Wtl-Specific Serum Antibodies in Patients with Leukemia 1

Vol. 7, 761s-765s, March 2001 (Suppl.) Clinical Cancer Research 761s

WTl-specific Serum Antibodies in Patients with Leukemia 1

Alexander Gaiger, Lauren Carter, Introduction Hildegard Greinix, Darrick Carter, Overexpressed oncogenic proteins can be considered as potential candidate antigens for cancer vaccines and T-cell ther- Patricia D. McNeill, Raymond L. Houghton, apy (1-3), particularly if the oncogenic proteins are important Charisa D. Cornellison, Thomas S. Vedvick, for maintenance of the malignancy, have restricted tissue dis- Yasir A. W. Skeiky, and Martin A. Cheever 2 tribution, and are immunogenic. WT13 is an oncogenic protein Corixa Corporation, seattle, Washington 98104 [A. G., L. C., D. C., involved in transcriptional regulation, especially during fetal P. M., Y. S., R. H., C. C., T. V., M. A. C.], and First Department of development (4, 5). WT1 was originally identified in Wilms' Medicine, Division of Hematology, University of Vienna, A-1090 tumor (6, 7), but it is also oncogenic and overexpressed in many Vienna, Austria [A. G., H. G.] human leukemias including the majority of AMLs and CMLs (8-15). WT1 overexpression appears to often be an important Abstract factor for leukemogenesis and for the viability of leukemic blasts (16-19). WT1 is an oncogenic protein expressed by the Wilms' During fetal development, WTI is normally expressed in a tumor gene and overexpressed in the majority of acute time- and tissue-specific manner and is involved in the regula- myelogenous leukemias (AMLs) and chronic myelogenous tion of growth and differentiation via interaction with a variety leukemias (CMLs). The current study analyzed the sera of of different target genes (4-6). In the adult, WTI has a tissue patients with AML and CML for the presence of antibodies distribution restricted to low levels of expression in the nucleus to full-length and truncated WT1 proteins. Sixteen of 63 of some normal tissues, including hematopoietic precursor cells, patients (25%) with AML had serum antibodies reactive kidney, and gonad cells (5, 20). The level of expression in with WT1/full-length protein. Serum antibodies from all 16 hematopoietic precursor cells is substantially less than the level were also reactive with WT1/NHz-terminal protein. By of overexpression in a substantial proportion of leukemias. marked contrast, only 2 had reactivity to WT1/COOH- Thus, WT1 protein is abundant in leukemia cells compared with terminal protein. Thus, the level of immunological tolerance normal hematopoietic cells (12, 20). WT1 is expressed in the to the COOH terminus may be higher than to the NH a adult kidney, but expression is limited to the kidney podocyte. terminus. The WTl/COOH-terminal protein contains four The extent to which the kidney podocyte is an immunological zinc finger domains with homology to other self-proteins. By restricted site is unknown. Of note, in mice immunized to WT1, implication, these homologies may be related to the in- kidney toxicity has not been observed (3). creased immunological tolerance. Results in patients with There is substantial evidence that WTI is immunogenic in CML were similar with antibodies reactive to WT1/fuli- humans and in mice. Human WTl-specific CTLs can be gen- length protein detectable in serum of 15 of 81 patients erated by priming to peptides in vitro and can kill leukemic cells (19%). Antibodies reactive with WT1/NHe-terminal protein that overexpress WT1 (1, 2). WTl-specific CTLs can have were present in the serum of all 15, whereas antibodies exquisite specificity for leukemic progenitor cells. In one study, CTL-specific for WT1 killed leukemic cells, leaving normal reactive with WT1/COOH-terminal protein were present in bone marrow progenitor cells intact (2). Murine WTI is virtu- only 3. By contrast to results in leukemia patients, antibodies ally identical to human WT1; thus, many aspects of vaccine reactive with WT1/full-length protein were detected in only studies in mice can be extrapolated to humans. In mice, immu- 2 of 96 normal individuals. The greater incidence of anti- nization with peptide fragments of WT1 can elicit Ab, helper T body in leukemia patients provides strong evidence that cell, and CTL responses specific for WTI without apparent immunization to the WT1 protein occurred as a result of toxicity to the small number of normal tissues that express WT1, patients bearing malignancy that expresses WT1. These including the kidney (3). Two murine cancers that "naturally" data provide further stimulus to test therapeutic vaccines overexpress WT1 have been identified. Immunization of mice directed against WT1 with increased expectation that the with murine cancer cells that "naturally" overexpress WT1 can vaccines will be able to elicit and/or boost an immune re- induce antibodies specific for WT1, demonstrating that WT1 sponse to WT1. can be immunogenic in the context of cancer cells (3). Studies demonstrating that priming in vitro can generate human WTl-specific CTLs (1, 2) imply that WT1 vaccines can be immunogenic in humans. Previous studies had demonstrated the presence of Ab to the NH2-terminal portion of the WT1

