoptotic proteins such as
Bcl-2Bcl-2

Activity mediated by tyrosine kinase receptors, VEGFR 1 andActivity 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

23. RITUXIMAB (Rituxan)RITUXIMAB (Rituxan)
11stst
therapeutic mAb approved by FDA in 1997therapeutic 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 CD20Contains the human IgG1 and murine variable regions that target CD20
B-cell antigenB-cell antigen
CD20 antigen function: cell cycle progressionCD20 antigen function: cell cycle progression
Binding Rituximab to CD-20 causes: autophosphorylation, activation ofBinding 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 lymphomasResponse rates of 50% to 70% in follicular lymphomas
Response rates of 90% to 100% when used in combination withResponse rates of 90% to 100% when used in combination with
various chemotherpay proceduresvarious chemotherpay procedures
Concluded that the dose of 4, once-weekly 375 mg/m squared IVConcluded that the dose of 4, once-weekly 375 mg/m squared IV
infusions of Rituximab was safe and effective in patients withinfusions of Rituximab was safe and effective in patients with
relapse or refractory B non-Hodgkin’s lymphomarelapse or refractory B non-Hodgkin’s lymphoma

24. Toxic effects of RituximabToxic effects of Rituximab
Short-lived mildShort-lived mild
reactions to infusionreactions to infusion
after first treatment:after first treatment:
fever, chills, rigors,fever, chills, rigors,
rash, and nausearash, and nausea

25. Factors affecting pharmacokineticFactors affecting pharmacokinetic
parametersparameters
Circulating target antigens (which can lead toCirculating target antigens (which can lead to
rapid clearance)rapid clearance)
Antigen-antibody internalization in cells (whichAntigen-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 rapidFragments have shorter half-lives and more rapid
clearance rates than their full-sized immunoglobulinsclearance rates than their full-sized immunoglobulins

26. FUTUREFUTURE
Researchers hope to define the optimalResearchers hope to define the optimal
combinations of the use of mAb withcombinations of the use of mAb with
conventional chemotherapeutic agentsconventional chemotherapeutic agents
and with radiation therapyand with radiation therapy
Determine best therapy candidates andDetermine best therapy candidates and
expand clinical trials to other tumor typesexpand clinical trials to other tumor types

27. THANK YOUTHANK YOU