1. New Cancer Therapies Based on Thyrosine Kinase Inhibitors
Axel Ullrich
Max-Planck-Institute of Biochemistry
Martinsried, Germany
November 7, 2008

3. Targeted Cancer Therapy „The Dream of the Magic Bullet“
Side Effect-Free Cure of Cancer

4. Paul Ehrlich 1854 - 1915 Father of Chemotherapy
Salvarsan for Treatment
of Syphilis
Nobel Prize 1908
“Magic Bullet Concept”
Ehrlich in his office

5. Signalling Pathways

6. Human Epidermal Growth Factor Receptor (EGFR)
Size: Da
Length: 1186 aa MW
mRNA: 5.8 / 10.5 kb
Downward J, Yarden Y, Mayes E, Scrace G, Totty N, Stockwell P, Ullrich A, Schlessinger J, and Waterfield MD (1984). Close similarity of epidermal growth factor receptor and v-erb-B oncogene protein sequences. Nature
Ullrich A, Coussens L, Hayflick JS, Dull TJ, Gray A, Tam AW, Lee J, Yarden Y, Libermann TA, Schlessinger J, Downward J, Bye J, Whittle N, Waterfield MD, and Seeburg PH (1984). Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells. Nature

7. EGFR / v-erbB EGF-R v-erbB-H v-erbB-ES4 F/S699 F/S699 T/K718 I/V705
H/R811
Q/L840
S/I932
Δ1034 Δ
Y/N1091

8. RTK-derived Oncogene Products
neu
v-fms
M/V 11
R/Q 91
S/L 301
S/A 374
Y/H 461
P/Q 505
T/A 587
P/L 680
G/D 711
v-erbB-H v-erbB-ES v-kit
F/S699
T/K718
H/R811
Q/L840
S/I932
Δ1034 Δ
Y/N1091
F/S699
I/V705
Δ AY
G/D
Δ 34 aa Δ 71 aa Δ 40 aa Δ 40aa

9. Human EGF Receptor-Related Receptor HER2 / neu / c-erbB2
Size: Da
Length: 1234 aa MW
mRNA: 4.8 kb
King CR, Kraus MH, and Aaronson SA. (1985) Amplification of a Novel v-erbB-related Gene in a Human Mammary Carcinoma. Science 229,
Coussens L, Yang-Feng TL, Liao YC, Chen E, Gray A, McGrath J, Seeburg PH, Libermann TA, Schlessinger J, Francke U, Levinson A, and Ullrich A. (1985) Tyrosine kinase receptor with extensive homology to EGF receptor shares chromosomal localization with neu oncogene. Science 230,
Schechter AL, Hung MC, Vaidyanathan L, Weinberg RA, Yang-Feng TL, Francke U, Ullrich A, and Coussens L (1985). The neu gene: An erbB-homologous gene distinct from and unlinked to the gene encoding the EGF receptor. Science

10. “The Herceptin Story” Oncogene Homology-Based Target Discovery
Genentech:
R. Hudziak
M. Shepard
B. Fendley
L.Coussens
P. Carter
Herceptin Development Team
Clinical Collaborator:
D. Slamon, UCLA

12. HERCEPTIN History Cloning of EGFR cDNA Relation to V-erbB 1984
Slamon et al.
Science
HER2 gene
amplification
in breast cancer
and correlation
with disease
progression
1987
1985
HER2
Sequence published
Coussens et al.
1989
Hudziak et al.
MCB
Anti-tumor effect
of MAb 4D5 and 2C4
Phase I
Rhu MAb
1992
1993
Phase II
Phase III
1995
Approval
In Europe
2000
1998
FDA
2002
MAb 2C4 In
Development

15. HERCEPTIN Efficacy of Monotherapy in HER2/neu +++ Patients Low (15%)
Reminder: Cancer is NOT a Monogenic Disease
Moving Disease Target due to Genetic Plasticity of Tumor Cells
Combination Therapy
HERCEPTIN +
- Anthracyclines
- Taxotere
- Platinum Salts
- other MABs
- etc