This work was supported by NIH National Cancer Institute Grant R37 CA30558 (to M. A. C.) and NIH National Cancer Institute Small Busi- ness Innovation Research Grant R43 CA81752 (to A. G.). 2 To whom requests for reprints should be addressed, at Corixa Corpo- 3 The abbreviations used are: WT1, Wilms' tumor 1; AML, acute ration, 1124 Colombia Street, Suite 200, Seattle, WA 98104. Phone: myelogenous leukemia; CML, chronic myelogenous leukemia; TRX, (206) 754-5791; Fax: (206)754-5715; E-mail: [email protected]. thioredoxin; Ab, antibody.

Table 1 Characteristics of recombinant WT1 proteins used for serological analysis Recombinant protein WT1 amino acid position Molecular weight WT 1/full-length Ra 12-WT 1 full-length fusion protein 1--449 85,000 WT1/NH2 terminus TRX-WT1 NH2 terminal fusion protein 1-249 50,000 WT1/COOH terminus WTI COOH-terminal protein 267-449 20,000

protein in the sera of three patients with AML by Western blot CA) as described (22). Proteins were analyzed by SDS-PAGE, analyses (3). The current study was undertaken to confirm and and fractions containing the protein of interest were pooled and extend those findings. Antibodies reactive to WT1 were shown dialyzed overnight against excess 10 mM Tris (pH 8.0). Dialy- to be present in the sera of many patients with AML and CML. sates were brought to 8 M urea and loaded onto a Source Q anion The demonstration of antibodies specific for WT1 provides exchange resin (Amersham Pharmacia Biotech, Uppsala Swe- direct evidence that WT1 is ilnmunogenic in humans. The den) equilibrated in buffer A. Proteins were eluted in buffer A presence of antibodies provides substantial evidence for the with a gradient from 0 to 1 M NaCI. Fractions containing the concurrent presence of WTl-specific helper T cells in the same proteins of interest were pooled, dialyzed overnight against individuals. The demonstrated presence of existent immunity to excess 10 mM Tris (pH 8.0), and stored at -80~ for further use. WT1 adds great credence to the prospect that vaccination of The identity of the WT1 proteins was confirmed by NH 2- humans with formulations of WTI will be able to elicit and terminal sequencing. boost Ab and T-cell immunity to WT1. ELISA. Sera from adult patients with de novo AML or CML were studied for the presence of WTl-specific Ab. Fifty Materials and Methods txl/well of a 5 lxg/ml recombinant protein in coating buffer (15 Recombinant Proteins. For protein expression, cDNA mM NazCO3, 30 mM NaHCO 3, pH 9.6) was adsorbed to TC constructs representing the human WT1/full-length (amino ac- microwell plates 12 • 8 (Nunc, Roskilde, Denmark) overnight ids 1-449), the NH2-terminal (amino acids 1-249; denoted at 4~ Plates were washed with PBS/0.5% Tween 20 and WT1/NHz-terminal), and COOH-terminal (amino acids 267- blocked for 2 h at room temperature with 200 ~l/well of 1% 449; denoted WT1/COOH-terminal) regions were subcloned BSA/PBS/0.1% Tween 20. After washing, 50 ixl/well of serum into a modified pET28 vector. Because of the insolubility of the dilutions, ranging from 1:50 to 1:20,000, in 1% PBS/BSA/0.1% recombinant WT1/full-length and WT1/NHz-terminal proteins Tween 20 were added and incubated overnight at 4~ Plates as well as issues with expression in Escherichia coli, it became were washed, and 100 txl/well diluted secondary Ab/l% PBS/ necessary to express these two proteins as fusion proteins. Ral2 