Anticancer Drugs

Some basic Facts about Cancer

• Cancer cells have lost the normal regulatory mechanisms that

control cell growth and multiplication
• Cancer cell have lost their ability to differentiate (that means to
specialize)
• Benign cancer cell stay at the same place
• Malignant cancer cells invade new tissues to set up secondary
tumors, a process known as metastasis
• Chemicals causing cancer are called mutagens
• Cancer can be caused by chemicals, life style (smoking), and
viruses
• genes that are related to cause cancer are called oncogenes.
Genes that become onogenic upon mutation are called proto-
oncogenes.
 The Hallmarks of Cancer

• Self-sufficiency in growth signals (e.g. via activation of the H-Ras

oncogene)
• Insensitivity to growth inhibitory (anti-growth) signals (lose
retinoblastoma suppressor)
• Evasion of programmed cell death (apoptosis) (produce IGF
survival factors)
• limitless replicative potential (turn on telomerase)
• sustained angiogenesis (produce VEGF inducer)
• tissue invasion and metastasis (inactivate E-cadherin)
• inactivation of systems that regulate in response to DNA damage
(e.g. p53)
 Anti-Cancer Strategies

Gleevec Iressa Erbitux (ab) Avastin (ab)

sales of kinase inhibitors

Some current and prospective modalities of cancer chemotherapy

Category Function Examples
Antimetabolites Interfere with intermediary metabolism of proliferating cells Methotrexate, 5-fluorouracil
Monoclonal antibodies Target cancer cells that express specific antigen Herceptin (Genentech), Zevalin (IDEC
Pharmaceuticals/Schering-Plough)
Mitosis inhibitors Target microtubules and associated proteins required in cell division Taxol
Steroid hormones Block steroid- and hormone-dependent growth of certain tumours Tamoxifen, flutamide
Alkylating/cross-linking agents Damage DNA and result in death of growing cells Endoxan, cisplatin, cyclophosphamide
Signal-transduction agents Modulate communication between cells Gleevec (Novartis), Tarceva (OSI), Iressa
(AstraZeneca), LY900003 (ISIS 3521) (Eli Lilly)
Angiogenesis inhibitors Block blood-vessel formation to tumour Avastin (Genentech)
Histone-deacetylase inhibitors Affect transcription of genes SAHA (Aton Pharma)
Telomerase inhibitors Affect telomere maintenance required for tumour growth BIBR1532 (Boehringer Ingelheim)
Antitumour antibiotics Bind DNA to prevent DNA and/or RNA synthesis Etoposide, doxorubicin
Phases of the Cell Cycle

• G1 phase (gap 1): Cell grows in size

and prepares to copy its DNA in
response to various growth factors
• S phase (synthesis): Replication of
DNA, copying of the chromosome
• G2 phase (gap 2): Preparation for
cell division. Check copied DNA
and repair damaged copies.
• M phase (mitosis): Formation of the
mitotic spindle, and separation into
two individual cells (cell division).
Control of Cell Cycle Progression by CDKs

• Progression through the cell cycle is controlled

by cyclin-dependent kinases (CDKs).

• Binding of cyclin with its associated kinase

triggers to move the cell cycle to another
phase

• inhibitory proteins are present that can modify

the effect of cyclins. These include p15 and
p16, that block activity of the cyclin D-CDK
complex. Another regulator is p21, that is
controlled by the tumor suppressor protein p53.

• over-active cyclins or CDKs have been

associated with many tumors. Excessive
production of cyclins or CDKs or insufficient
production of CDK inhibitors leads to disruption
of the normal regulation of the cell cycle.
 Cell Death

• Necrosis is the uncontrolled (pathological) cell death. In contrast with

apoptosis, cleanup of cell debris by phagocytes of the immune system is
generally more difficult. There are many causes of necrosis including injury,
infection, cancer, infarction, toxins and inflammation. Necrosis can arise
from lack of proper care to a wound site. Usually cell outlines do not stay
intact, and cell debris is released into the environment

• Apoptosis is the programmed cell death. It is used by organisms to control

the number of cells and tissue size. The cells during apoptosis shrink, but no
uncontrolled release of cell debris into the environment occurs. The
immune system usually “cleans up” the dying cells, and the content is
recycled.

Apoptosis is triggered by an extracellular signal to the CD95 receptor. In
response to that signal a set of cysteine proteases, called caspases are
activated, that are largely responsible for the morphological changes
observed.
 Routes for Apoptosis

•Two pathways for activation: i) at the plasma membrane via external

ligands upon binding to the death receptor or ii) via the mitochondrial
pathway
•Binding of external ligands such as tumor necrosis factor receptor (TNFα)
to Fas receptors of the TNF superfamily induces receptor oligomerization
and formation of a death-inducing signaling complex. This complex
recruits, via the adaptor molecule FADD (Fas-associated death domain)
multiple Pro-caspase-8 molecules, resulting in caspase-8 activation that
finally results in caspase-3 activation
•In the mitochondrial pathway release of apoptogenic factors such as
cytochrome c, Apaf-1, caspase-9-containing apoptosome complex and
inhibitors-of-apoposis proteins trigger caspase-3 activation
• Links between the two pathways exist. For example, caspsase-8 results
in cleavage of Bid, a Bcl-2 family protein, which translocates to the
mitochondria to release cytochrome c.
Apoptosis
 Regulators of Apoptosis

• The Bcl-2 family of factors regulate caspase activation either

negatively ( e.g. Bcl-2, Bcl-XL, MCL1) or positively (e.g. Bcl-XS, Bax,
BAD, BAK, BID)
• The inhibitors of apoptosis proteins (IAP) retard apoptosis
• Upstream modulators are oncogenes such as c-myc, that
activates apoptosis in a manner important in cancer therapy
• the tumor suppressor p53 induces apoptosis under certain
circumstances
Triggers Regulators Executioners
• DNA damage • p53 • Apaf-1
• cytokine star vation • death domain factors • caspases
• hypoxia • Bcl-2 family
• detachment • Myc/oncogenes
• temperature • cytokine-responsive kinases
• death receptor • cytochrome c
 Telomeres