16. The EGFR Family Signalling Network Transcription factors
TGFa (1)
EGF (1)
Epiregulin (1,4)
Beta- cellulin (1)
HB-EGF (1,4)
Amphi- regulin (1)
NRG1 (3,4)
NRG2 (4)
NRG3 (4)
NRG4 (4)
LPA Thrombin ET, etc.
Ligands
Cytokines a b a b
Input layer 2 2 4 2 1
Receptor dimers 1 1 4 3 2 4 4 1 2 3 4 1 3 3 3
SRC
JAK
CBL
SHC
PLC
p21-GDP
CRK
P13K
VAV
GRB7
SHP2
GAP
GRB2
NCK
Adaptors & enzymes
SOS
p21-GTP
RAC
Hidden layers
AKT
RAF
PAK
Cascades
MEK
JNKK
ABL
PKC
BAD
S6K
MAPK
JNK
Transcription factors
JUN
SP1
MYC
FOS
ELK
EGR1
STAT
Output layer
Growth
Migration
Apoptosis
Adhesion
Differentiation
Yarden and Sliwkowski (2001) Nature Rev. Mol. Cell Biol, 2,

17. Mechanism-Based Target Identification HER3
Wallasch et al. and Ullrich EMBO J HRG-dependent regulation of HER2/neu oncogenic signaling by heterodimerization with HER3
Holbro et al. and Hynes PNAS 2003 The ErbB2/ErbB3 heterodimer functions as an oncogenic unit: ErbB2 requires ErbB3 to drive breast tumor cell proliferation
Htun van der Horst et al. and Ullrich IJC 2005 Anti-HER3 MAbs inhibit HER3-mediated signaling in breast cancer cell lines resistant to anti-HER2 antibodies

18. HER3 is a Determinant for Poor Prognosis in Melanoma and a Target for Therapeutic Intervention
Markus Reschke
Daniela Mihic-Probst
Edward Htun van der Horst
Pjotr Knyazev
Peter J. Wild
Markus Hutterer
Stefanie Meyer
Reinhard Dummer
Holger Moch
Martin Treder (U3 Pharma)
AMGEN

19. HER3 protein expression in primary melanoma and metastases

20. HER3 Expression Confers Poor Prognosis for Melanoma Patients
Kaplan-Meier Curve
Multivariate Analysis
HER3 p=0.044
(Hazard ratio: 2,636)
Metastases p=0.000
(Hazard ratio: 22,251)
Other variables
sex
age
tumor thickness (p=0.065)
HER3 low
HER3 high
Cumulative Survival
p = 0.014
Overall Survival

21. Treatment with Human Anti-HER3 mAb Significantly Reduces pHER3 Levels in BxPC3 Tumor Xenografts
IgG1
(25 mpk per mouse)
Anti-HER3 mAb
pHER3
-Actin
U3Pharma/AMGEN

22. Treatment with Human Anti-HER3 mAbs Results in Inhibition of BxPC-3 Pancreatic Carcinoma Xenografts
100
200
300
400
500
600
700
800
900 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44
hIgG1 , 25 mg/kg 2X/wk
Anti-HER3 mAb C , 25 mg/kg 2X/wk
Anti-HER3 mAb A, 25 mg/kg 2X/wk
Anti-HER3 mAb B, 25 mg/kg 2X/wk
Gemcitabine, 80mg/kg weekly
p< vs. hIgG1 Control
Time (days)
RMANOVA Analysis
Blinded Study n=10 per group
U3Pharma/AMGEN

23. Conclusion: HER3- Specific MAbs
Have an HER2-independent Inhibitory Effect in Melanoma and Pancreatic Cancer Cell Models
May Be Valuable Therapeutic Agents Alone as Well as in Combination with Herceptin, Lapatinib and other Drugs