BSA/0.1% Tween 20 were added (donkey antihuman IgG- is the COOH-terminal fragment of a secreted Mycobacterium horseradish peroxidase; Jackson-Immunochem, West Grove, tuberculosis protein, denoted as MTB32B (21). The WT1 full- PA) and incubated for 2 h at room temperature. Plates were length region was subcloned into a modified pET28 vector that washed, incubated with 50 ~l/well of substrate solution (TMB has a 5' histidine-tag, followed by the Ral2-WT1/full-length peroxidase/substrate; Kirkegaard and Perry Laboratories, MA) fusion region, followed by a 3' histidine-tag. The WT1/NH 2- for 10 rain at room temperature, stopped with 50 Ixl/well 1N terminal region was subcloned into a modified pET28 vector H2804, and immediately read (Cyto-Fluor 2350; Millipore, that has a 5' histidine-tag, followed by the TRX-WT1/NH2- Bedford, MA). terminal fusion region, followed by a 3' histidine-tag. The For the serological survey, human sera were tested by WT 1/COOH-terminal coding region was subcloned into a mod- ELISA over a range of serial dilutions from 1:50 to 1:20,000. A ified pET28 vector without a fusion partner containing only the positive reaction is defined as an absorbance of a 1:500 diluted 5' histidine-tag. Table 1 lists the three WT1 proteins included in serum that exceeds the mean absorbance of sera from normal this study. donors (n = 96) by three (WT1/full-length and WT1/COOH- Recombinant BL21 pLysS E. coli (Stratagene, La Jolla, terminal proteins) SDs. Because of a higher background in CA) containing the expression constructs for the Ral2-WT1/ normal donors to the WT1/NHz-terminal protein, a positive full-length, TRX-WT1/NHz-terminal, or the WT1/COOH- reaction to the WT1/NH 2 terminus is defined as an absorbance terminal proteins were grown overnight and induced with iso- of 1:500 diluted serum that exceeds the mean absorbance of sera propyl-[3-D-thiogalactoside. All of the proteins behaved simi- from normal donors by 4 SDs. To verify that the patient Ab larly and were purified following essentially the same protocol. response was directed against WT1 and not to the Ral2 or TRX Cells were harvested and lysed by incubation in 10 mM Tris (pH fusion part of the protein or possible E. coli contaminant pro- 8.0) with Complete Protease Inhibitor Tablets (Boehringer teins, controls included the Ral2 and TRX protein alone puri- Mannheim Biochemicals, Indianapolis IN) at 37~ followed by fied in a similar manner. Samples that showed reactivity against repeated rounds of sonication. Inclusion bodies obtained were the Ral2 and/or TRX proteins were excluded from the analysis. washed twice with 10 mM Tris (pH 8.0)/0.5% 3-[(3-cholamido- propyl)dimethylamino]-1-propanesulfonateand solubilized in 8 Results M urea containing 10 rnM Tris at pH 8.0 (buffer A). Proteins WTl-specific Abs in Sera of Normal Individuals. To were then purified by metal chelate affinity chromatography evaluate for the presence of immunity to WT1, we analyzed for over nickel-nitrilotriaeetic acid resin (Qiagen, Inc., Valencia, the prevalence of Ab to recombinant full-length and truncated

Table 2 WTl-specific serum Abs in patients with AML and CML

WT1/full- WT1/NH 2 WT1/COOH 1.6 length terminus terminus Normal individuals (n = 96) 2/96 (2%) 1/96 (1%) 1/96 (1%) AML patients (n = 63) 16/63 (25%) 16/63 (25%) 2/63 (3%) 1o2 9 CML patients (n = 81) 15/81 (19%) 15/81 (19%) 3/81 (4%) OD 0.8 84