• Cancer cells are often called

immortal since there seems to be no
limit for how often they can divide
• Life-time of normal cells is limited to
50-60 cell divisions. This is regulated
by telomeres. The telomeres are at
the 3’ end of the chromosomes.
After each replication about 50-100
base pairs are lost
• At some point telomeres are too
short to be effective and the DNA
becomes unstable thereby limiting A few short telomeres

replication. Cancer cells possess an

enzyme called telomerase which Critically short telomeres

maintains the length of the

telomeres and thereby allows more
DNA replications.
Apoptosis Cancer (1 in 10,000,000)
 Tissue Invasion

• In malignant cancers cells cancer cells break away from the primary
tumor site, invade a blood or lymphatic vessel, to form metastasis sites
• Usually, cells only stick to similar cells. The signature on the cell-surface
is transmitted via cell-adhesion molecules (e.g. cadherins). Moreover,
cells are connected to each other via mounting them on the
extracellular matrix (EM).
• Adhesion to the EM involves molecules called integrins.
• The protein matrix metalloproteinase degrades the extracellular
matrix, and therefore is important for leaving the site of the primary
tumor and attaching to the secondary site.
• If a non-cancer cell is detached from the extracellular matrix it stops
growing and apoptosis is triggered.
• In metastized cells cell adhesion molecules are missing, so that they
can leave the site of the primary tumor.
 Angiogenesis

• Tumors are quickly growing tissue that need to have good blood
supply.
• Angiogenesis refers to the formation of new blood vessels
• Tumor cells release growth factors such as vascular endothelial
growth factor (VEGF) or fibroplast growth factor (FGF-2) leading to
sprouting and extension of existing capillaries
• In healthy tissue repair of injured tissues is controlled by
angiogenesis inhibitors such as angiostatin and thrombospondin
• Blood vessels arising from angiogenesis are abnormal in that they
are disorganized in structure and leaky.
• These cells display integrins on their surface to protect the newly
formed cells from apoptosis
• Before angiogenesis can start the basement membrane around
the blood vessel has to be broken down (carried out by matrix
metalloproteinases (MMPs))
Strategies for Anti-Cancer Therapeutics
Intrinsic Tumor Suppression: p53

•In response to DNA damage,

oncogene activation or other
harmful events the tumor
suppressor gene p53 is induced
•various kinases phosporylate
p53 which help stabilizing it.
Activated p53 results in DNA
binding and transcriptional
activation
•MDM2 serves to down-regulate
p53, which in turn is regulated by
p14ARF
•p53 triggers cell-cycle arrest in
untransformed cells via cell-cycle
regulators such as CDKs
•it also triggers apoptosis in
transformed cells via Bax
•in most tumor cells p53 is
mutated and inactivated
 Drugs directly interacting with DNA

Intercalating agents

Mechanism of action

• Contain planar aromatic or heteroaromatic ring systems

• Planar systems slip between the layers of nucleic acid pairs and disrupt the
shape of the helix
• Preference is often shown for the minor or major groove
• Intercalation prevents replication and transcription
• Intercalation inhibits topoisomerase II (an enzyme that relieves the strain in
the DNA helix by temporarily cleaving the DNA chain and crossing an intact
strand through the broken strand.
 Intercalating agents

Dactinomycin Doxorubicin (Adriamycin)

(Extra binding to sugar phosphate (Extra binding to sugar

backbone by cyclic peptide) phosphate backbone by NH3)
Intercalating reagents (II)

•During replication, supercoiled DNA is

unwound by the helicase. The thereby
created tension is removed by the
topoisomerase II, that cuts and rejoins
the DNA strands.
•When doxorubicin is bound to the
DNA it stabilizes the DNA-topoII complex
at the point where the enzyme is
covalently bound
 Natural Products in Cancer Therapy:
Bleomycins

• intercalate via the bithiazole moiety

• the N-atoms of the primary amines, pyrimidine ring and imidazole ring
chelate Fe, which is involved in the formation of superoxide radicals,
which subsequently act to cut DNA between purine and pyrimidine
nucleotides
 Drugs directly interacting with DNA
Alkylating agents
• Contain highly electrophilic groups
• Form covalent bonds to nucleophilic groups in DNA
• Attack N-1 and N-3 of adenine and N-3 of cytosine, and in
particular N-7 of guanine bases
• Prevent replication and transcription
• Useful as anti-tumour agents
• Toxic side effects (e.g. alkylation of proteins)

Intrastrand cross linking Interstrand cross linking

 Mechanism of action of Chlormethine

•Chlormethine •Aziridine ion

•Crosslinked DNA

•too reactive to survive oral route, putting an aromatic moiety instead of the
Me group lowers reactivity (and moreover, mimics the amino acid Phe)
 Alkylating Drugs: Mitomycin C
Converted to alkylating agent in the
Mitomycin C body

Reduction

Alkylating agent

Crosslinked DNA
 Nitrosoureas

• Lomustine and carmustine are lipid-soluble and can cross the

blood-brain barrier
• The drugs decompose to form alkylating and carbamoylating

•the formed isocyanate reacts with lysine NH3

groups thereby inactivating DNA repair enzymes
•the alkylating agent reacts first with O-6 of
guanine followed with N-3 of cytosine of the
other strand
 Pt-Alkylating agents

• Binds to DNA in regions rich in guanine units

• Intrastrand links rather than interstrand
• Inhibits transcription
• In solution the Cl- ligands are exchanged against water to result
in positively charged ligands that bind to the DNA (to N-7 or O-6 of
adjacent guanine groups)
• Results in localized unwinding of the DNA
 NH3
NH3
H 3N Cl NH3 NH3
Pt Pt
Pt
H3N Cl