24. From PCR-Based Discovery of a Novel RTK to a Multi-Targeted Cancer Drug “Flk-1/VEGFR2 as Target in Anti-Angiogenesis Therapy”
MPI for Biochemistry
Munich
MPI Biochemistry, Martinsried, Germany
Birgit Millauer
Werner Risau
SUGEN
Laura Shawver
Jerry McMahon
Annie Fong
Development Team
Collaborators
Alex Levitzki, Hebrew University, Jerusalem
György Kéri, Vichem, Budapest
Pharmacia Inc.
Pfizer Inc.
SUGEN
San Francisco

25. RTK Subclasses EGFR HER2/ neu HER2 HER4 I-R IGF-1R IRR PDGFRα PDGFRβ
CSF-1R
KIT
FLK2/FLT3
VEGFR1
VEGFR2
VEGFR3
FGFR-1
FGFR-2
FGFR-3
FGFR-4
CCK4
TRKA
TRKB
TRKC
MET
RON
EPHA1-
EPHA8
EPHB1-
EPHB7
AXL
MER
TYRO3
TIE
TEK
RYK
DDR-1
DDR-2
RET
ROS
LTK
ALK
ROR1
ROR2
MUSK
MDK4
AATYK
AATYK2
AATYK3

26. Terman et al.
Biochem Biophys Res Commun 1992
Quinn et al.
Proc Natl Acad Sci USA 1993
Millauer et al.
Cell 1993

28. Flk-1/VEGFR2: Validation as Anti-Angiogenesis Drug Development Target
Receptor for VEGF
Specifically Expressed in Endothelial Cells
Essential for Tumor Angiogenesis
DEVELOPMENT OF SELECTIVE FLK-1 KINASE
SMALL MOLECULE INHIBITORS

29. Chemical Structure of SU5416 - An Inhibitor of VEGF Receptor Kinase
SU [2, 4-dimethylpyrrol-5-yl methylidenyl]-2-indolinone

30. SU5 416 Response in AIDS Kaposi’s Sarcoma
Protocol , Patient #003, R-P
Before treatment with SU After treatment with SU5416

31. Patient 018/JZH: SU5416 Response Facial Lesions

32. SU5416 Highly Selective Efficacious in Mouse Models Anti - Metastatic
Sub – Optimal Pharmacological Properties
Efficacious in Phase I Kaposi-Sarcoma Trial
Colon Cancer Trial Terminated

33. Biochemical Effects of SU6668 a VEGFR-2, PDGFR, & FGFR RTK Inhibitor
Flk-1
PDGFRb
FGFR-1
EGFR Ki
Km (ATP) Ki
Km (ATP) Ki
Km (ATP)
IC50
SU5416
SU6668
0.16
2.1
0.43
0.32
0.008
6.2
19.5
1.2
2.6
>100
As is shown in this table, SU6668 is very potent against PDGF receptor tyrosine kinase.
SU6668 is more potent at inhibiting FGFR than SU5416 with only a slight loss in activity against Flk-1/KDR.
Mean Ki and Km values are shown (mM)
Both compounds exhibit competitive (with respect to ATP) inhibition
Both compounds also inhibit ligand-dependent phosphorylation of c-Kit

34. SU11248 A Multitargeted Kinase Inhibitor
Receptor
Ligand
Tumor Expression
PDGFR
PDGF
Glioma, HCC, NSCLC, OvCa, CaP, melanoma, CMML*, MDS*
VEGFR
VEGF
CRC, OvCa, RCC, Melanoma, NSCLC, Sarcomas, Breast, NET
KIT
SCF
GIST*, AML*, Melanoma, SCLC, Breast, OvCa, Cervix, Mastocytoma*
FLT3
FLT3 Ligand
AML*, MDS*, T-ALL, Neurological
* Proliferation driven by mutant receptor

35. Anti-Angiogenic Activity
SU11248
Oral Multi-Targeted Receptor Tyrosine Kinase Inhibitor with Anti-Tumor and
Anti-Angiogenic Activity

