A monoclonal antibody reactive with a tumor antigen that is expressed upon tumor progression

336

I..P 5

Targeting in immune effector cells in cancer 16 .03

24 June 1997 - Poster presentations

1 Directidentification of MHC class I bound

CDllc+ CD13+ CD40+ CD54+ CDsO+ CD88+ DR+ CD4+ CD14+ Total 55 92 40 87 34 33 88 17 cl CDla+ 85 99 99 99 76 97 99 10

tumor-associated peptide antigens of a renal carcinoma ceil line T. Flad', H. K&acher*, R. Kaufmann 3, B. Spengler3, H. Meyer4, M. Blilggel 4, C.A. MOllerI. ’ Secf. Transplant. Immunot. Med. Clinic, Univ. 72076 Tibingen, Germany, *Center of Medical and Natural sciences, Univ 72074 TUbingen, Germany, 3/nstitute of Laser Medicine Univ 40001 Dijsseldod Gennany, 4/nst. Ibr Physiol. Chem., Unix 44780 Boohum, Germany introduction: Renal cell carcinoma belongs to the group of human malignancies where spontaneous remission, regression on immunomodulation by cytokines and isolation of tumor reactive T cells in single patients suggest that the immune system might be capable tc mount an effective response to specific tumor antigens. Therefore the AO201, BO801, Cw7 homozygotous renal carcinoma cell line A-498 (ATCC) was used to ldentii MHC class I associated peptides as potential target antigens of cytotoxic T-lymphocytes (CTL). MaterfaIr and Methods: HIA-B and C antigens as well as total HLA class I molecules were isolated by affinity chromatographie using a specific monoclonal antibody (mAb) against HLA-B/C antigens (TH4) and the W8/32. HL mAb against HLA A, B, C. Related peptides were released by subsequent acid elution and further purified by microbore reversed phase-HPLC. Sequencing of various peptides was achieved for the first time by the MALDI-Post-Source Decay (PSD) technique as an analytical tool to obtain sequence information from exceedingly small amounts (100-30 fmoles) of peptides even if present in rather heterogenous samples. HIA A2 positive T2 cells were used to identify HLA A2 derived peptides in specific binding assays. Reauftaz A large number of peptides with the HlA B8 respectively the HlA A2 binding motif could be identified. Most of these peptides could be correlated with proteins also expected from normal human cells. However a few others were found to be processed from very distinct proteins which am suspected to be highly related to cancer physiology e.g. the tpr protein known to be involved in tumorigenic rearrangement with the met or raf genes. Conciuslon: For the first time MALDI-PSD mass spectrometry has been employed to characterize and identify ligand peptides bound to the broadly distributed HLA class I molecules A0201 and 80801 on renal carcinoma cells. Expression studies of the respective proteins in different renal carcinomas and normal tissue as well as in vitro stimulation of CTL with synthetic peptkfes are currently performed to further define their tumor specificity and immunogenicity.

P.5.16.04

Generation of dendritic ceils (DC) from CD&I+ ceils of patients with malignant diseases. Comparison wlih DC from bone niitrrow and peripherai blood stem ceils of heaithy donors

Ph. Guardiola, M.N. Lacassagne, L. Dal Cortivo, P. Rohtiich, M. Benbunan, J.P. Marolleau. ETS Saint-Louis, Paris, Fmnoe Introduction: This study was initiated to assess the capacity of patients with malignant diseases to generate DC, and to compare them with DC obtained from peripheral blood (PB) or bone marrow (BM) stem cells of healthy pts. Materials and mods: PB (n = 24) and BM (n = 5) CD34+ cells were selected from health (HP - n = IO), mveloma (MM - n = ll), lymphoma (NHL - n = 4), or breast cancer (BC - n’= 4jadult pti, using imm;n&&gnetic bead svstem. Cells were cultured in 96-well plates with RPM1 10% FCS, GM-CSF 20 n-&ml, TNFa 20 ngIml, SCF 25 @ml, IL4 100 U/ml. Single and dual cofor flow cytometric analysis were perfoti from day 5 to d 18 of cultures. Results were confirmed with the analysis of CDla+ and CDla-cell fractions, sorted at d 12 with a FACS Vantage. Mixed leukocyte reaction was used to assay the functional capacities of the different kind of cells obtained at d 12 (whole population of cells, CDla+ and CDla-sorted cells). Reauh After CD34 positive selection, the CD34+ cells median purity was 98% (range, 30-99.4). Day 12 mean expansion of PB CD34+ cells was 7 (range: O-17) with a significant difference between NHL and 1) MM (13.7 vs 3.2, p = 0.025) and 2) HP (13.7 vs 85, p = 0.0001). For HP, BM stem cell expansion was higher than the one of PBSC (4 vs 24 - p = 0.037). The overall mean percentage of CDla+ cells was 11% at d5, reaching 40.5% at d12, reducing to 32% at d18. The mean percentages of d12 CDla+ cells obtained from PB did not vary significantly according to the underlying disease ff any, but we observed high variations between each pt: %CDla+celLs