Cisplatin

Guanine N7 position

H
H ..
N O N
7 Replication inhibition
C N H N G N
Transcription inhibition
N N Cell-cycle arrest
O DNA repair
H N Cell death
H
 DNA chain cutters

Calicheamicin γ1I
Antitumor agent

• Generates DNA diradical

• DNA diradical reacts with oxygen
• Results in chain cutting
 siRNA-based anti-cancer approaches
a c e
Transferrin

siRNA Single-stranded
siRNA
targeting RRM2 DNA targeting
targeting PKN3
BCL2

~75 nm ~100 nm ~130 nm

b d
siRNA-I
targeting VEGF

siRNA
siRNA-II targeting PLK1
targeting KSP

80–100 nm ~80 nm
Figure 1 | Schematic illustrations of the siRNA-based therapeutics used in Phase I trials to treat patients with
solid cancers. a | CALAA-01 is a polymer-based nanoparticle containing a targeting ligandNatureonReviews | Drug
its surface Discovery
(the human
protein transferrin) and a small interfering RNA (siRNA) that targets the M2 subunit of ribonucleotide reductase (RRM2).
b | ALN-VSP is a lipid-based nanoparticle that contains two different siRNAs that target vascular endothelial growth
factor A (VEGFA) and kinesin spindle protein (KSP). c | Atu27 is a lipid-based nanoparticles that contains an siRNA that
targets protein kinase N3 (PKN3). d | TKM-PLK1 is a lipid-based nanoparticle that contains an siRNA that targets
polo-like kinase 1 (PLK1). e | PNT2258 is a lipid-based nanoparticle that contains single-stranded DNA (rather than
siRNA) that targets BCL2.

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | DECEMBER 2015 | 847

 Antimetabolites
• inhibit the synthesis of DNA or nucleotide building blocks
• Dihydrofolate reductase inhibitors

methotrexate

source of one-C unit for methylations of

deoxyuridinemonophosphate (dUMP) to form
deoxythymidinemonophosphate (dTMP)
 Antimetabolites
• Thymidylate synthase inhibitors

•5-Fluorouracil acts as an suicide inhibitor

 Antimetabolites
• Inhibitors of ribonucleotide reductase
• enzyme converts ribonucleotide diphosphates into desoxyribonucleotide
diphosphates, inhibited by Hydroxycarbamide

•adenosine deaminase inhibitors, e.g. Pentostatin

Pentostatin
 Antimetabolites:

Purine antagonists DNA polymerase inhibitors

 Hormone-based Anti-Cancer Therapies

• steroid hormones bind to nuclear receptors and act as transcription factors

• if the cancer requires a specific hormone, a hormone resulting in the opposite
effect can be administered
• Used: glucocorticoides (hormones involved in the biosynthesis of glucose, e.g.
prednisolone), oestrogens, progestins (e.g. medroxyprogesterone acetate),
analogues of the luteinizing hormone-releasing hormones (LHRH)
 Drugs acting on structural proteins
•mitosis is a ordered series of events in which identical copies of the
genome are moved to discrete locations within the dividing cell
•The mitotic spindle is very important for that event. The filaments in the
mitotic spindle are formed from microtubule

•microtubule are cytoskeletal elements present in all eukaryotic cells.

•they are composed of α- and β-subunits
•both, formation (polymerization) and destruction (depolymerization) of
microtubules are important for proper execution of cell division
•drugs interfering with microtubule polymerization/depolymerization interfere with
mitosis , cause cell-cycle arrest and trigger apoptosis
Drugs acting on structural proteins
Inhibitors of tubulin polymerization

•vinca alkaloids from the Madagascar periwinkle plant

•can be substrate for the P-glycoprotein efflux pump
 Drugs acting on structural proteins
Inhibitors of tubulin depolymerization

Epothilone
Taxol

• Taxol is harvested from the bark of the yew trees

• binds to the β-subunit of tubulin and accelerates polymerization
• the resultant microtubules are stabilized, inhibiting
depolymerization
• cell cycle is halted at the G2/M stage
• prepared semi-synthetically from a compound from yee needles
• cannot be taken orally
• causes multidrug resistance (substrate for p-glycoprotein)
• ephothilones are bacterial metabolites. They are not substrate for
P-GP
Signaling pathways important for Cancer

p53 Pathway RB1 Pathway RAS Pathway PI3K Pathway

p15INK4B,
p14ARF IGF1, EGF, PDGF
p16INK4A

HDM2 CDK4 IGF1R, EGFR, PDGFR

p53 Cyclin D1 NF1 RAS PI3K

PTEN

RB1 MAPK AKT

Apoptosis Cell-cycle arrest Proliferation Survival

• Ras: (Rat sarcoma) Ras protein family members belong to the GTPases, and
are involved in transmitting signals within cells. When Ras is 'switched on' it
subsequently switches on other proteins, that turn on genes involved in cell
growth, differentiation and survival. As a result, mutations in ras genes can lead
to the production of permanently activated Ras proteins. Because these
signals result in cell growth and division, overactive Ras signaling can
ultimately lead to cancer.
• p53/HDM2: Tumor suppressor. p53 has many mechanisms of anticancer
function and plays a role in apoptosis, genomic stability, and inhibition of
angiogenesis. It is modulated by MDM2.
• PIK3: phosphatidylinositol-3-kinase. PI3Ks are a family of signal transducer
enzymes capable of phosphorylating the 3 hydroxyl of the inositol ring of
phosphatidylinositol. PI 3-kinases have been linked to cell growth, proliferation,
differentiation, motility, survival and intracellular trafficking.The class IA PI 3-
kinase p110α is mutated in many cancers. PI 3-kinase activity contributes
significantly to cellular transformation and the development of cancer.
• AKT: Protein kinase B (PKB, Akt) is a serine/threonine-specific protein kinase
that plays a key role in multiple cellular processes such as glucose metabolism,
apoptosis, cell proliferation, transcription and cell migration.
• MAPK: Mitogen-activated protein kinases (MAPK) are protein kinases that are
specific to the amino acids serine, threonine, and tyrosine. They regulate cell
functions including proliferation, gene expression, differentiation, mitosis, cell
survival, and apoptosis
 Farnesyltransferase inhibitors

