Regulators of Apoptosis

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(Regulators of Apoptosis) Specific
T-cells Mads Hald Andersen NCEV Forlì
2007
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Tumour associated antigens (more than 200
described)
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Ideal vaccination targets

• Specifically and broadly expressed among tumors
• Important for tumor cell growth and/or survival
• (lowering the risk of escape variant selection)

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Inhibition of apoptosis
Inhibition of apoptosis enhances the survival of
cancer cells and facilitates their escape from
cytotoxic therapies.   Two gene families of
apoptosis regulators   The BCL2 family comprises
molecules with pro- or anti-apoptotic function.
Anti-apoptotic members include Bcl-2, Bcl-X(L)
and Mcl-1   Another family that regulates cell
death is the inhibitor of apoptosis (IAP)
proteins, e.g. survivin and livin.
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Why target IAP...?
Some IAPs are expressed specifically - or at
significantly higher levels - in cancer cells
Some IAP are expressed by tumor cells of
different origin (lung, breast, skin,
heamatological tumors, colon, ect)
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Characterization of novel tumor antigens- methods
Scanning of selected proteins for antigenic
peptides Binding analyses using synthetic IAP
derived peptides Affinity maturation by amino
acid substitution Detection of spontaneous
peptide specific T cell responses in blood or
TIL from cancer patients
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Peptide/HLA restriction
..LTLAKHTISSDYVIPIGTYGQMKEKCDICTDEYMGGQHPTN. T
LEGFASPLTGIADASQSSMHNALHIYMNGTMSQVQGSAND VLTAL
LAGLVSLLCRHKRK
HLA-A1
HLA-A2
HLA-A3
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Analyses of spontaneous immune reactivity
Responding patient
No peptide Peptide
Not responding patient
Isolation of lymphocytes
In vitro peptide specific culture
Blood sample
No peptide Peptide
Elispot
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HLA-A2 restricted survivin responses
No peptide
Sur9 peptide
Sur1 peptide
Sur1M2 peptide
Patient 1
Patient 2
Patient 3
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Survivin specific T cells cancer patients
Spontaneous anti-survivin T-cell reactivity has
been described in cancer patients suffering from
breast cancer, colon cancer, lymphoma, leukaemia,
multiple myeloma, neuroblastoma and melanoma..
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?
immunological control of the disease
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SuMo-Sec-01(Ethics Cie 07/03 PEI 0899/01)
second line therapy of advanced solid tumors by
vaccination with SURVIVIN peptides in Montanide
ISA-51 Ongoing study
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SuMo-Sec-01 liver metastasis of pancreatic cancer
After nine month of vaccination
Before vaccination
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Table. CTL epitopes from Bcl-2 family members
Andersen MH et al., Nature Reviews (drug
discovery). 2005
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Additional regulators of apoptosis targets
Inhibition of apoptosis enhances the survival of
cancer cells and facilitates their escape from
cytotoxic therapies.   Two gene families of
apoptosis regulators   The BCL2 family comprises
molecules with pro- or anti-apoptotic function.
Anti-apoptotic members include Bcl-2, Bcl-X(L)
and Mcl-1   Another family that regulates cell
death is the inhibitor of apoptosis (IAP)
proteins, e.g. survivin and livin.
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Livin/ML-IAP (Melanoma inhibitor of apoptosis
protein)
Belongs to the inhibitor of apoptosis protein
family Overexpressed in melanoma cells
(and...???)
Andersen MH et al., J.Invest.Dermatol 2004
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Mcl-1
A
No peptide
Mcl-1(95-103)
No peptide
Mcl-1(300-309)
HLA-A3 breast cancer patient
HLA-A3 pancreatic cancer patient
B
Andersen MH et al., leukemia 2005
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Bcl-2
Overexpressed in many cancers Increased
resistance to apoptotic stimuli, eg.
chemotherapy Correlates with a poor prognosis in
many malignancies Spontaneous immune
reactivity have been detected against bcl-2
in patients suffering from cancers of
different origen (pancreatic cancer,
breast cancer, AML, CLL) Bcl-2 specific CTL
are capable of killing

HLA-matched tumor cell targets
Andersen MH et al., Blood. 2005
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Bcl-X(L)
B
Spontaneous immune reactivity have been detected
against bcl-X in patients suffering from cancers
of different origen, e.g. pancreatic cancer,
breast cancer, melanoma
Andersen MH et al., Journal of Immunology 2005
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Regulators of apoptosis proteins specific T-cell
clones
Regulators of apoptosis proteins specific T-cell
clones
Isolation and expansion of peptide specific
T-cells
Isolation and expansion of peptide specific
T-cells
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Cloning and expansion of T-cells
Specific T-cells
Test specificity
Lymphocyte mix PHA IL-2
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Cloning of T-cells killing of cancer cells of
different origin
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Cloning of T-cells Killing of enriched tumor cells
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Specific lysis of different cancer cell lines by
Bcl-2, Survivin and Bcl-X(L) specific
clones Some target cells are killed most
efficiently by Bcl-2 CTL, some by Survivin CTL
and some by Bcl-X(L) specific CTL ND not done
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Serial targeting Targeting of both the Bcl-2
protein family and IAPs would be particularly
attractive since they execute their
anti-apoptotic function though different
mechanisms. Thus, although regulators of
apoptosis proteins are upregulated in almost all
cancers the amount of each protein differs in
individual patients suffering from the same
disease. One should take the expression profile
and prognostic significance and conventional
treatments of the particular proteins into
consideration Bcl-2 and Bcl-XL are dually
overexpressed in e.g. malignant melanoma,
prostate cancer, Small Cell Lung Cancer
Survivin and Bcl-2 are dually overexpressed in
e.g. breast cancer, neuroblastoma, gastric
cancer, colorectal cancer, and high-grade
non-Hodgkins lymphoma
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MART-1 specific CTL clone lysed 8/16 Gp100
specific clone lysed 11/16 Bcl-2 specific clone
lysed 16/16 Survivin specific clone lysed 15/16
Cloning of T-cells killing of melanoma cells
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Cloning of T-cells absence of MART-1 peptide on
cell surface
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Staining with MART-1/HLA-A2 specific antibody
absence of MART-1 peptide on cell surface
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Conclusions
The resistance of killing by the MART-1 specific
T cell clones could be overcome by pulsing the
melanoma cells with the respective T-cell
epitopes . Our data emphasize that the selected
tumor antigens and/or epitopes are critical for
the outcome of anti-cancer immunotherapy.
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Conclusions
IAP/Bcl-2 reactive T cells are present in
patient suffering from hematological
malignancies, melanoma, breast cancer,
pancreatic cancer, ect. IAP/Bcl-2 specific T
cells are capable of killing HLA matched tumor
cell targets IAP/Bcl-2 specific cells are
present in the tumor lesions IAP/Bcl-2
derived antigenic peptides may serve as a
widely applicable targets for anti-cancer
immunotherapy
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Perspectives
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Acknowledgements
Cancer patients.......!
Jürgen C. Becker Dept.
of Dermatology University Hospital
Würzburg Germany
Tumor Immunology Group Danish Cancer
Society Copenhagen Tania Kølgaard Lynn
Wenandy Rikke Bæk Sørensen Rikke Sick
Andersen Tina Seremet Merete Jonassen Mads Hald
Andersen Per thor Straten
Inge Marie Svane Department
of Hematology Hospital Herlev
Denmark
Lars Østergaard Pedersen Lundbeck Valby Denmark
Center for Cancer Immune Therapy (CCIT)