36. From Mono- to Multi-Targeted Kinase Inhibitors

37. Eric Raymond et al., 2002 NCI/EORTC/AACR
SU11248 Exhibits Cytoreductive Activity in Diverse Tumors of Patients in Phase I Clinical Trials
Baseline
Week 4
Patient with metastatic renal cell carcinoma
Phase I trials ongoing at multiple international sites
Patients heavily pre-treated, with progressive disease at entry
Confirmed partial responses observed
SU11248 is well tolerated
most common toxicities seen in patients are fatigue, GI, and hematologic toxicity
Eric Raymond et al., 2002 NCI/EORTC/AACR

38. Sunitinib in Metastatic RCC
Before Treatment
After 4 weeks of Sunitinib
Lung Lesion Response in RCC;
Courtesy of Dr. Ronald Bukowski, Cleveland Clinic Foundation
Sunitinib is New First-Line Therapy in Metastatic Clear Cell Renal
Cell Carcinoma (RCC)
Adverse Side Effects are Tolerable

39. FDA approves SUTENT for Treatment of Gleevec-Resistant GIST and RCC
SU 11248/SUTENT
January 26, 2006
FDA approves SUTENT for Treatment of Gleevec-Resistant GIST and RCC
July 19,2006
EMEA Approval for Europe

40. SUTENT History Flk-1 shown to be VEGF-R
(Millauer et al., Quinn et al.)
Dominant negative VEGFR-2
inhibits tumor angiogenesis and-growth in vivo
(Millauer et al.)
SU5416 inhibits tumor growth
in vivo (Fong et al.)
SU11248 orally active
multi-targeted drug
(O‘Farrell et al.)
SUTENT approval by FDA
and EMEA (Pfizer)

42. and Metastasis Formation
Receptor-Tyrosine-Kinases Drivers of Cancer Progression
Activated
Receptor
Ligand
Binding
Receptor- Tyrosine-Kinase
Cell Membrane Y P
Tyrosine
Kinase
Domain
Signal Transduction
Migration
Proliferation
Survival
Tumor Growth
and Metastasis Formation

43. Multi-Targeting = Blocking Multiple Tyrosine Kinases Simultaneously
Ligand
Binding
Activated
Receptors Y P Y P
Receptor
Tyrosine
Kinase
Sunitinib   
Proliferation
Survival
Migration
Tumor Growth
and Metastasis Formation

44. Sunitinib

45. Sunitinib  
Tumor
Angiogenesis   

46. SUTENT/Sunitinib going forward….
Comprehensive Kinase Interaction Profile
Cell Line-Characteristic Biological Responses
Identification of Therapeutically Relevant Kinase Targets (Clinical Response Prediction Signature)
Identification of Kinase Targets Responsible for Side Effects (Fatigue, Cardiotoxicity etc.)
Resistance Formation

47. Molecular Targets in Cancer
Receptor Tyrosine Kinase
RAS-GDP
RAF-1
SHC
BAD
SOS
GRB-2
14-3-3
Nucleus
BCLXL
Mitochondria
SRC P
STAT1+3
RAS-GTP
SAPK
AKT
RAS-GAP
DOK ?
MYC
PI3K
MEK1/2
MAPK
mTOR
SU11248 (Pfizer)
Zarnestra (JNJ)
CT2584 (CTI)
RAD001 (Novartis)
SKI 606(Wyeth)
BAY (Bayer)
Imatinib (Novartis)
BMS (Bristol-MS)

48. Future of Individualized Cancer Therapy
Diagnostic Data
Traditional Pathological Parameter
Tumor Gene Expression Analysis
Oncogene Mutation Profile
Tumor Markers
Germline SNP Profile

