presentationsdiapharmapptapoptosis2'ppt

1
THE M30-APOPTOSENSETM ASSAY
PEVIVA AB
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THE M30-APOPTOSENSETM ASSAY
THE M30-APOPTOSENSETM ASSAY IS AN
APOPTOSIS-SPECIFIC 96-WELL FORMAT ELISA USES
THE M30-ANTIBODY WHICH RECOGNIZES A
NEO-EPITOPE ON CYTOKERATIN 18 EXPOSED AFTER
CASPASE-CLEAVAGE IS SPECIFIC FOR EPITHELIAL
CELLS (Leers et al., J Pathology 187, 567, 1999)
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WHAT ARE CYTOKERATINS?
CELLS CONTAIN A CYTOSKELETON
INTERMEDIATEFILAMENTS
MICROTUBULI
ACTIN FILAMENTS
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THERE ARE DIFFERENT INTERMEDIATE FILAMENTS IN
DIFFERENT CELL TYPES EPITHELIAL CELLS
HAVE CYTOKERATINS
MICROTUBULI
CYTOKERATIN
ACTIN FILAMENTS
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CELLS OF SIMPLE EPITHELIA HAVE CYTOKERATIN-18
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THE MOST FREQUENT TYPES OF CANCER STEM FROM CELLS
OF SIMPLE EPITHELIA AND EXPRESS
CYTOKERATIN-18 LUNG BREAST OVARIAN
PROSTATE COLON
7

• CYTOKERATIN 18 IS EXPRESSED IN HIGH
• AMOUNTS IN MAJOR TYPES OF CANCERS
• IS NOT EXPRESSED IN MOUSE NIH-3T3
• FIBROBLASTS, JURKAT, U929 CELLS

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• CYTOKERATIN 18 IS EXPRESSED IN HIGH
• AMOUNTS IN MAJOR TYPES OF CANCERS
• IS NOT EXPRESSED IN MOUSE NIH-3T3
• FIBROBLASTS, JURKAT, U929 CELLS
• (FOR CLINICAL RESEARCH - MAJOR ADVANTAGE
• THAT CYTOKERATIN-18 IS NOT EXPRESSED IN BONE
• MARROW)

