Principles of “Targeted Therapy”

• Thanks to recent acquisitions in the field of molecular biology it has been possible to identify new biological targets expressed by tumor cells

• Using drugs directed against these targets it is possible to hit, in a “targeted way” these cells

• One of the most studied target is the Epidermal Growth Factor Receptor (EGFR)

TARGETED-THERAPY

• Signal transduction inhibitors

• Proteasomes inhibitors (Multiple Myeloma)

• Cox-2 inhibitors (Colon Poliposis)

• Vaccines, immunotherapy and gene therapy

TARGETED THERAPY IN CANCER

Nat Rev Cancer. 2009 Jan;9(1):28-39.

Targeting cancer with small molecule kinase inhibitors.

Zhang J, Yang PL, Gray NS.

Deregulation of kinase activity has emerged as a major mechanism by which cancer cells evade normal physiological constraints on growth and survival. To date, 11 kinase inhibitors have received US Food and Drug Administration approval as cancer treatments, and there are considerable efforts to develop selective small molecule inhibitors for a host of other kinases that are implicated in cancer and other diseases. Herein we discuss the current challenges in the field, such as designing selective inhibitors and developing strategies to overcome resistance mutations. This Review provides a broad overview of some of the approaches currently used to discover and characterize new kinase inhibitors.

FURTHER READING

MetastasisProliferation

Cell-cycle progression

pY

pYK K

pY

M

G1S

G2

Growth factor

SIGNAL TRANSDUCTION INHIBITORS

• EGFR Inhibitors– Gefitinib (Iressa)– Erlotinib (Tarceva)

• EGFR Monoclonal antibodies– Cetuximab (Erbitux)

• VEGF Monoclonal antibodies– Bevacizumab (Avastin)

TARGETED THERAPIES

EGF VEGFBevacizumab

GefitinibErlonitib

AZD(TK inhibitor)

Cetuximab

myc cyclin D1

Jun Fos

RRRR

RR

K

KK

Gene transcriptionGene transcription

RR

K

EGFR SIGNALING

myc cyclin D1

Jun Fos

RRRR

KRR

K

RR

K

MAPKMAPK

MEKMEK

RASRAS RAFRAF

SOSSOS

GRB2GRB2PI3-KPI3-K

AKTAKT

Proliferation

Inhibition of apoptosis

KPPPP

PPPP

PPPP

MetastasisAngiogenesis

GEFITINIB

Gene transcriptionGene transcription

EGFR SIGNALING

Iressa received accelerated FDA approval in May 2003 as a 3rd line therapy in NSCLC

based on the IDEAL-2 study.

GEFITINIB (IRESSA®)

0

10

20

30

40

5060

70

80

90

100

Pazienti

Perce

ntuale

di ri

spos

ta

Totale

Donne conadenocarcinomanon fumatrici

0

10

20

30

40

50

60

70

80

90

100

Pazienti

Perce

ntuale

di ri

spos

ta

Uomini

Donne

Adenocarcinoma

Non fumatori

IRESSA®: results in different subgroups of patients with NSLC

pre-treatment post-treatment

RESULTS (I)

pre-treatment post-treatment

RESULTS (II)

• The efficacy of GEFITINIB is limited to a subset of patients (nonsmoker women affected by adenocarcinoma)

• The overall response rate is 7%• In nonsmoker women with adenocarcinoma the response

rate reaches 50%• Gefitinib toxicity is lower with respect to that observed

with conventional chemotherapy

CONCLUSIONS

myc cyclin D1

Jun Fos

RRRR

KRR

K

RR

K

MAPKMAPK

MEKMEK

RASRAS RAFRAF

SOSSOS

GRB2GRB2PI3-KPI3-K

AKTAKT

Proliferation

Inhibition of apoptosis

KPPPP

PPPP

PPPP

MetastasisAngiogenesis

ERLOTINIB

Gene transcriptionGene transcription

EGFR SIGNALING

Tarceva

(n=488)

Placebo

(n=243)

Resp. Rate 8.9% <1%

Median survival

6.7 mo 4.7 mo

PFS* 2.2 mo 1.8 mo

Shepherd FA et al. NEJM 2005

Most common AE: rash, diarrhea

*sopravvivenza libera da progressione

ERLOTINIB (TARCEVA®) IN NSLC

Tarceva received FDA approval in 2004 as a 2nd or 3rd line therapy in NSCLC based on Shepherd’s trial.

* Progression Free Survaival

• TALENT trial - Cisplatin/gemcitabine + (tarceva or placebo) - Phase III– 1172 pts., previously untreated

Tarceva Placebo

Med. survival (days)

301 309

TTP* (days) 167 179

Gatzemeier U. ASCO 2004 Abstract

*intervallo per ripresa di progressione tumorale

* Time To Progression

ERLOTINIB (TARCEVA®) IN NSLC

Group RR (%) p

Women v men 19 v 3 0.001

Japanese v

non-Japanese27.5 v 10.4 0.0023

Adenocarcinoma v others

13 v 4 0.046

BAC* v adeno 38 v 14 <0.001

Never smoker v current/former

36 v 8 <0.001

Fukuoka JCO 2003;21:2237-46. Kris JAMA 2003;290:2149-58. Miller JCO 2004;22:1103-09.* Bronchioloalveolar carcinoma

PREDICTORS OF RESPONSE TO TK INHIBITORS IN NSCLC

Pao et al. JCO 2005; 23:2556-2568.