49. Future of Individualized Cancer Therapy
Multi-Targeted
Kinase Inhibitors (Gleevec, SUTENT, Nexavar….)
Traditional
Chemotherapy
(Taxol,
Anthracyclines…..)
Target Specific Monoclonal Antibodies
(Herceptin, HER3 Ab…..)
Combinatorial Treatment
Hormonal therapy
Radiotherapy

50. Acknowledgements SUTENT Response Prediction Project
Michaela Bairlein
Henrik Daub (MPI for Biochemistry)
Cooperations:
György Kéri (Vichem, Hungary)
Singapore OncoGenome Project (Singapore)
Matthias Mann (Dept. Proteomics, MPI for Biochemistry)

51. Acknowledgements Axl Project
Yixiang Zhang
Pjotr Knyazev
Yuri Cheburkin
Yuri Knyazev
Henrik Daub MPIB-DM
Kirti Sharma MPIB-DM
Vichem Chemie, Hungary
György Kéri
László Örfi
Istvan Szabadkai

52. THANK YOU FOR YOUR ATTENTION

53. Michaela Bairlein et al. 2008
Proteomic Characterization of SUTENT/Sunitinib Maleate Activity on Cancer Cell Lines
Michaela Bairlein et al. 2008

54. Target-Identification via Affinity Chromatography and Mass Spectrometry
modified SUTENT
SUTENT
provided by G. Kéri
Coupling to
ECH-Sepharose S1 S2
In-gel
Trypsin
digestion
FPLC- affinity chromatography
LC/MS
(LTQ)-Orbitrap
Coomassie Gel
cell lysate
Eluates 1-3
10 cancer cell lines of different tissue origin
(3x breast, 1x pancreas, 1x prostate, 2x glioblastoma, 3x melanoma)
- 2 affinity columns in series (0.3 & 3 mM ligand concentration)
- elution: 1. specific: 1 mM free SUTENT; 10 mM ADP; 10 mM AMP-PNP
2. SDS (0,5%, RT)
- tryptic ”in gel digestion”  LC/MS
- qualitative data analysis in MASCOT  target- comparison between columns & cell lines

55. Targets bound to 0.3mM-column:
Kinase Affinity Spectrum in Cancer Cell Lines
breast cancer cell lines  identified kinases 38 24 40
0.3mM
column
3mM
Hs578T (102 kinases in total) 36 23 56
MDA-MB-435S (115 kinases in total) 52 37
MDA-MB-231 (129 kinases in total)
Targets bound to 0.3mM-column:
e.g.: PDGFRa/b
cKit
VEGFR2
CSF-1R
AuroraA
AuroraB
Ron
Axl
Ret
Nek2
FAK
Cdc2
RSK1/2

56. Receptor Tyrosine Kinases (RTKs)
SUTENT-Responsive
Receptor Tyrosine Kinases (RTKs)
= identified RTKs (0.3mM SUTENT column)

57. Summary
SUTENT…
… is a multi-targeted kinase-inhibitor with an interactome of >100 kinases
… has an inhibitory effect on cancer cell proliferation
… induces cancer cell apoptosis
… interferes with cancer cell migration and invasiveness
… inhibits autophosphorylation of several RTKs in cellular kinase assays

58. Gas/Axl System
Hafizi S, et al.. Cytokine Growth Factor Rev. 2006, 17(4):

59. Gene Expression Analysis towards Target Identification AXL
Overexpression of the RTK Axl correlates with cancer cell line invasivity of breast cancer cell lines
Basis for the development of an anti-metastasic cancer drug

60. Identification of AXL as a Gene Characteristic for Invasive Breast Cancer
Non-invasive
normal

61. Discovery of Axl Tyrosine Kinase Inhibitors
Yixiang Zhang et al. 2008

62. Screening for Axl Tyrosine Kinase Inhibitors
Nested Chemical Library
First compound library
(216 compounds)
(18 chemical families)
Identify active chemical family
Second compound library
(99 compounds)
(7 chemical families)
Identify active compounds
Third compound library
(220 compounds)
(106 compounds belong to Quinazoline family)
Structure-activity relationship of compounds
Cooperation with György Kéri (Vichem, Hungary)

63. Active Compounds
Some compounds of quinoline, quinazoline, pyrimidines, quinoxaline and Indolin-2-ones families have inhibitory activities against auto-phosphorylation of AXL (IC50<10µM).