• The RAS signaling protein is involved in cancer. Mutations in RAS are

found in 30% of cancer cells. Mutant RAS is constitutively active.
• RAS signaling requires linking RAS to the inner membrane leaflet. This is
done by adding a carbon chain by the farnesyl transferase.
 Design of Farnesyltransferase inhibitors

•substrate has Cys-aa-X architecture


(a=V,L,I; X=M,E,S)

•masking of C-terminal free acid

•removal of susceptibility to peptide

bond cleavage by proteases •both carboxyl acid and thiol
masked as prodrugs
 Tyrosine Kinase Inhibitors

• Small molecule tyrosine kinase inhibitors (or TKIs) – generic names end in “-nib”
• Generally oral
• Side effects vary, depending on which enzymes they inhibit (what their target is)
• Several are effective against cancers resistant to most previous therapies

Generic Name Brand Name Cancer

Imatinib Gleevec CML, GIST, others

Dasatinib Sprycel CML, ALL

Nilotinib Tasigna CML

Gefitinib Iressa Lung

Erlotinib Tarceva Lung, Pancreas

Lapatinib Tykerb Breast

Sorafenib Nexavar Kidney, Liver

Sunitinib Sutent Kidney

 Strategies for targeting kinases
Small molecule
inhibitors of ligand–
Proteolysis Anti-receptor Anti-ligand receptor binding
mAbs mAbs (unarmed
or armed) Ligand–toxin
e conjugate

Protein

a ATP

b
ATP competitors
Protein

c
RNA
Antisense to receptor or ligand
d Nucleus

DNA

•inhibition of ATP binding (a) • antibodies against the ligand-binding epitope

• antibodies against the bound ligand
•inhibition of kinase folding (b)
•inhibition of translation (siRNA, c) • ATP competitors
•inhibition of transcription (antisense, d) •
ligand antagonists
Dancey et al. Nature Rev Drug Discov. 2 (2003),296
 Inhibitors of Growth Factors:
Targeting the EGF receptors, a TK receptor
• Overexpression of altered Epidermal Growth Factor receptors results (EGFR,
ERBB family) in formation of a oncogene.
Ligand (EGF, TGFα)
Cl
EGFR
F

O NH
Autophosphorylation morpholine groups
N O
N improves solubility
TK TK P TK TK P
Gefitinib O N

Gefitinib (ZD1839)
Activation of signal-transduction ATP competitor
cascades (for example, MAPK)
IC50 (EGFR) = 0.033 μM
IC50 (ERBB2) >3.7 μM
IC50 (KDR) >3.7 μM
Cell Invasion and
Apoptosis Angiogenesis IC50 (FLT-1) >100 μM
proliferation metastasis

metabolite of I metabolite of I
lead
Inhibitor of the Abelson Tyrosine Kinase (BCR-
ABL)

• the BCR-ABL kinase is the sole oncogene responsible in rare

blood cancer
• Inhibition of autophosphorylation of BCR-ABL by Gleevec
• treatment of BCR-ABL transformed cell-lines with Gleevec results
in dose-dependent reduction of tumor growth
• the anti-tumor effect is specific for BCR-ABL expressing cells
• Gleevec re-activates apoptosis in BCR-ABL cells
 Angiogenesis Inhibitors
Targeting the VEGF receptor, a protein tyrosine

•elevated levels of fibroplast growth factors

(FGF) and vascular endothelial growth factor
(VEGF) receptor are associated with
angiogenesis
• VEGF is regulated by multiple cytokines, e.g.
the transforming growth factor (TGF-β), the
epidermal growth factor (EGF) and the platelet-
derived growth factor (PDGF)
•inhibitors mostly target the ATP binding site

other kinase targets: platelet-derived growth
factors (PDGF-R), mitogen-activated protein
kinases (MAPK), insulin growth factor 1 receptor
(IGF-1R), protein kinase B (PKB), c-Src tyrosine
kinase, inositol triphosphate kinase (IP3K)
 Inhibitors of cyclin-dependent kinases, S/T kinases
• CDKs are important for the CHK1
Growth factors
G0
control of the cell-cycle (mainly
at G1/G2 depending on DNA
Cdc25
Cdk4,6 / Cyclin D1, D2, D3
G1 E2F
damage for example) Mitosis Cdk2 / Cyclin E
Cdk1 / Cyclin B p53
CHK2
BRCA1

• Ser/Thr kinases Cdc25

G2
S phase
• they are activated by cyclins Cdk3
Cdk5
Cdk2 / Cyclin A
and inhibited by cyclin-
Cdk1 / Cyclin A Cdk7 Non-cell cycle
Cdk8 functions
Cdk9
dependent kinase inhibitors Current Opinion in Pharmacology
 Matrix Metalloproteinase Inhibitors

• MMP are zinc-dependent enzymes (proteases)

• extremely destructive enzymes involved in the remodelling of the extracellular matrix
or the connective tissue
• MMPs comprise callagenases, gelatinases, stromelysins and the membrane type
(MT)
• They inhibit angiogenesis
• collagenase cleaves between glycine and isoleucine

marimastat
 Proteasome Inhibitors

• is the unit for degradation of damaged or misfolded proteins, but

also degrades protein involved in regulation
• protein marked for degradation are labeled with ubiquitin
• inhibiting the proteasome leads to accumulation of regulatory
proteins such as the apoptosis promoter Bax
• Accumulation of regulatory proteins leads to cell crisis and
triggers apoptosis