Some patients have significant initial clinical response to TK Inhibitors, but then develop progressive disease– Secondary mutation found (4/7 pts.)

• Substitution of methionine for threonine at position 790

• Get steric hindrance of TK Inhibitor binding

Pao et al. 2005; PLoS Med 2:1-11.Kobayashi et al. 2005; NEJM 352:786-92.

ACQUIRED RESISTANCE TO TK INHIBITORS

OTHER MUTATIONS

Pao et al. JCO 2005; 23:2556-2568.

Mutation

• Mutations in K-ras– Occur in 15-30% of lung adenocarcinomas– High incidence in heavy smokers

• Predict a poor response to TK inhibitors

• EGFR and K-ras mutations are mutually exclusive

Ahrendt SA et al. Cancer 2001; 92:1525-30.Kosaka T et al. Cancer Res 2004; 64: 8919-23.Eberhard DA et al. JCO 2005;23:5900-09.

MUTATIONS IN K-ras

• Many centers have started testing for the most common mutations in EGFR

• Problem: • What to do if negative for mutations?

– Some pts. with responses to Gefitinib/Erlotinib have no detectable EGFR mutation

MUTATIONAL PROFILING

ANTI EGFR MONOCLONAL ANTIBODIES

Pao et al. JCO 2005; 23:2556-2568.

mAb (CETUXIMAB)

RR (%)

(CI)

Median survival

(mo.)

Median TTP* (mo.)

Cisplatin/Vinor.20

(7.6-32.4)7.0 4.2

Cisplatin/Vinor.

+Erbitux

32

(17.5-46.0)8.3 4.7

Rosell 2004 ASCO Abstract

CETUXIMAB WITH CHEMO: RESULTS

RR: response rateTTP: time to progression

Survivalin circulationIntravasation

Invasion

initiationof growth

Sustainedgrowth

Primarytumour

Clinicallyevident metastasis

Adapted from Chambers A, Matrisian L. J Natl Cancer Inst 1997;89:1260–70

ANGIOGENESIS:MULTIPLE STEPS IN CANCER BIOLOGY

Sustained Growth requires blood vessels

Primarytumor

Metastasis

VEGF

EGF

Multiple steps in cancer biology Heading towards Personalized Care

ANGIOGENESIS

ANGIOGENESIS IN TUMORS

Endostatin is a natural antiangiogenic protein that inhibits the growth of blood vessels

Vaccines targeting tumor angiogenesis—a novel strategy for cancer immunotherapy Y. Okaji et al, 2006

ANGIOGENESIS INHIBITION

Nature Reviews Drug Discovery 4, 448-449 (2005)

Anti-angiogenesi (II)Anti-angiogenesi (II)

Bevacizumab (Avastin, Genetech): Vascular Endothelial Growth factor (VEGF) inhibitor.Phase III Trial in NSCLC:- Survival of 12.5 months in Avastatina/CHT arm vs. 10.5 months in conventional CHT arm - 2 years survival: 22% vs. 17%- Adverse events: 5% major bleedings, 1% mortality

Avastin + ChemoAvastin + Chemo

Carbo/Taxol

(n=32)

Carbo/Taxol + Avastin

(n=35)

Median survival

(mo.)14.9 17.7 (NS)

RR (%) 18.8 31.5

TTP* (mo.) 4.2 7.4Johnson et al., JCO 2004.Johnson et al., JCO 2004.(19/32 pts. crossed over)

Avastin in combination with Tarceva appears to have a synergistic effect*intervallo per ripresa di progressione tumorale

Animal Model to study angiogenesis

Growth Factor Reduced BD Matrigel Matrix

Storage -30°C

Melting +4°C

Solidification +37°C

Matrigel is an extracellular matrix produced by murine sarcoma Engelbreth-Holm-Swarm (EHS), consisting predominantly of laminin (60%), collagen IV (30%),heparan sulphate (5%), proteoglycans (3 %) and entactin (1%).

• Matrigel subcutaneous injection (the matrix can be mixed with different molecules to be studied).

• After the injection, thanks to body temperature, the matrix solidifies and leads to the formation of “plugs”. With appropriate stimuli the angiogenic process starts and develops into the plugs.

Model for in vivo study of angiogenesis using Matrigel Matrix

MATRIGEL PLUGS

Reperimento dei plugs dopo 7 giorni dall’inoculo

sezione addominale lungo la linea mediana

isolamento masserelle dai tessuti

conservazione del materiale in apposite biocassette

Allestimento sezioni istologiche fissazione in formalina

inclusione in paraffina

taglio con microtomo

allestimento vetrino

colorazione

RESULTS EVALUATION

NEGATIVE CONTROLS

MATRIGEL MATRIGEL + Gas 6