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THE M30 MONOCLONAL ANTIBODY BINDS TO
CYTOKERATIN-18 CLEAVED AT ARG396 BY CASPASES
M30 Polycl M21
40
HEAD COIL COIL COIL
TAIL
1
429
30
CPM BOUND (10-3)
396
20
396
238
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- - -
CASPASE 3
www.diapharma.com
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WHAT ARE CASPASES?
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WHAT ARE CASPASES? CASPASES ARE A CLASS OF
PROTEASES THAT CLEAVE PROTEINS C-TERMINALLY OF
ASPARTIC ACID RESIDUES AT THE SEQUENCE Asp X X
Asp
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WHAT ARE CASPASES? CASPASES ARE A CLASS OF
PROTEASES THAT CLEAVE PROTEINS C-TERMINALLY OF
ASPARTIC ACID RESIDUES AT THE SEQUENCE Asp X X
Asp CASPASES ARE ACTIVATED DURING APOPTOSIS
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caspases
PRO-CASPASE-1 PRO-CASPASE-4 PRO-CASPASE-5 PRO-CASP
ASE-11
QACXG
NON-APOPTOTIC
QACXG
CASPASE-12
PRO-CASPASE-12
QACXG
CASPASE-9
PRO-CASPASE-9
INITIATORS
QACXG
CASPASE-8
PRO-CASPASE-8
QACXG
CASPASE-10
PRO-CASPASE-10
QACXG
CASPASE-3
PRO-CASPASE-3
QACXG
CASPASE-2
PRO-CASPASE-2
EFFECTORS
QACXG
CASPASE-6
PRO-CASPASE-6
QACXG
CASPASE-7
PRO-CASPASE-7
CARD CASPASE RECRUITMENT DOMAIN
DED DEATH EFFECTOR DOMAIN
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Inactive proenzyme
(20 kDa)
(10 kDa)
Asp-X
Asp-X
Proenzyme is cleaved at caspase cleavage
sequences (Asp-X)
Catalytic sites
2 large and 2 small subunits combine to form
the active tetrameric enzyme
Active caspase
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CASPASES ARE USUALLY ACTIVATED AFTERRELEASE OF
CYTOCHROME C FROM MITOCHONDRIA
BH-3 BCL-2 LIKE APAF-1 CASPASE
BH-3 (i.e. Bid, Bim)
CYTOCHROME C
Bak, Bax
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CASPASES ARE USUALLY ACTIVATED AFTERRELEASE OF
CYTOCHROME C FROM MITOCHONDRIA
BH-3 BCL-2 LIKE APAF-1 CASPASE
BH-3 (i.e. Bid, Bim)
CYTOCHROME C
Bak, Bax
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Steroids DNA-damage
Cytokine deprivation
Ca2 flux Taxol
Bik
Noxa
Bad
Bim
tBid
Bid
Caspase-8
Apaf-1
Death ligands
Caspase-9
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THE M30 MONOCLONAL ANTIBODY BINDS TO
CYTOKERATIN-18 CLEAVED AT ARG396 BY CASPASES
M30 Polycl M21
40
HEAD COIL COIL COIL
TAIL
1
429
30
CPM BOUND (10-3)
396
20
396
238
10
- - -
CASPASE 3
www.diapharma.com
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DEVELOPMENT OF AN ELISA ASSAY M30-APOPTOSENSE
SANDWICH ELISA
HRP
M5E
CK18 frag.
M30
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DETECTION OF APOPTOSIS
PACLITAXEL-INDUCED APOPTOSIS OF HUMAN BREAST
CANCER CELLS
24 HRS
48 HRS
2500
2000
M30 U/L
1500
1000
500
0
PACLITAXEL zVAD
- -
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THE M30-APOPTOSENSETM ASSAY
THE ASSAY EASY TO PERFORM INSENSITIVE TO
INCUBATION TEMPERATURE OR SPEED OF AGITATION OF
THE PLATES THE SAMPLES PLASMA OR SERUM SAMPLES
CAN BE USED FREEZE-THAWING DOES NOT AFFECT THE
ANTIGEN HEMOLYSIS DOES NOT AFFECT THE
ASSAY The M30-ApoptosenseTM ELISA is for
Research Use Only
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THE M30-APOPTOSENSE ASSAY HAS TWO MAIN POTENTIAL
APPLICATIONS

