Lynn Wenandy

1
Naturally and vaccine induced T-cell responses in
cancer patients Is hTERTp540 a Tumor-Associated
Antigen (TAA) recognized by Cytotoxic T
Lymphocytes (CTL)?

• Lynn Wenandy

2

• TAA as Targets for Anticancer Strategies
• Survivin (Sur) and Telomerase (hTERT)
• High numbers of peptides defined
• Monitoring/characterizing CD8 T-cell responses
• Renal cell carcinoma (RCC) patients
• Phase I/II clinical trial using autologous DCs
  pulsed with
• Sur/hTERT peptides

www.http//clinicaltrials.gov Identifier
NCT00197860
3
Methods

• Indirect/Direct ELISPOT

Sorting
2. FACS
Expansion
3. Cloning
4. Cytotoxicity assay
Killing of tumor cells
5. Clonotype mapping
4
Immune reactivity against Sur derived peptides by
Indirect ELISpot
5 RCC patients
5
Immune reactivity against hTERT derived peptides
by Indirect ELISpot
5 RCC patients
6
Immune reactivity against hTERTp988 by ELISpot
after in vitro stimulation
UR414.11
No peptide Peptide
Pre 4th
6th 9th 10th
7
Cloning of hTERTp540 specific CD8 T cells

• FACS

• Direct ELISPOT

Single cell Sorting/Cloning
CD8
4.1
T cell
hTERTp540 specific cells/5.10e5 PBMC
hTERT540
No peptide
HLA-A20201 /hTERTp540 pentamer
4.1

• Cloning
• Cytokines from PBL mix, IL-2, PHA

Expansion
T cell
T cell
T cell
T cell
T celle
T cell
T cell
T cell
T elle
Are the expanded cells capable of killing
specific tumor cells???
8
Perforin ELISPOT
Functionality ? ?
? (Cytotoxic
assay)Specificity Yes
Yes Yes (Facs)
9
Conclusions

• Single/weak spontaneous responses
• Induction of Sur/hTERT specific anti-tumor
  responses by DC-based vaccination
• No pattern varying reactivity (despite textbook
  example)
• Generation of hTERTp540 specific cells

10
We are Tania Køllgaard, Rikke Bæk Sørensen,
Rikke Sick Andersen, Inge Marie Svane, Annika
Berntsen, Tina Seremet, Merete Jonassen, Mads
Hald Andersen, Per thor Straten Center for
Cancer Immune Therapy Department of
Hematology University Hospital
Herlev Denmark
Supported by Danish Cancer Society Grant
BFR04/097 Ministry of Research Luxembourg National
research Fond Luxembourg Lions Luxembourg
11
Survivin

• IAP (Inhibitor of Apoptosis Protein)
• Apoptosis/Cell division
• Attractive candidate
• Absent in most somatic tissues
• Expressed in the majority of human tumors
• Drug resistance, e.g. chemotherapy
• Reduced risk of antigen-escape tumor cell variants

12
Telomerase

• Ribonucleoprotein
• RNA template hTR, catalytic subunit hTERT
• (Human telomerase reverse transcriptase)
• Attractive candidate
• Silent in most somatic tissues
• Expressed in the majority of human tumors
• Reduced risk of antigen-escape tumor cell
  variants