Myeloma

Lymphoma

BreastCancer

Healthy

P=

Mean%

41.7 204

41.8 6

44.3 10.5

34.7 21.3

0.860

std &w%

The percentage of CDla+ cells generated from PB and BM CD34+ cells of the same HD were not significantly different 35% (PB) vs 47% (BM) (p = 0.44). Viability of d12 cells was 88%. Day 12 cytometric analysis results were as follows:

CDla-

17

76

25

54

25

10

36

20

CDla+ cells were also WAG+ (99%), but CD6-. There was no significant difference between groups of pts according to the pathology or the origin of d12 cells for the expression of CDllc. CD40, CD80. and DR. To confirm these results, d12 cells were sorted according to the expression of CDla. CDla+ cells (purity 295%) expressed CDllC (710/o),CD13 (69%). CD40 (91%), CD80 (98%), DR (990/o),and few were CD4+ (18%). Moreover some CDla- cells were, as found with dual staining, CD4+ (20%), CDllc+ (15%), CD40+ (46%). DR+ (82%), but they were mainly CD80- (94%). Using allogeneic MLR, d12 DC were efficient stimulators of naive T cells in a dose dependent way (from Iti to IO5 DC per 5 x 104 T cells). The number of cpm was not different between groups of pts. whatever the orfgin of cells was. Over 3 experiments, d12 CDla+ sorted cells did not appear to be more efficient stimulator cells than CD1a- or the overall unsorted d12 cells (p = 0.746, power of the test do%). Conclusion: DC can be obtained from pts with malignant diseases, with phenotypic and functtonal characteristics identical to those generated from healthy pts. Nevertheless, the percentage of CDla+ cells was highly heterogenous among each group of pts. Furthermore, among the d12 CDla- cells, a subset of cells which expressed costimulatoty and MHC class-II molecules were able to stimulate allogeneic T cells efficiently, leading to the hypothesis that DC studies should not be restricted to the analysis of the CDla+ cells contingent, since a subset of CD1a-, perhaps also CDllc- cells certainly belongs to the functional spectrum of the dendrftic cells.

P.5.16.05

A monocionai antibody reactive wlth a tumor antigen that is expressed upon tumor progression

P. Rottien, T. Verfaillie, H. Dooms, M. Desmedt, R. Contrems, W. Fiers, J. Grooten. Laboratory of Molecular Biology! Flanders Interunivetsity Institute for Biotechnology and University of Ghent, B-9ooo Ghent, Belgium Introduction: Immortalization, accompanied by loss of growth control, is a critical step in the process of neoplastic transformation. However, besides these features, a developing tumor needs to escape immune rejection as well as to adapt to its microenvironment in order to progress to malignancy. We provide evidence that this tumor progression is accompanied by expression of new genes, resulting in the presence on the cell surface of novel tumor-associated antigens. Materialsand Methods:Our studies were performed with the murfne cell line EL4/13 that was grown in vitrc as a suspension culture and as an established tumor in C57BU6 mice; the differential gene expression between EL40 3 maintained as described above was performed with a PCR-based suppression subtractive hybridization (SSH) method. Ftesufts: Thus, we isolated a moncclonal antibody, IIBI, which recognizes a tumor antigen that is expressed on the surface of EL403 thymoma cells upon tumor growth in susceptible animals but that is absent on in vitro cultured cells. Hence, this acquisition of a new antigen upon tumor growth illustrates the phenotypic changes which immortalized cells undergo in their response to micrOenvironmentand progression to a tumor. Ttfton X-100 extraction revealed that the IIBI antigen is associated with the detergent-insoluble cytoskeleton fraction. lmmunoprecipitation and immunoblotting revealed a band of approximately 250 kDa. In an effort to isolate the gene encoding the II81 structure, cDNA isolated from cultured EL4/13 cells was subtracted from cDNA derived from cells grown as a tumor inoculum. Thii yielded several distinct cDNA clones that apparently am specifically induced during tumor progression. Concfuslon: Besides the characterization of the gene encoding the IlBl antigen, the identification of these differential expressed clones indicates that distinct genes are involved in the adaptation of a transformed cell to its microenvironment, which will allow us to evaluate their role in escape from immune surveillance. Additionally, the diiemntial expression of tumor associated antigens upon tumor growth may provide new tools for immunotherapy.

I..P 5

16.06

1interieukin-1 , -2 and -12 allow the development of antitumor autoiogous cytotoxic 1 lymphocytes in non-Hodgkin’s iymphomas

L. Chaperot’, M.-C. Jacob’. J.-P. Molens’, J.-J. Sotto”, J.-C. Bensa’, J. Plumas’. ’ ETSIs&eet Saw/e. BP35.38701 La Tronohe Cedex, France, *Service d’H6matologie. CHU, 38701 La Tmnche, Franc8 Introduction: Non-hodgkin’s lymphomas derived from B cells are mostly incurable with conventional strategies. The use of antitumor cytotoxic T lymphocytes could be one way to improve the outcome of this pathology. We had previously shown that malignant B cells were able to stimulate the cytotoxic and prolifer-