• SCREENING/CHARACTERIZATION OF
• PRO-APOPTOTIC COMPOUNDS
• SERUM SAMPLES FOR STUDYING PROGRESS
• OF APOPTOSIS

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M30-APOPTOSENSE WORKS IN THE 96-WELL FORMAT
DOSE RESPONSE CURVES
1000
400
400
M30-ANTIGEN (U/L)
300
300
M30-ANTIGEN (U/L)
M30-ANTIGEN (U/L)
500
200
200
100
100
0
80
STS
3
145
73
5
10
20
40
CTR
0
7
15
30
CISPLATIN (mM)
TAXOL (??)
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M30-APOPTOSENSE WORKS IN THE 96-WELL FORMAT
TIME COURSE KINETICS
4,00
3,00
A450
2,00
1,00
0,00
4
6
(HOURS)
1
0,5
2
Drug
1
2
3
4
Cells HCT116 colon cancer
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THE DIFFICULTY OF CATCHING APOPTOTIC CELLS
FAST ACTING DRUG
24H
SLOW ACTING DRUG
24H
MEASURING CASPASE-ACTIVITY IN CELLS CAN BE
DIFFICULT/MISLEADING
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VIABLE CELLS
MEDIUM
CELLS
APOPTOTIC CELLS
FRAGMENTS/DEBRIS
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THE DIFFICULTY OF CATCHING APOPTOTIC
CELLS. ADVANTAGE OF THE M30-APOPTOSENSE ASSAY
POSSIBLE TO DETECT ACCUMULATION OF
CASPASE-CLEAVED CK18 OVER TIME, TYPICALLY 24
HOURS POSSIBLE TO SCORE BOTH FAST AND SLOW
ACTING DRUGS Add drug to cells in 200 ml
medium At 24 hours add non-ionice detergent
(NP40 or Triton X-100) to medium, final
concentration 0.5 Assay 25 ml in triplicate
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PRO-APOPTOTIC AGENTSCREENING USING
M30-APOPTOSENSE
A
A
96-WELL PLATE WITH COLLECTION OF DRUGS
96-WELL PLATE WITH TARGET CELLS INCUBATE 24 HOURS
M30-APOPTOSENSE PLATE
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PRO-APOPTOTIC AGENT SCREENING USING
M30-APOPTOSENSE
A
A
96-WELL PLATE WITH COLLECTION OF DRUG
M30-APOPTOSENSE PLATE
96-WELL PLATE WITH TARGET CELLS INCUBATE 24 HOURS
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NCI 60 cell line screen Cells plated in 96 well
plates Drugs added at different
concentrations Number of cells remaining after 48
hours determined by MTT Results available at
www.dtp.nci.nih.gov as LC50 values 144 000 DRUGS
ON 60 CELL LINES Note measures ceased
proliferation, not specifically apoptosis
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THE M30-APOPTOSENSETM ASSAY CAN BE USED FOR IN
VITRO DRUG SCREENING 500 DRUGS FROM THE NCI DRUG
LIBRARY HAVE BEEN SCREENED USING THE HUMAN BREAST
CANCER CELL LINE MDA-MB-231 16 STRONGLY
PRO-APOPTOTIC DRUGS WERE IDENTIFIED 2
DRUGS WERE FOUND TO SYNERGIZE WITH CISPLATIN
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SCREENING FOR PRO-APOPTOTIC DRUGS IN THE 96-WELL
FORMAT
MDA-MB-231 CELLS, 5 mM OF DRUG (NCI DIVERSITY
SET)
750
500
M30-ANTIGEN (U/L)
250
1
10
20
30
40
50
60
70
CTR CISP
DRUG
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33
ELLIPTICINE - AN ALKALOID FROM THE LEAVES OF
OCHROSIA ELLIPTICA LABILL, A TREE GROWING IN
AUSTRALIA AND OCEANIA
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ELLIPTICINE - MECHANISM OF ACTION
Cytoplasmic target (not lysosomes or
mitochondria) Induces endoplasmatic reticulum
stress (link to apoptosis?) Rapid activation
of Bak and of caspase-3 Involvement of
PKCd (t????????????????????????????? within 2
months)
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MANY OF OUR CURRENT CANCER THERAPEUTICS ARE FROM
PLANTS
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Anti-Cancer Agents

• Plants have figured prominently in folk remedies
  for cancer
• Over 3000 plant species had been used
• Ancient records from Egypt, India, Greece, and
  Rome all contain direction for plant use to treat
  various cancers

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SEARCH FOR CANCER DRUGS

• In the late 1950's National Cancer Institute
  began to search for plants with anti-cancer
  properties
• Thousands of plants were screened, and several
  have become standard chemotherapy for different
  forms of cancers
• In early 1980s program was discontinued because
  few new compounds developed
• Problem may have been the screening assay because
  only using a mouse leukemia cell line
• A new screening strategy using 60 human cell
  lines from nine cancer types (leukemia, lung,
  colon, CNS, melanoma, ovarian, renal, prostate,
  and breast) was initiated in 1985.

