Immune surveillance: The immune system can recognize and destroy nascent malignant cells

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1 Immune surveillance: The immune system can recognize and destroy nascent malignant cells Control Escape APC T C T H B NKT NK Innate Tumor T cells are believed to play a major role in controlling tumor growth.

2 T cell-based Immunotherapy Activated CTL CD8+ T cell precursor Tumor CD4+ T cell TA HLA class I TA peptide complex Immunization APC Tumor Cell lysate TA TA derived peptides Peptide-pulsed APC Tumor HSP

3 T cell-based immunotherapy clinical trials: lessons learned Immunization strategies have been successful in eliciting tumor antigen-specific CTL in at least a proportion of the immunized patients Disappointing clinical response rates have been obtained A tumor antigen-specific CTL immune response is often not accompanied by a clinical response

4 Why does a TA-specific CTL immune response not correlate with clinical response in patients with malignant disease treated with immunotherapy? defects in CTL resistance of tumor cells to CTL recognition

5 HLA class I antigen-peptide complex expression is necessary for tumor antigen derived recognition by CTL TA derived peptide CTL TCR Target cell HLA class I Killing TA derived peptide HLA class I peptide HLA class I No killing

6 How are HLA class I antigen-tumor antigen peptide complexes generated? Antigen processing and presentation pathway DRiPs Cytosolic peptidases cytosol Protein Tumor antigen (TA) Ub Proteasome/ Immunoproteasome Peptide TAP Translocon DRiPs ERp57 Tapasin Calnexin Calnexin Heavy chain Calreticulin ERAP1 ER Cell membrane Kill No killing TCR TA-specific CTL

7 Immunohistochemical staining of formalin fixed, paraffin embedded malignant lesions by HLA class I specific mab Heavy chain β 2 -microglobulin Heterogeneous expression Loss of expression in undifferentiated cells Serial Sections of a Breast Carcinoma Lesion

8 Different frequency of HLA class I antigen downregulation in different tumor types Monomorphic Polymorphic

9 Correlation of LMP2 and tapasin expression with HLA class I antigen expression in primary laryngeal squamous cell carcinoma lesions 1 LMP2 TAP1 6 % Positive 2 1 tapasin r=.41 p=.55 calnexin r=.23 p= r=.7 p< r=.25 p= HLA class I antigen

10 Association of APM component and HLA class I antigen expression with CD8+ T cell infiltration in primary laryngeal squamous cell carcinoma lesions 1 LMP2 TAP1 6 % Positive 2 1 tapasin r=.45 p=.21 r=.43 p=.37 HLA class I antigen 6 2 r=.56 p<.1 r=.53 p< CD8 + T cell infiltration

11 Association of HLA class I antigen expression and CD8+ T cell infiltration in primary laryngeal squamous carcinoma lesions with poor survival Cause-specific survival HLA class I antigen Positive vs. negative: p=.2 Positive vs. heterogeneous: p=.42 Heterogeneous vs. negative: p=.1 β 2 m Positive vs. negative: p=.52 Positive vs. heterogeneous: p=.52 Heterogeneous vs. negative: p= Months

12 Restoration by IFN-γ of recognition of SCCHN PCI 13 cells by HLA class I antigen restricted, TA-specific CTL. IFN-γ spots / 3, cells CTL + Tumor CTL + Tumor + BB7.2 CTL + Tumor + L243 PCI 13 PCI 13 + IFN-γ

13 Relationship between upregulation of TAP1 and tapasin level and recognition of IFN-γ treated SCCHN cells PCI-13 and SCC4 by HLA class I antigen restricted, TA-specific CTL PCI-13 PCI-13 SCC4 IFN-γ spots SCC4 TAP1 Tapasin PCI-13 cells SCC4 cells HLA A*21

14 Phage display antibody libraries V H V L A sailor Thewas went toa man frsee see We are fa Immunoglobulin Semi-synthetic single chain fragment of antibody variable region (scfv) phage displayed scfv

15 Panning phage display antibody libraries with HLA class I allele-ta peptide complexes B. Binding C. Wash A. Incubation Phage bound HLA class I allele-ta peptide complexes D. Elution scfv library HLA class I allele-ta peptide complex Eluted phage E. Amplification

16 Enriched phage displayed scfv recognize purified HLA-A*21-peptide complexes HLA-A2-MART (ELAGIGILTV) HLA-A2-HER2/neu (RLLQETELV) HLA-A2-HER2/neu (KIFGSLAFL) 45 nm scfv

17 Isolation of unique HLA class I allele-ta peptide complex specific scfv Clone Heavy chains a Light chains Family Segment CDR3 Family Segment HLA-A*21-MART specific scfv CDR3 8.3 VH3 DP-45 ARSSSLCTWGQ Vκ2 DPK-15 MQALQTQC 24.3 VH3 DP-45 ARSSSLCTWGQ Vλ3 DPL-16 NSRDSSGF 25.3 VH3 DP-45 ARSSSLCTWGQ Vκ3 DPK-1D QQYDNLPS HLA-A*21-HER2/neu specific scfv VH3 DP-13*1 AGPAGAGPWGQ Vκ2 DPK-29*1 MQSIQLHT VH3 DP-13*1 AGPAGAGPWGQ Vλ3 DPL-19*1 NSRDSSGNHPDV HLA-A*21-HER2/neu specific scfv VH3 DP-23*1 ARGEFRTYFPT Vκ1 DPK-39*1 QQANSFLSST