0A WT1 proteins in the sera of normal individuals and patients with leukemia. Ab reactivity was determined by ELISA reactivity to WT1/full-length protein, WT1/NH2-terminal protein, and WT1/ Fig. 1 Serum Ab reactivity to WTl in 63 patients with AML. Reac- COOH-terminal protein. The proteins used are defined in Table tivity of serum Ab to WT1/full-length protein was evaluated by ELISA 1. A positive reaction was defined as an absorbance of a 1:500 in patients with AML. The first and second columns represent the diluted serum that exceeds the mean absorbance of all sera from positive and negative controls, respectively. Commercially obtained WTl-specific Ab WT180 was used for the positive control (column 1). normal donors (n = 96) by either 3 SDs (WT1/fulMength Serum obtained from a normal individual was used for the negative protein and WT1/COOH-terminal protein) or 4 SDs (WT1/NH 2- control (column 2). The next 63 lanes represent results using sera from terminal protein). The ELISA for WT1/NH2-terminal protein each individual patient. The absorbance values depicted were from had a higher background in normal individuals and leukemia ELISA using a 1:500 serum dilution. The figure includes cumulative data from three separate experiments. patients. Thus, the cutoff was set at a higher level. Summary results (Table 2) show that 2 of 96 normal donors have serum antibodies reactive with WT1/full-length protein. 2.5 One of these individuals had Ab to WT1/NH2-terminal protein, I WTl/FulMength and one had Ab to WT1/COOH-terminal protein. WTl-specific Abs in Sera of Patients with AML. The ] WT1/N4ermlnus presence of immunity to WTt in patients with AML was sim- TRX ilarly analyzed in the same experiments. Summary results (Ta- A-- [] R~,2 ble 2) show that 16 of 63 patients (25%) with AML had serum ...... OD . ,ll~ antibodies reactive with WT1/full-length protein. Serum from all of the 16 patients with reactivity to WT1/full-length protein 1.0 l~ also reacted with the WTl/NH2-terminal protein. By marked | contrast, only 2 of 63 patients (3%) had reactivity to the WT1/ COOH-terminal protein. 0 Fig. 1 depicts serum reactivity to WT1/full-length protein WTI80 Normal AML-I AML-2 for each of the 63 individual patients. The WT1/full-length protein was expressed as a fusion protein with Ral2, a foreign Fig. 2 Serum Ab reactivity to WT1 proteins and control proteins in two patients with AML. Reactivity of serum Ab to WT1/full-length, protein. The WTl/NH2-terminal protein was expressed in E. WT1/NH2-terminal, TRX, and Ral2 proteins was evaluated by ELISA coli as a fusion protein with TRX, another foreign protein. To in two patients with AML. The absorbance values depicted were from verify that the patient Ab response was directed against WT1 ELISA using a 1:500 serum dilution. Positive control (WT180) was and not to the Ra12 or the TRX fusion portion of the protein or WTl-specific Ab WT 180. Negative control (Normal) was serum from a normal individual. AML-1 and AML-2 denote serum from two of the possible E. coli contaminant proteins, controls included the individual patients in Fig. 1 with demonstrated Ab reactivity to WT1/ Ral2 and TRX protein alone (Fig. 2). Reactivity against the full-length. The WT1/fulMength protein was expressed as a fusion Ra12, TRX fusion, or possible E. coli contaminant proteins was protein with Ral2. The WT1/NH2-terminal protein was expressed as a minimal, if at all present in all of the positive patients tested, fusion protein with TRX. The control Ral2 and TRX proteins were purified in a similar manner. The results confirm that the serum Ab including the two representative patients depicted. reactivity against the WT1 fi~sion proteins is directed against the WT1 WTl-specific Abs in Sera of Patients with CML. Stud- portions of the protein. ies of immunity to WT1 in patients with CML provided similar results (Table 2). In total, 15 of 81 patients (19%) with CML had serum antibodies reactive with WT1/full-length protein, and all 15 had serum antibodies reactive with the WT1/NH 2 terminus. Discussion Only 3 of 81 patients (4%) had reactivity to the WT1/COOH- The data presented demonstrate that Ab responses to WT1 terminal protein. are detectable in some patients with AML and CML. Sera from Fig. 3 depicts serum reactivity to WT1/full-length protein 144 leukemia patients and 96 normal volunteers were examined for each of the 81 individual patients. Fig. 4 shows a lack of and demonstrated the presence of Ab to WT1 in 25% of AML reactivity to TRX and Ral2 proteins in two representative and 19% of CML patients but only 2% of normal individuals. individual sera to confirm that the patient Ab response was The greater incidence of Ab in leukemia patients provides directed against WT1. strong evidence that immunization to the WT1 protein occurred