64. SKI-606 (Wyeth, clinical phase II)
Derivative of NA80x1 with Stronger Inhibitory Activity on Axl Phosphorylation, Migration and Invasion
NA80x1
SKI-606 (Wyeth, clinical phase II)

65. Inhibitory Effects of SKI-606 on the Phosphorylation of Receptor Tyrosine Kinase AXL
Kinase assay
IC50=0.56±0.08µM
MDA-MB-435s cells
Hs578T cells, IC50 = 0.34± 0.04µM

66. Cellular Specificity Profile of Compounds
VI17525 and VI17614
Potential targets: 32 kinases
Src family members
Abl family members
Axl
8 more tyrosine kinases (FAK, 4 Eph family members)
9 STE group of kinases (6 MAP4K/STE20 kinase family members and 2 MAP2K family members
Other kinases

67. Summary AXL:
NA80x1 and SKI-606 are Submicromolar AXL Kinase Inhibitors
They Inhibit More than 32 Other Tyr- and Ser/Thr Kinases
SKI-606 is in Phase II Clinical Trials for CML and other Cancers (Wyeth)
Starting Point for Medicinal Chemistry Towards a Tyrosine Kinase Inhibitor with an Even Better Target Inhibition Profile

68. Genentech Inc.
CMM Singapore
A*STAR
MPI for Biochemistry
Munich
Biopolis

69. NA80x1 A 3-quinolinecarbonitrile Compound
with Strong Inhibitory Activity on Axl Phosphorylation,
Cell Migration and Invasion

70. Inhibitory Effects of NA80x1 on the Phosphorylation of Receptor Tyrosine Kinase AXL
Kinase assay
IC50= 12.67± 0.45µM
MDA-MB-435s cells
Hs578T cells, IC50=4,11± 1,47µM

71. Axl is a Promising Target for Invasive Breast Cancer Therapy
AXL is expressed in highly invasive breast cancer cells, but not in breast cancer cells of low invasivity
Ectopic expression of AXL is sufficient to confer a highly invasive phenotype to weakly invasive MCF7 breast cancer cells
Experimental inhibition of AXL signaling by dominant-negative mutant, Axl shRNA or Axl antibody decreases motility and invasivity of highly invasive breast cancer cells
Overexpression of dominant-negative Axl suppresses brain tumor cell growth and invasivity, and prolongs survival in a mouse xenograft model Vajkoczy P, et al.. PNAS, 2006

72. Henrik Daub et al. KinaTor Technology Definition of Kinase Inhibitor Specificity
Affinity column
enrichment factors in the order of 10‘000 signalling proteins < 2% of cellular proteome

73. RTK Axl : Relevance for Cancer
In 1991, first identified as Axl/Ufo/Ark
Janssen et al – O’Bryan et al –
Rescigno et al. 1991
Axl overexpression has been reported in several types of human cancers, including glioblastoma, colon, prostate, melanoma, thyroid, breast and lung carcinomas

74. Targeted Cancer Drug Development Kinase Inhibitors
So far:
Identification of Potential Targets
OncoGenomis, Oncoproteomics, Rational Pathway Analysis
Validation in vitro / Model Systems
Clinical Validation
Development of Interfering Agent in vitro /whole cell screening
Medicinal Chemistry Optimization
Preclinical / Clinical Testing

75. Targeted Cancer Drug Development Kinase Inhibitors
New:
Structurally Diverse Kinase Inhibitor Library
HCS of Multiple Cancer Cell Lines
Hit / Lead Kinase Inhibition Profile
Medicinal Chemistry
Preclinical / Clinical Testing