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.
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
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
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
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
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
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
Different frequency of HLA class I antigen downregulation in different tumor types Monomorphic Polymorphic
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=.12 6 2 r=.7 p<.1 2 6 1 r=.25 p=.1 2 6 1 HLA class I antigen
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<.1 8 16 8 16 CD8 + T cell infiltration
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=.17 2 6 1 14 Months
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 6 4 2 CTL + Tumor CTL + Tumor + BB7.2 CTL + Tumor + L243 PCI 13 PCI 13 + IFN-γ
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
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
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
Enriched phage displayed scfv recognize purified HLA-A*21-peptide complexes HLA-A2-MART1 27-35 (ELAGIGILTV) HLA-A2-HER2/neu 689-697 (RLLQETELV) HLA-A2-HER2/neu 369-377 (KIFGSLAFL) OD @ 45 nm 3 2 1 2.3.5 1.3.13 8.3 scfv
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-MART1 27-35 -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 369-377 -specific scfv 2.3.5 VH3 DP-13*1 AGPAGAGPWGQ Vκ2 DPK-29*1 MQSIQLHT 2.4.38 VH3 DP-13*1 AGPAGAGPWGQ Vλ3 DPL-19*1 NSRDSSGNHPDV HLA-A*21-HER2/neu 689-697 -specific scfv 1.3.13 VH3 DP-23*1 ARGEFRTYFPT Vκ1 DPK-39*1 QQANSFLSST
scfv 8.3 does not bind to MART1 27-35 peptide alone 1 8 % Inhibition 6 4 2 HLA-A*21-MART1 27-35 MART1 27-35 HLA-A*21-HER2/neu 369-377 :1 1:1 25:1 5:1 1:1 2:1 4:1 8:1 Molar ratio (inhibitor:scfv)
scfv 8.3 binds to a determinant located on the α1/α2 domains of HLA-A*21 and MART1 27-35 peptide α1 α2 α1 58 62 82 - β2m - α3 α2 169 17 145 162 149 161 127 142 138 TM 17 - Intracellular tail
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 - +
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
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 2.3.5 tetramer (A2/HER2/neu) 7 MFI 5 3 Tetramer kd=.2 nm Monomer kd=4 nm 1.1.3.6.11.22.34.45.53.66 scfv tetramer (nm)
Heterogeneous HLA-A*21 surface expression and intracellular MART1 protein expression in human melanoma cells 4 HLA-A*21 1 6 7 3 4 7 6 1 8 2 2 5 6 7 7 2 9 3 5 2 1 MART1 - IFN-γ + IFN-γ 51 526 537 553 624 677 836 111 188 112 1182 1195 128 1286 1287 1317 133 1379 1383 1495 152 Fold change over background
Heterogeneous APM component expression in human melanoma cells - IFN-γ + IFN-γ 3 TAP1 TAP2 2 1 4 2 3 calreticulin 2 1 tapasin 51 526 537 553 624 677 836 111 188 112 1182 1195 128 1286 1287 1317 133 1379 1383 1495 152 51 526 537 553 624 677 836 111 188 112 1182 1195 128 1286 1287 1317 133 1379 1383 1495 152 Fold change over background
Heterogeneous HLA-A*21-MART1 27-35 peptide complex expression on human melanoma cells 4 HLA-A*21-MART1 27-35 2 Fold change over background 51 526 537 553 624 677 836 111 188 112 1182 1195 128 1286 1287 1317 133 1379 1383 1495 152 - IFN-γ + IFN-γ HLA-A*21 (+)
Lack of correlation between HLA-A*21, MART1 and HLA-A*21-MART1 27-35 peptide complex expression 4 HLA-A*21 MART1 2 2 4 2 1 - IFN-γ + IFN-γ 1 6 4 7 2 2 5 6 HLA-A*21-MART1 27-35 4 HLA-A*21-MART1 27-35 2 Fold change over background 51 624 188 112 1182 1195 128 1495 152 51 624 188 112 1182 1195 128 1495 152
Lack of relationship between HLA-A2 antigen and HLA- A2 antigen-her2/neu 369-377 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
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
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
Reactivity of scfv 8.3 with peptide pulsed T2 cells is dependant on scfv & MART1 27-35 concentration Mean fluorescence intensity 6 4 2 T2-MART1 27-35 T2-HER2/neu 369-377 [peptide] [scfv] kd=3 49 245 123 61 3 15 8 4 2 1 65 13 194 258 323 97 1612 2528 29 355 peptide (µm) scfv (nm)
scfv 8.3 mimics the reactivity of HLA-A*21- MART1 27-35 -specific TCR 64 MART1 27-35 : AAGIGILTV CTL (+) E AAGIGILTV CTL (+) EL AAGIGILTV CTL (+) A AAGIGILTV CTL (+) AL AGIGILTV CTL (+) 1 1 2 1 4 Cell count 64 Y AAGIGILTV CTL (+) 1 1 2 1 4 Log fluorescence intensity (PE) HER2/neu 369-377 : F AAGIGILTV AL GIGILTV KIFGSLAFL CTL (+) CTL (-) CTL (+) 1 1 2 1 4 1 1 2 1 4 1 1 2 1 4 Antibody scfv 8.3 tetramer mab CR11-351 mab 9E1 scfv 2.3.5 tetramer
Enriched phage displayed scfv recognize peptide pulsed T2 cells T2-HER2/neu 369-377 T2-MART1 27-35 64 Antibody Specificity Cell count 1 1 2 1 4 1 1 2 1 4 Log fluorescence intensity (PE) scfv 8.3 HLA-A2-MART1 27-35 scfv 2.3.5 HLA-A2-HER2/neu 369-377 mab CR11-351 HLA-A2, -A24, -A28 mab 9E1 JH1 c-myc scfv JH1 Human VEGF
HLA class I allele-ta peptide complex specific scfv tetramers retain their specificity 64 188 MART1(+) HER2/neu(-) LG2 MART1(-) HER2/neu(-) 553 MART1(+) HER2/neu(-) SCC4 MART1(-) HER2/neu(+) 64 188 A2/MART1 27-35 188 A2/HER2/neu 369-377 1 1 2 1 4 1 1 2 1 4 Antibody Specificity Cell count 1 1 2 1 4 1 1 2 1 4 Log fluorescence intensity (PE) scfv 8.3 HLA-A2-MART1 27-35 mab CR11-351 HLA-A2, -A24, -A28 mab 9E1 c-myc scfv 2.3.5 HLA-A2-HER2/neu 369-377
Lack of correlation between APM component and HLA-A*21-MART1 27-35 peptide complex expression 4 TAP1 Proteasome subunit (LMP2) 16 Fold change over background 2 4 2 HLA-A*21-MART1 27-35 8 4 2 HLA-A*21-MART1 27-35 - IFN-γ + IFN-γ 51 624 188 112 1182 1195 128 1495 152 51 624 188 112 1182 1195 128 1495 152