Bortezomib Aclarubicin
 Antibody Cancer Therapy
• The killing of tumour cells using monoclonal antibodies (mAbs) can result from direct
action of the antibody (through receptor blockade, for example), immune-mediated cell
killing mechanisms, payload delivery, and specific effects of an antibody on the tumour
vasculature and stroma.
• Tumour antigens that have been successfully targeted include epidermal growth factor
receptor (EGFR), ERBB2, vascular endothelial growth factor (VEGF), cytotoxic T lymphocyte-
associated antigen 4 (CTLA4), CD20, CD30 and CD52.
• Serological, genomic, proteomic and bioinformatic databases have also been used to
identify antigens and receptors that are overexpressed in tumour cell populations or that are
linked to gene mutations identified as driving cancer cell proliferation (tumor markers).
• A major objective for the clinical evaluation of mAbs has been determining the toxicity
and therapeutic efficacy of the antibody alone or as a delivery system for radioisotopes or
other toxic agents. It is also crucial to assess its in vivo specificity by determining its
biodistribution in patients and to assess the ratio of antibody uptake in the tumour versus
normal tissues.
• Twelve antibodies (2012) have received approval from the US FDA for the treatment of
various solid tumours and haematological malignancies, and a large number of additional
therapeutic antibodies are currently being tested in early stage and late-stage clinical trials.
 Antibody Cancer Therapy

Weiner et al., Nature Rev. Cancer 15, 361–370 (2015)

 Antibody Applications

Scott, Nature Rev. Cancer, 12 (2012), 278

 Antibody Modifications

Weiner et al., Nature Rev. Cancer 15, 361–370 (2015)

 Nomenclature of Monoclonal Antibodys

• Last syllable is always –mab

• Next to last syllable
▪ -u- human (100%) : Panitumumab
▪ -zu- humanized (95%) : Trastuzumab
▪ -xi- chimeric (65%) : Rituximab
▪ -o- mouse, -a- rat, -e- hamster, -i- primate : Tositumomab
• Previous syllable
– -tu(m)- for tumor in general [-ma(r)- breast, -pr(o)- prostate,
-co(l)- colon, etc.]
– -ci(r)- for circulatory : Bevacizumab
Scott, Nature Rev. Cancer, 12 (2012),
 Cytotoxic T-cell Targeting

bispecific antibody
HEA125xCD3

carcinoma cell
cytotoxic
T lymphocyte

Ep-
CAM CD3

Kill
 Antibody Targets

Targets of VEGF Bevacizumab Tumour vasculature

anti-angiogenic mAbs
VEGFR IM-2C6 and CDP791 Epithelium-derived solid tumours
Integrin αVβ3 Etaracizumab Tumour vasculature
Integrin  α5β1 Volociximab Tumour vasculature
Growth and EGFR Cetuximab, panitumumab, Glioma, lung, breast, colon, and head and neck
differentiation nimotuzumab and 806 tumours
signalling
ERBB2 Trastuzumab and pertuzumab Breast, colon, lung, ovarian and prostate tumours
ERBB3 MM-121 Breast, colon, lung, ovarian and prostate, tumours
MET AMG 102, METMAB and SCH 900105 Breast, ovary and lung tumours
IGF1R AVE1642, IMC -A12, MK-0646, R1507 Glioma, lung, breast, head and neck, prostate and
and CP 751871 thyroid cancer
EPHA3 KB004 and IIIA4 Lung, kidney and colon tumours, melanoma, glioma
and haematological malignancies
TRAILR1 Mapatumumab (HGS-ETR1) Colon, lung and pancreas tumours and
haematological malignancies
TRAILR2 HGS-ETR2 and CS -1008
RANKL Denosumab Prostate cancer and bone metastases
Stromal and FAP Sibrotuzumab and F19 Colon, breast, lung, pancreas, and head and neck
extracellular matrix tumours
antigens
Tenascin 81C6 Glioma, breast and prostate tumours
CAIX, carbonic anhydrase IX; CEA, carcinoembryonic antigen; EGFR, epidermal growth factor receptor; EpCAM, epithelial cell adhesion molecule; EPHA3, ephrin
receptor A3; FAP, fibroblast activation protein; gpA33, glycoprotein A33; IGF1R, insulin-like growth factor 1 receptor; Le y, Lewis Y antigen; mAbs, monoclonal
antibodies; PSMA, prostate-specific membrane antigen; RANKL, receptor activator of nuclear factor -κB ligand; TAG-72, tumour-associated glycoprotein 72;
TRAILR, tumour necrosis factor-related apoptosis-inducing ligand receptor; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor.

Scott, Nature Rev. Cancer, 12 (2012), 278

Table 2 | Tumour-associated antigens targeted by therapeutic monoclonal antibodies in oncology
Antigen category Examples of antigens Examples of therapeutic mAbs Tumour types expressing antigen
raised against these targets
Haematopoietic CD20 Rituximab Non-Hodgkin’s lymphoma
differentiation
antigens Ibritumomab tiuxetan and tositumomab Lymphoma
CD30 Brentuximab vedotin Hodgkin’s lymphoma
CD33 Gemtuzumab ozogamicin Acute myelogenous leukaemia
CD52 Alemtuzumab Chronic lymphocytic leukaemia
Glycoproteins EpCAM IGN101 and adecatumumab Epithelial tumours (breast, colon and lung)
expressed by solid
tumours CEA Labetuzumab Breast, colon and lung tumours
gpA33 huA33 Colorectal carcinoma
Mucins Pemtumomab and oregovomab Breast, colon, lung and ovarian tumours
TAG-72 CC49 (minretumomab) Breast, colon and lung tumours
CAIX cG250 Renal cell carcinoma
PSMA J591 Prostate carcinoma
Folate-binding protein MOv18 and MORAb-003 (farletuzumab) Ovarian tumours
Glycolipids Gangliosides (such as 3F8, ch14.18 and KW-2871 Neuroectodermal tumours and some epithelial
GD2, GD3 and GM2) tumours
Carbohydrates Ley hu3S193 and IgN311 Breast, colon, lung and prostate tumours
Scott, Nature Rev. Cancer, 12 (2012), 278
blocks binding site receptor can still receptor cannot
of EGF ligand heterodimerize heterodimerize
 A short primer on the immune system