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NCI 60 cell line screen Cells plated in 96 well
plates Drugs added at different
concentrations Number of cells remaining after 48
hours determined by MTT (proliferation
test) Results available at www.dtp.nci.nih.gov
as LC50 values 144 000 DRUGS ON 60 CELL
LINES Note measures ceased proliferation, not
specifically apoptosis
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FALL 2002 SCREEN THE NCI MECHANISTIC DIVERSITY
SET, 879 COMPOUNDS TOXIC TO TUMOR CELLS 1 HOW
MANY COMPOUNDS WILL INDUCE APOPTOSIS? HOW
DO LC50 FROM PROLIFERATION TESTS AND LC50
FOR APOPTOSENSE COMPARE?
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THE M30-APOPTOSENSETM ASSAY
PACLITAXEL TREATED HUMAN BREAST CANCER CELLS gt
APPROX. 10-5 U M30-ANTIGEN IS GENERATED/CELL
APOPTOSIS IN A TUMOR WITH 109 CELLS 10
APOPTOSIS WILL GENERATE 108 x 10-5 U 103 U IN
A PLASMA VOLUME OF 3 L 330 U/L THE SERUM LEVEL
OF HEALTHY SUBJECTS IS 154 U/L INCREASES OF 50
U/L ARE EASILY DETECTED
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THE M30-APOPTOSENSETM ASSAY
M30-ANTIGEN LEVELS IN SERA median (25 -
75) NORMAL 153.9 (150-5 - 159.6) U/L (n
42) PRIMARY BREAST CA 165.7 (136.9 - 192.8) U/L
(n 152) RECURRENT BREAST CA 181.8 (140.9 -
225.1) U/L (n 68) (P lt 0.005) HIGHER M30
ANTIGEN LEVELS IN SERA FROM PATIENTS WITH ER(-)
TUMORS COMPARED TO ER()
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THE M30-APOPTOSENSETM ASSAY
M30-ANTIGEN LEVELS IN SERA mean median HEAL
THY 159,5 U/L 153,9 U/L (n 42) RECURRENT
BREAST CA 295,2 U/L 181.8 U/L (n 42) LUNG CA
293,3 U/L 149,4 U/L (n 13) LIVER
CA 294.6 U/L 181,7 U/L (n 14) GASTRIC
CA 156,7 U/L 151,7 U/L (n 32)
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THE M30-APOPTOSENSETM ASSAY
THE FIRST DEMONSTRATION OF A CIRCULATING APOPTOSIS
PRODUCT IN THE SERUM OF CANCER PATIENTS
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THE M30-APOPTOSENSETM ASSAY
THE ASSAY HAS A POTENTIAL USE FOR EVALUATION
OF PERFORMANCE OF PRO-APOPTOTIC ANTI-CANCER
AGENTS IN THE RESEARCH FIELD.
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1
2
3
1
2
0
0
1
2
0
0
1
0
0
0
1
0
0
0
Progression
M30
8
0
0
8
0
0
Nucleosomes (AU)
6
0
0
6
0
0
?
4
0
0
4
0
0
2
0
0
2
0
0
0
0
0
1
0
2
0
3
0
4
0
5
0
6
0
7
0
8
0
9
0
Days
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THE M30-APOPTOSENSETM ASSAY
EVALUATION METHOD (PEAK VALUE - PRETREATMENT
VALUE) (PRETREATMENT VALUE)
(x 100)
M30-ANTIGEN
DAYS
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THE M30-APOPTOSENSETM ASSAY
TAXOTERE/FEC TREATED REC BREAST CA (n
52) NECESSARY TO DIVIDE INTO TWO GROUPS BASED ON
PRE-TREATMENT VALUES lt 200 U/L (32 PATIENTS
62 98 OF NORMAL BELOW THIS LEVEL) gt 200 U/L
(20 PATIENTS 38) The M30-Apoptosense TM ELISA
is for RESEARCH USE ONLY
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TAXOTERE/FEC TREATED REC BREAST CA (n 32) lt
200 U/L PRE-TREATMENT
100
P lt 0.02)
PD (n 3) NC (n 15) PR (n 14)
INDEX
50
PD NC PR
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M30-APOPTOSENSETM
NOTE M30-APOPTOSENSETM IS AN APOPTOSIS
MARKER, NOT ATUMOR MARKER
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M30-APOPTOSENSETM
SUMMARY THE M30-APOPTOSENSETM ASSAY IS
SPECIFIC FOR APOPTOSIS OF EPITHELIAL CELLS
THE SENSITIVITY OF THE ASSAY IS SUFFICIENT TO
DETECT TUMOR APOPTOSIS IN SERUM LARGE
PROSPECTIVE STUDIES NEED TO BE CARRIED OUT -
DIFFERENT PRO-APOPTOTIC AGENTS AND DISEASES