1.6 • WTl/Fu|l-length

1,t ~ WT1/N-termin~Ls 1.2 1.2- TRx OD ] Ral2 0.8 0.8

0.4 0.4

O~ ...... Fig. 3 Serum Ab reactivity to WT1 in 81 patients with CML. Reac- WT 180 Normal CML-1 CML-2 tivity of serum Ab to WT1/full-length protein was evaluated by EL1SA in patients with AML. The first and second columns represent the Fig. 4 Serum Ab reactivity to WT1 proteins and control proteins in positive and negative controls, respectively. Commercially obtained two patients with CML. Reactivity of serum Ab to WT1/full-length, WTl-specific Ab WT180 was used for the positive control (column 1). WT 1/NHe-terminal, TRX and Ra 12 proteins was evaluated by ELISA in Serum from a normal individual was used for the negative control two patients with CML. The absorbance values depicted were from (column 2). The next 81 lanes represent results using sera from each ELISA using a 1:500 serum dilution. Positive control (WT180) was individual patient. The absorbance values depicted were from ELISA WT1 specific Ab WT180. Negative control (Normal) was serum from a using a 1:500 serum dilution. The figure includes cumulative data from normal individual. CML-1 and CML-2 denote serum from two of the three separate experiments. individual patients in Fig. 3 with demonstrated Ab reactivity to WT1/ full-length. The WT1/full-length protein was expressed as a fusion protein with Ral2. The WT1/NH2-terminal protein was expressed as a fusion protein with TRX. The control Ral2 and TRX proteins were purified in a similar manner. The results confirm that the serum Ab as a result of patients bearing malignancy that expresses, or at reactivity against the WT1 fusion proteins is directed against the WT1 some time expressed, WT1. portions of the protein. A central problem with targeting oncogenic proteins with cancer vaccines is that the oncogenic proteins are most often "self' proteins. Attempting to elicit immune responses directed be able to elicit an immune response to WT1. Whether that against oncogenic proteins raises the reciprocal problems of immune response can therapeutically impact on the outcome of immunological tolerance and autoimmune toxicity (23). Immu- leukemia will be the essential question. The extent to which nological tolerance to self-proteins can inhibit or dampen the helper T cell and CTL responses can be elicited in humans level of immune response. Reciprocally, if an effective immune should be evaluated. response is elicited, the result might be antoimmune toxicity The WTI protein is a transcription factor that is composed against those tissues that normally express the target protein. of two functional domains: a proline-glutamine-rich domain at The observed Ab responses to WT1 most probably result from the NH 2 terminus, and a zinc finger domain composed of four patients becoming immune to WT1 on their own leukemia cells zinc fingers at the COOH terminus with homology to the EGR1/ and provide direct evidence that WTI can be immunogenic, Spl family of transcription factors (24-26). The majority of the despite being a self-protein. In prior murine experiments elici- Ab detected was reactive with epitopes within the NH 2 termi- tation of an immune response to murine WTI did not result in nus. Only a small subgroup of patients showed a weak Ab autoimmune toxicity, in particular to bone marrow or kidney, response to the COOH terminus. This is consistent with obser- i.e., the normal tissues that express some level of WT1 protein vations in the animal model, where immunization with peptides (3). The current study was not designed to examine WT1 Ab- derived from the NH 2 terminus elicited Ab, helper T cell, and positive patients for autoimmune manifestations. However, au- CTL responses, whereas none of the peptides tested from the toimmune toxicity directed against WTl-positive tissues, such COOH terminus elicited Ab or T-cell responses (3). WTI is a as kidney, is not a commonly recognized syndrome associated self-protein. One possible implication is that the NH 2 terminus with leukemia. Thus, it is unlikely that the observed WT1- is unique and thus more immunogenic. By contrast, the COOH specific antibodies are substantially toxic. terminus is more highly homologous to other self-proteins and is The presence of Ab to WT1 strongly implies that concur- thus less immunogenic, i.e., the subject of a greater degree of rent helper T cell responses are also present in the same patients. immunological tolerance. Of note, the four zinc-finger domains WT 1 is an internal protein. Thus, CTL responses are likely to be within the COOH terminus have high homology to EGR family the most effective in terms of leukemia therapy and the most members (25). Data presented in this study indicate that toler- toxic arm of immunity. The presence of antibodies to WT1 does ance might vary between different portions of a protein, possibly not speak directly to the likelihood of existent CTL responses. dependent upon sequence homologies and functional domains. However, helper T cells, if present, can potentially provide the The current studies did not address important issues con- necessary help for vaccine induction of CTL responses. Thus, cerning possible correlations of Ab responses with levels of these data provide further stimulus to test therapeutic vaccines WT1 gene and protein expression and clinical characteristics directed against WT1 with the expectation that the vaccines will such as French-American-British subcategory and response to