Innate immune system Adaptive immune system

Response is non-specific Pathogen and antigen specific response

Exposure leads to immediate maximal Lag time between exposure and maximal
response response

Cell-mediated and humoral components Cell-mediated and humoral components

No immunological memory Exposure leads to immunological memory

Found in nearly all forms of life Found only in certain vertebrates

Cellular and Humoral Immunity
Innate and Adaptive Immunity

Abbas & Litchman, Basic Immunology

Phases of Adaptive Immune Response
 Allergen

Airway lumen

Regional lymph nodes (or local mucosa)

Source
of early IL-4
Epithelial
cell MHC class II
Dendritic cell molecule
Migration of
T-cell receptor
dendritic cells
Allergen uptake
and processing Allergen-non- T H 2 cell
by dendritic cells specific IgE
CD28
Naive CD40 ligand
Mast cell Notch IL-4
T cell
Jagged Allergen- IL-13 CD40
derived CD80 or
peptide CD86
FcεRI
Allergen-specific IgE
IgE production
B cell

Airway smooth muscle

Galli et al., Nature 454 (2008),445

T-cell mediated immune response

• T-cell triggered immune reaction

involves formation of antigen, T-cell
receptor and MHC (major
histocompatibility complex)
• it presents fragments from proteins
and peptides processed by the cell
(no need to be surface exposed)
• The APC-T-cell complex then triggers
the immune response (production of
cytokines, cell-lysing factors etc.)
 Types of Antibodies
Name Types Description

Found in mucosal areas, such as the gut, respiratory tract

IgA 2 and urogenital tract, and prevents colonization by
pathogens. Also found in saliva, tears, and breast milk.

Functions mainly as an antigen receptor on B cells that have

IgD 1 not been exposed to antigens. It has been shown to activate
basophils and mast cells to produce antimicrobial factors.

Binds to allergens and triggers histamine release from mast

IgE 1 cells and basophils, and is involved in allergy. Also protects
against parasitic worms.

In its four forms, provides the majority of antibody-based

immunity against invading pathogens. The only antibody
IgG 4
capable of crossing the placenta to give passive immunity to
the fetus.
Expressed on the surface of B cells (monomer) and in a
secreted form (pentamer) with very high avidity. Eliminates
IgM 1
pathogens in the early stages of B cell-mediated (humoral)
immunity before there is sufficient IgG.
source: Wikipedia

Some daughter cells of the activated B cells undergo isotype switching, a mechanism that causes
the production of antibodies to change from IgM or IgD to the other antibody isotypes, IgE, IgA, or
IgG, that have defined roles in the immune system
 Antibody/T-cell Response
• After entry antigens are engulfed (mostly by dendritic cells), and proteins
degraded internally. Fragments are presented on the surface in association with
the major histocompatibility complex (MHC). Non-self proteins or capsular
polysaccharides activate B-cells via the B-cell receptor (BCR)
• Initial antibodies are predominantly low-affinity immunoglobulin-M (IgM) abs.
• Re-exposure triggers a more rapid immune response. Antibodies are predominantly
IgG or IgA class. They have in general much higher affinity.
• Likewise there is a 10’000-fold higher number of cytotoxic T-cells
• The receptor on B-cells is membrane bound immunoglobulin (BCR), that can
recognise entire proteins. In contrast the T-cell receptors (TCRs) recognise only small
peptides. B- and C-cells recognise different epitopes.
• Within the naive (unchallenged) B- and T-cell population cells capable of
recognising all epitopes are present.
• Upon challenge by an antigen only the corresponding B-cells that can recognise
the antigen are rapidly multiplied (clonal expansion). Likewise T-cells undergo
similar activation
• Antibody binding affinity is increased (affinity maturation) . During this process many
mutations in the hypervariable loops occur (somatic hypermutation). During this
process antibody class switching may occur.
 Cancer Immunotherapy:
Blockade of Immune Checkpoints
• Tumors result in many mutations in gene products, that can be recognised as
non-self and trigger a immune response for their clearance
• Immune checkpoints refer to inhibitory pathways of the immune system that
are crucial for maintaining self-tolerance and modulating the duration and
amplitude of physiological immune responses in peripheral tissues in order to
minimize collateral tissue damage.
• Tumors misuse immune-checkpoint to evade the immune system clearance, in
particular to avoid tumor-antigen specific T-cell responses
• Immune checkpoints are often initiated by ligand-receptor interactions, and
these can be blocked by antibodies, or modulated by recombinant forms of
the ligands or receptors.
• These antibodies do not target the tumor cell, but target molecules involved in
regulation of T cells, the soldiers of the immune system.
• The goal of the immune checkpoint therapy is not to activate the immune
system to attack particular targets on tumor cells, but rather to remove
inhibitory pathways that block effective antitumor T cell responses.
 Blockade of Immune Checkpoints (II)

