WT1 Full Length Protein Vaccination Shows High Immunogenicity and In Vivo Anti-Tumour Activity.
Authors: Cilloni Daniela , Defilippi Ilaria , Nicoli Paolo , Bracco Enrico , Reviglione Raffaella , Roetto Antonella , Bondi Alessandro , Di Giovanni Daniela , Decklic Erika , Girola Elisa , Pellegrino Rosa Maria , Arruga Francesca , Messa Francesca , Saglio Giuseppe 
Published: October 2019

Abstract
The Wilms’ tumor gene is overexpressed in many types of haematological malignancies including acute myeloid leukaemia (AML), acute lymphoid leukaemia (ALL), chronic myeloid leukaemia (CML) and Ph negative myeloproliferative disorders. WT1 holds great promise for immunotherapy of leukaemia since it is expressed at high levels in blast cells but not in normal tissues, it is involved in the maintenance of malignant phenotype and it is spontaneously immunogenic. Many clinical trials using MHC class I-restricted WT1 peptide, have been recently performed in patients affected by AML, MDS, lung and breast cancer with satisfactory clinical results and without relevant toxicity. The first aim of the study was to analyze the natural immunity against WT1 in different types of haematological malignancies and the second was to set up a vaccination approach in the mouse model using the WT1 full length protein and to test the safety and efficacy of this vaccine. Using a dot blot technique, we analyzed for the presence of WT1 specific antibodies 107 patients including: 18 AML, 20 MDS, 22 Multiple Myeloma, 14 CML, 12 Idiopatic Myelofibrosis (IM), 6 CMML, and 15 ALL and 30 healthy subjects as control. For the second aim, we purified the WT1 full length protein. Complete coding sequence was cloned in a pGEX bacterial expression vector for the production of the fusion protein GST-WT1 that was subsequently purified by glutathione conjugated beads. Twenty C57BL7/6 mice were immunized with 50 μg of purified WT1 protein and 50 μL of Freund adjuvant every 15 days, for a total of 3 immunizations, 20 mice were injected with GST and adjuvant and 20 with PBS alone as control. 2 weeks after the last administration, 10 vaccinated mice and 10 controls have been injected with 200.000 TRAMP-C2 cells. Ten mice for each group were sacrificed and lymphocytes, cultured in presence of IL-2 to perform a cytotoxicity assay. Antibodies against WT1 have been evaluated. Toxicity on normal tissues has been rouled out using the histochemical analysis of different tissues.
Results: Dot blot analysis demonstrated the presence of significant levels of WT1 IgG antibodies in 81% and IgM in 76% of the patients but not in normal subjects so demonstrating that WT1 is highly immunogenic. Particularly 88% of AML, 85% of MDS, 63% of MM, 100% of CML, 83% of IF and 66% of CMML presented high levels of antibodies. After mice vaccination, no organ toxicity has been observed. Importantly, immunized mice showed high levels of both, IgM and IgG antibodies with a mean value of luminescence of 690000 in WT1 injected mice vs 7500 in control mice and high levels of CTLs against WT1 as compared to mice injected with PBS or GST alone. Importantly, in control mice the mean size of the tumor was 1,6±0,4 cm, by contrast in vaccinated mice the tumor was unable to develop. Data obtained from this study clearly demonstrated that WT1 is immunogenic. The full length protein vaccination approach is able to elicit both, umoral and cytotoxic response and it results in a significant antitumor effect in vivo. Although further studies are required to compare the efficacy of this approach to the peptide based vaccine, WT1 full length protein could represent a valid vaccination strategy allowing to overcome the HLA restriction limit of the peptide approach.