ANTI-CANCER THERAPY

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

2. Conventional Anti-Cancer Therapy
Conventional Anti-Cancer Therapy
Chemotherapy: Imperfect
Chemotherapy: Imperfect

Systematic nature of cytoxicity
Systematic nature of cytoxicity

Agents lack intrinsic anti-tumor selectivity
Agents 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 cell
particular 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 cells
viable tumor cells

3. HISTORY
HISTORY
Emil von Behring in 1890
Emil von Behring in 1890

Discovered antibodies
Discovered 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 substances
substances in pathogenic substances
Kohler and Milstein in 1975
Kohler and Milstein in 1975

Discovery of monoclonal antibodies (mAb) directed
Discovery of monoclonal antibodies (mAb) directed
against well-characterized antigens
against well-characterized antigens

Use of DNA bio-engineered technologies within last
Use of DNA bio-engineered technologies within last
25 years
25 years

4. Rationale
Rationale
mAb as efficient carriers for delivery of anti-
mAb as efficient carriers for delivery of anti-
tumor agents
tumor 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 tumors
accumulation 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 tissue
molecules 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 tumors
accumulate in solid tumors

5. Patho-physiology of Tumor Tissue
Patho-physiology of Tumor Tissue
Angiogenesis
Angiogenesis
Hypervasculature
Hypervasculature
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****

6. Genetic Engineering
Genetic 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 forms
modified, or recombinant forms
Use of phage display antibody libraries or transgenic
Use of phage display antibody libraries or transgenic
animals
animals

Identify animals that make desired antibodies
Identify 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
antibodies
antibodies

Perform cell fusions to generate clones and isolate stable
Perform cell fusions to generate clones and isolate stable
clones, making mAb
clones, making mAb

Most mAb used in the clinical setting were generated
Most mAb used in the clinical setting were generated
in mice
in mice

7. Structure of Antibody
Structure of Antibody
Presently, all intact therapeutic antibodies are murine
Presently, all intact therapeutic antibodies are murine
immunoglobulins of the IgG class
immunoglobulins 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 bond
light 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 portion
a mouse Fab portion and a human Fc portion
3 main components
3 main components

Two identical Fabs (fragment-antigen binding site): the arms of
Two identical Fabs (fragment-antigen binding site): the arms of
the Y
the Y

An Fc (for fragment crystallizable), the stem of the Y
An 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 cells
eliminate 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)

8. IgG structure
IgG structure

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

10. 1.
1. Antigen Cross-Linking
Antigen Cross-Linking
Target growth factor receptor
Target growth factor receptor

Antagonize ligand-receptor signaling
Antagonize 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 growth
Results in arrest of tumor cell growth

11. 2.
2. Activation of death receptors
Activation 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 receptors
specific receptors

Response: intracellular Ca II ions increase
Response: 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)

12. APOPTOSIS PATHWAY
APOPTOSIS PATHWAY

13. 3.
3. Delivery of Cytotoxic Agents
Delivery of Cytotoxic Agents
Physically link antibodies to toxic
Physically link antibodies to toxic
substances for delivery
substances 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
surfaces
surfaces

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

15. TOXIN IMMUNOCONJUGATES
TOXIN IMMUNOCONJUGATES
Cell surface antigen must internalize upon mAb binding
Cell surface antigen must internalize upon mAb binding
When drug is released, it interferes with protein
When drug is released, it interferes with protein
synthesis to induce apoptosis
synthesis to induce apoptosis
3 methods to attach cytotoxic drug to variable regions of
3 methods to attach cytotoxic drug to variable regions of
mAb
mAb

a. Couple drug to lysine moieties in the mAb
a. 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 derivatives
drug 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 mAb
interchain disulfides near the Fc region of the mAb

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

17. Immunoconjugate
Immunoconjugate
BR96-doxorubicin conjugate
BR96-doxorubicin conjugate
(BR96-DOX)
(BR96-DOX)

Promising toxin-
Promising toxin-
immunoconjugate
immunoconjugate

mouse/human chimeric mAb
mouse/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 ovary
breast, colon, lung, and ovary

Disulfide reduction attaches
Disulfide reduction attaches
mAb to drug, BR96
mAb to drug, BR96

Dose that can be safely
Dose that can be safely
administered every 3 weeks is
administered every 3 weeks is
insufficient
insufficient

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

19. Why are these toxin-
Why are these toxin-
immunoconjugates unsuccessful?
immunoconjugates unsuccessful?
Cause gastrointestinal toxicity
Cause gastrointestinal toxicity
Inner 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 cells
tumor cells

Restricts the amount of cells that can be
Restricts the amount of cells that can be
effectively targeted by antibody conjugates
effectively 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 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 enzyme
genes and the gene coding the enzyme
Convert subsequently administered anti-cancer
Convert subsequently administered anti-cancer
pro-drugs into active anti-tumor agents
pro-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 drug
active drug

22. Anti-growth factor mAb Therapy
Anti-growth factor mAb Therapy
Angiogenesis
Angiogenesis

Formation of nascent blood vessels
Formation of nascent blood vessels
VEGF
VEGF

One of the most upregulated antigens in cancer
One 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-2
Bcl-2

Activity mediated by tyrosine kinase receptors, VEGFR 1 and
Activity mediated by tyrosine kinase receptors, VEGFR 1 and
VEGFR 2
VEGFR 2

Functions indirectly as survival factor for tumor cells
Functions indirectly as survival factor for tumor cells
Inhibit VEGF signaling
Inhibit VEGF signaling

Block the receptor
Block the receptor

Inhibits tumor growth and metastasis
Inhibits tumor growth and metastasis

Deprives tumors of nutrient-providing blood vessels
Deprives tumors of nutrient-providing blood vessels

23. RITUXIMAB (Rituxan)
RITUXIMAB (Rituxan)
1
1st
st
therapeutic mAb approved by FDA in 1997
therapeutic mAb approved by FDA in 1997

High-level expression of the gene encoding Rituximab was found
High-level expression of the gene encoding Rituximab was found

a mouse-chimeric mAb
a 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 antigen
B-cell antigen
CD20 antigen function: cell cycle progression
CD20 antigen function: cell cycle progression
Binding 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 apoptosis
induces 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 procedures
various chemotherpay procedures
Concluded 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 lymphoma
relapse or refractory B non-Hodgkin’s lymphoma

24. Toxic effects of Rituximab
Toxic 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 nausea
rash, and nausea

25. Factors affecting pharmacokinetic
Factors affecting pharmacokinetic
parameters
parameters
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 region
Antibody 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 immunoglobulins
clearance rates than their full-sized immunoglobulins

26. FUTURE
FUTURE
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 therapy
and with radiation therapy
Determine best therapy candidates and
Determine best therapy candidates and
expand clinical trials to other tumor types
expand clinical trials to other tumor types