• An important immune-checkpoint is cytotoxic T-lymphocyte associated

antigen 4(CTLA4), that down-regulates T-cell activation.
• The programmed cell-death protein 1 (PD1) limits T-cell effector function in
tissues. By up regulating ligands for PD1, tumor cells block anti-tumor
immune response in the tumor microenvironment.
• Immunotherapy- induced tumour destruction, in contrast, is often delayed
or even preceded by a period of apparent tumour growth.
• Only a fraction of patient responds to the blockade of immune checkpoints
• In these cases the tumor microenvironment may be not immunogenic.
Combination therapy can then still create an immunogenic
microenvironment that responds to immune checkpoint therapy.
• many of these therapies are limited by toxicity…
CTLA-4 PD1

Kyi, FEBS Lett. 588 (2014) 368–376

 CTLA4

The cytotoxic T-lymphocyte-associated antigen 4 (CTLA4)-mediated immune checkpoint

is induced in T cells at the time of their initial response to antigen. The level of CTLA4
induction depends on the amplitude of the initial T cell receptor (TCR)-mediated
signalling. High-affinity ligands induce higher levels of CTLA4, which dampens the
amplitude of the initial response. Naive and memory T cells express high levels of cell
surface CD28 but do not express CTLA4 on their surface. Instead, CTLA4 is sequestered in
intracellular vesicles. After the TCR is triggered by antigen encounter, CTLA4 is transported
to the cell surface. The stronger the stimulation through the TCR (and CD28), the greater
the amount of CTLA4 that is deposited on the T cell surface. Therefore, CTLA4 functions as
a signal dampener to maintain a consistent level of T cell activation in the face of widely
varying concentrations and affinities of ligand for the TCR.
Pardoll, Nature Rev. Cancer 12 (2012), 252
 PD1

The major role of the programmed cell death protein 1 (PD1) pathway is not at the
initial T cell activation stage but rather to regulate inflammatory responses in tissues by
effector T cells recognizing antigen in peripheral tissues. Activated T cells upregulate
PD1 and continue to express it in tissues. Inflammatory signals in the tissues induce the
expression of PD1 ligands, which downregulate the activity of T cells and thus limit
collateral tissue damage in response to a microorganism infection in that tissue. The
best characterized signal for PD1 ligand 1 (PDL1; also known as B7-H1) induction is
interferon-γ (IFNγ), which is predominantly produced by T helper 1 (TH1) cells.

Pardoll, Nature Rev. Cancer 12 (2012), 252

 Cancer Immunotherapy II:
Reengineering T-cells: CAR T Cells

• Doctors collect a patient's T cells

• they place a protein on the outside
of the (cytotoxic) cells
• the protein is either a T-cell receptor
or a chimeric antigen receptor
(CAR)
• the engineered T cells are then
injected back into the patient.
• the added protein has two roles: it
guides the T cell directly to the
tumour, and on arrival, it triggers
the T cell's fighting power to attack
the cancer cells.
 NCI information
• T cells interact via their T-cell receptors with major histocompatibility
complex (MHC) proteins, that present tumour antigens on the surface
of the tumour cells
• T cells can be reengineered to present antibody-like molecules (CAR
cells) on their surface. The antibodies are often single-chain ABs
directed against the tumour cell antigens.
 74
BioProcess International, Nov 2017
 at rimiducid — the same small About two-thirds of tumour antigens exist inside the cell, beyondmolecule
the reachitofused
CARto boostThese
T cells. proliferation in
on the major histocompatibility complex (MHC) (see FIG. 1). To harness this process
antigens can be made immunogenic by tapping the T cell receptor BPX-601 — their
(TCR), via can drive caspase 9 signalling
presentation
ed to boost proliferation in therapeutically, a patient’s T cells are extracted and transduced to express a TCR that recognizes
and CAR
on the major histocompatibility complex (MHC) (see FIG. 1). To harness thisT cell
processapoptosis. The company
n drive caspase 9 signalling a tumour-specific antigen in the context of MHC, before being re-infused into the patient.
trialled
therapeutically, a patient’s T cells are extracted and transduced to express this so-called
a TCR caspacide technology
that recognizes
l apoptosis. The company Such TCR-edited therapies have the potential to target neoantigens — novel peptides that
a tumour-specific antigen in the context of MHC, before being in an adjunctive
re-infused into theT cell therapy in
patient.
-called caspacide technology arise from cancer-associated somatic mutations and that are unique to each patient’s tumour
Such TCR-edited therapies have the potential to target neoantigens — novel peptides
partial-match stem cellthat
transplantation and
ve T cell therapy in (Nat. Immunol. 18, 255–262; 2017).
arise from cancer-associated somatic mutations and that are unique showedto each
thatpatient’s
it couldtumour
induce the resolution of
stem cell transplantation and Several trials of TCR-engineered cells in solid tumours are ongoing at the US National Cancer
(Nat. Immunol. 18, 255–262; 2017). graft-versus-host disease and improve
could induce the resolution of Institute, including one that targets the cancer germline antigen NY-ESO-1 in sarcoma, breast
Several trials of TCR-engineered cells in solid tumours are ongoing at the US
outcomes Nationalwith
compared Cancer
historical controls.
ost disease and improve cancer, non-small-cell lung cancer and liver cancer.
Institute, including one that targets the cancer germline antigen NY-ESO-1 in sarcoma, breast
mpared with historical controls. Clinical trials of caspacide-controllable CAR Industry, too, is keeping its options open, with CAR T-specialist biotech companies including
cancer, non-small-cell lung cancer and liver cancer.
of caspacide-controllable CAR T cells are due to start this year. Kite Pharma, Juno Therapeutics and Bellicum Pharmaceuticals also running TCR programmes.
Industry, too, is keeping its options open, with CAR T-specialist biotech companies including
Safeguards “We take an agnostic approach. I don’t think there’s a clear winner and I don’t know if there ever
to start this year. Kite Pharma, Juno Therapeutics and Bellicum Pharmaceuticals also running TCR of some sort are required to
programmes.
accelerate progress
“We take an agnostic approach. I don’t think there’s a clear winner and I don’t know if there in theever
field, summarizes will be,” says Aaron Foster, Vice-President of Product Discovery at Bellicum. “The clinical data
of some sort are required to
will be,” says Aaron Foster, Vice-President of Product Discovery atFoster. “As you
Bellicum. “Thestart to push
clinical data the envelope in for TCRs in solid tumours has been a little better than what we’ve seen so far with CARs,” he
gress in the field, summarizes
for TCRs in solid tumours has been a little better than what we’veterms of far
seen so thewith
potency
CARs,” ofhe
your T cell product, adds.
u start to push the envelope in
adds. having a suicide gene is very important for TCR therapy specialist Adaptimmune Therapeutics is collaborating with GlaxoSmithKline on a
otency of your T cell product,
TCR therapy specialist Adaptimmune Therapeutics is collaborating with GlaxoSmithKline
exploring on a
those new technologies.” battery of clinical-stage programmes to test its NY-ESO-1-targeted TCR product in solid tumours
de gene is very important for including soft-tissue sarcoma and melanoma.
e new technologies.” battery of clinical-stage programmes to test its NY-ESO-1-targeted TCR product in solid tumours
including soft-tissue sarcoma and melanoma. But TCR-engineered therapies must be matched to the patient’s tissue type, as defined by their
Labour intensive
But TCR-engineered therapies must be matched to the patient’s tissue type, as defined MHC molecules. In addition, many tumour types can downregulate the MHC as part of their
sive CAR T cell therapies arebyalso
their
hard to
MHC molecules. In addition, many tumour types can downregulate the MHC as part of their immune evasion mechanism. “It’s attractive at first blush in terms of the relative number of
rapies are also hard to manufacture, presenting another challenge antigens that can be recognized, but there are some challenges,” says Bruce Levine, a professor
immune evasion mechanism. “It’s attractive at first blush in terms of the relative number of
presenting another challenge for resource- and time-strapped companies in cancer gene therapy at the University of Pennsylvania, Philadelphia.
antigens that can be recognized, but there are some challenges,” says Bruce Levine, a professor
and time-strapped companies and patients (turnaround times from T cell
in cancer gene therapy at the University of Pennsylvania, Philadelphia.
urnaround times from T cell