therapy in terms of disease-free or overall survival. The dem- Mannhalter, C., Haas, O. A., Lechner, K., Jaeger, U., and Gaiger, A. onstration of a high incidence of immunity should allow such Prognostic significance of WT1 gene expression at diagnosis in adult de issues to be readily evaluated in future studies. novo acute myeloid leukemia. Leukemia (Baltimore), 11: 639-643, 1997. 14. Gaiger, A., Linnerth, B., Mann, G., Schmid, D., Heinze, G., Tisljar, Acknowledgments K., Haas, O. A., Gadner, H., and Lion, T. Wilms' tumour gene (WT1) We thank T. Deuel and Z. Wang for providing a WT1 full-length expression at diagnosis has no prognostic relevance in childhood acute plasmid. lymphoblastic leukaemia treated by an intensive chemotherapy protocol. Eur. J. Haematol., 63: 86-93, 1999. References 15. Patmasiriwat, P., Fraizer, G., Kantarjian, H., and Saunders, G. F. 1. Ohminami, H., Yasukawa, M., and Fujita, S. HLA class I-restricted WT1 and GATA1 expression in myelodysplastic syndrome and acute lysis of leukemia cells by a CD8(+) cytotoxic T-lymphocyte clone leukemia. Leukemia (Baltimore), 13: 891-900, 1999. specific for WT1 peptide. Blood, 95: 286-293, 2000. 16. Algar, E. M., Khromykh, T., Smith, S. I., Blackburn, D. M., Bryson, 2. Gao, L., Bellantuono, I., Elsaesser, A., Marley, S. B., Gordon, M. Y., G. J., and Smith, P. J. A WT1 antisense oligonucleotide inhibits prolif- and Goodman, J. M. Selective elimination of leukemic CD34+ progen- eration and induces apoptosis in myeloid leukemia cell lines. Oncogene, itor cells by cytotoxic T lymphocytes specific for WT1. Blood, 95: 12: 1005-1014, 1996. 2198-2203, 2000. 17. Yamagami, T., Sugiyama, H., Inoue, K., Ogawa, H., Tatekawa, Y., 3. Gaiger, A., Reese, V., Disis, M. L., and Cheever, M. A. Immunity to Hirata, M., Kudoh, T., Akiyama, T., Murakami, A., Maekawa, T., and WT1 in the animal model and in patients with acute myeloid leukemia. 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Alexander Gaiger, Lauren Carter, Hildegard Greinix, et al.

Clin Cancer Res 2001;7:761s-765s.

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