18 scfv 8.3 does not bind to MART peptide alone 1 8 % Inhibition HLA-A*21-MART MART HLA-A*21-HER2/neu :1 1:1 25:1 5:1 1:1 2:1 4:1 8:1 Molar ratio (inhibitor:scfv)

19 scfv 8.3 binds to a determinant located on the α1/α2 domains of HLA-A*21 and MART peptide α1 α2 α β2m - α3 α TM 17 - Intracellular tail

20 Can we enhance the sensitivity of HLA class I allele-ta peptide complex specific probes? HLA class I-TA specific probe HLA-A2, TA specific CTL TCR Tumor HLA-A2-TA peptide complex HLA class I-TA specific probe CTL killing - +

21 Generation of HLA class I allele-ta peptide complex specific scfv tetramers scfv tetramer scfv tetramer V l V H VH V l V H V l Site specific biotinylation site Streptavidin V l V H V l V l VH VH V H V H V l V l Biotin Phycoerythrin VH Vl

22 scfv tetramerization enhances their ability to detect HLA class I allele-ta peptide complexes on target cells 9 scfv 8.3 tetramer (A2/MART1) scfv 8.3 monomer (A2/MART1) scfv tetramer (A2/HER2/neu) 7 MFI 5 3 Tetramer kd=.2 nm Monomer kd=4 nm scfv tetramer (nm)

23 Heterogeneous HLA-A*21 surface expression and intracellular MART1 protein expression in human melanoma cells 4 HLA-A* MART1 - IFN-γ + IFN-γ Fold change over background

24 Heterogeneous APM component expression in human melanoma cells - IFN-γ + IFN-γ 3 TAP1 TAP calreticulin 2 1 tapasin Fold change over background

25 Heterogeneous HLA-A*21-MART peptide complex expression on human melanoma cells 4 HLA-A*21-MART Fold change over background IFN-γ + IFN-γ HLA-A*21 (+)

26 Lack of correlation between HLA-A*21, MART1 and HLA-A*21-MART peptide complex expression 4 HLA-A*21 MART IFN-γ + IFN-γ HLA-A*21-MART HLA-A*21-MART Fold change over background

27 Lack of relationship between HLA-A2 antigen and HLA- A2 antigen-her2/neu peptide complex expression by SCCHN cell lines A complex B HLA-A2 PCI-13 SCC-4 PCI-3 T2+Peptide 1mM PCI-13+ Peptide 1mM

28 Conclusions The level of APM components and HLA class I antigens markedly vary in malignant cells Measure of the level of APM component and HLA antigen expression provides only limited information about their functional properties The level of HLA class I antigen-tumor antigen peptide complexes on tumor cells does not correlate with the level of APM components, HLA class I antigens and tumor antigens These results stress the need to measure the level of HLA class I antigen-tumor antigen peptide complexes on tumor cells to characterize their interactions with CTL

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30 HLA class I antigen-peptide complexes mediate recognition of target cells by cytotoxic T lymphocytes (CTL) Kill Target cell HLA class I antigen CTL peptide TCR

31 Reactivity of scfv 8.3 with peptide pulsed T2 cells is dependant on scfv & MART concentration Mean fluorescence intensity T2-MART T2-HER2/neu [peptide] [scfv] kd= peptide (µm) scfv (nm)

32 scfv 8.3 mimics the reactivity of HLA-A*21- MART specific TCR 64 MART : AAGIGILTV CTL (+) E AAGIGILTV CTL (+) EL AAGIGILTV CTL (+) A AAGIGILTV CTL (+) AL AGIGILTV CTL (+) Cell count 64 Y AAGIGILTV CTL (+) Log fluorescence intensity (PE) HER2/neu : F AAGIGILTV AL GIGILTV KIFGSLAFL CTL (+) CTL (-) CTL (+) Antibody scfv 8.3 tetramer mab CR mab 9E1 scfv tetramer

33 Enriched phage displayed scfv recognize peptide pulsed T2 cells T2-HER2/neu T2-MART Antibody Specificity Cell count Log fluorescence intensity (PE) scfv 8.3 HLA-A2-MART scfv HLA-A2-HER2/neu mab CR HLA-A2, -A24, -A28 mab 9E1 JH1 c-myc scfv JH1 Human VEGF

34 HLA class I allele-ta peptide complex specific scfv tetramers retain their specificity MART1(+) HER2/neu(-) LG2 MART1(-) HER2/neu(-) 553 MART1(+) HER2/neu(-) SCC4 MART1(-) HER2/neu(+) A2/MART A2/HER2/neu Antibody Specificity Cell count Log fluorescence intensity (PE) scfv 8.3 HLA-A2-MART mab CR HLA-A2, -A24, -A28 mab 9E1 c-myc scfv HLA-A2-HER2/neu

35 Lack of correlation between APM component and HLA-A*21-MART peptide complex expression 4 TAP1 Proteasome subunit (LMP2) 16 Fold change over background HLA-A*21-MART HLA-A*21-MART IFN-γ + IFN-γ