NATURE REVIEWS | DRUG DISCOVERY VOLUME 16 | MAY 2017 | 30

WS | DRUG DISCOVERY VOLUME 16 | MAY 2017 | 303
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 Antibody-Conjugates: Radionuclei
Representative monoclonal antibodies in advanced radioimmunotherapy clinical trials
Antibody Antibody Radionuclide Antigen Disease Clinical trial status
form
Tositumomab muIgG2a 131
I CD20 NHL Approved by FDA
(Bexxar) Monoclonal
Epratuzumab huIgG1 (LL2) 90
Y CD22 NHL Phase III antibody
(Lymphocide) conjugate
Labetuzumab huIgG1 90
Y CEA Colorectal, breast, lung, Pending Phase III
(CEA-Cide) pancreatic, stomach carcinoma
SCN CH3 SCN
chTNT-1/B chIgG1 131
I DNA Glioblastoma multiforme, Phase III N N
(Cotara) anaplastic astrocytoma
N CO2H N N CO2H N
I Lym-1
131
huIgG1 131
I HLA-DR10 NHL, CLL Phase II/III
(Oncolym) HO2C CO2H CO2H CO2H HO2C CO2H CO2H CO2H
Pemtumomab muIgG1 90
Y PEM Ovarian, gastric carcinoma Phase III
1B4M-DTPA CHX-A" DTPA
(Theragyn) MX-DTPA
Ibritumomab muIgG1 90
Y CD20 NHL Approved by FDA; Tiuxetan
tiuxetan (Zevalin) Phase IV
CEA, carcinoembryonic antigen; CLL, chronic lymphocytic leukaemia; huIg, human immunoglobulin; muIg, murine immunoglobulin; NCS
NHL, non-Hodgkin’s lymphoma; PEM, polymorphic epithelial mucin. HO2C CO2H
N N
HO2C CO2H
Therapeutic radionuclides N N
N N NCS
Radionuclide Type Half-life Emax (MeV) Mean range Imageable HO2C
N N
(mm) CO2H HO2C CO2H
Y
90
β 2.7 d 2.3 2.76 No
c-DOTA PA-DOTA
131
I β, γ 8.0 d 0.81 0.40 Yes
177
Lu β, γ 6.7 d 0.50 0.28 Yes
153
Sm β, γ 2.0 d 0.80 0.53 Yes
186
Re β, γ 3.8 d 1.1 0.92 Yes
188
Re β, γ 17.0 h 2.1 2.43 Yes
Cu
67
β, γ 2.6 d 0.57 0.6 Yes
225
Ac α, β 10 d 5.83 0.04–0.1 Yes
213
Bi α 45.7 min 5.87 0.04–0.1 Yes
212
Bi α 1.0 h 6.09 0.04–0.1 Yes α-particle
211
At α 7.2 h 5.87 0.04–0.1 Yes 50–80 μm range
5–8 MeV
212
Pb β 10.6 h 0.57 0.6 Yes
125
I Auger 60.1 d 0.35 0.001–0.02 Yes
123
I Auger 13.2 h 0.16 0.001–0.02 No
Ga
67
Auger, β, γ 3.3 d 0.18 0.001–0.02 Yes β–-particle
195m
Pt Auger 4.0 d 0.13 0.001–0.02 No 1–10 mm range
0.1–2.2 MeV

Milenic et al. Nature Rev Drug Discov. 3 (2004),488 Figure 3 | Comparison of path lengths and emission tracks of α- and β–-particle emissions used in antibody-targeted
Schrag D. N Engl J Med 2004;351:317-319.