Academic literature on the topic 'T-Cell WT1 Proteins'

Abstract Abstract 1993 Wilm's tumor protein-1 (WT1) is over-expressed in a number of solid and hematologic malignancies including multiple myeloma (MM). The emergence of WT1-specific T cells has been shown to correlate with better relapse-free survival after allogeneic stem cell transplantation in patients (pts) with hematologic malignancies, such as leukemia. In MM, the expression of WT1 in the bone marrow has been shown to correlate with numerous negative prognostic factors, including disease stage and M protein ratio. Taken together, these findings suggest that immunotherapeutic augmentation of WT1-specific immune responses, such as adoptive transfer of WT1-specific T cells, may be capable of eradicating minimal residual disease and preventing relapse in MM. Thus, we examined the significance of WT1-specific cellular immune responses in pts with relapsed MM and high-risk cytogenetics who are undergoing allogeneic T cell-depleted hematopoietic stem cell transplantation (TCD HSCT). In this study, pts were eligible to receive low doses of donor lymphocyte infusions (DLI, 5×105-1×106 CD3+/kg) no earlier than 5 months post TCD HSCT. WT1-specific T-cell frequencies were measured in freshly isolated peripheral blood and bone marrow specimens. Frequencies were detected by staining for intracellular IFN-γ production in response to WT1 peptides, and/or by tetramer analysis, where available. Of 17 pts evaluated, all pts exhibited low frequencies of WT1-specific T-cell responses pre TCD HSCT. Ten of these pts received DLI post TCD HSCT. All 10 pts developed WT1-specific T cell responses post DLI. These increments in WT1-specific T-cell frequencies were associated with reduction in circulating myeloma proteins in all pts. Long-term evaluation demonstrated fluctuations in persisting WT1-specific T-cell frequencies following DLI. In one representative patient, a peak of 3.5% (72/ml) WT1-specific CD8+ T cells were detected in the peripheral blood by staining with the tetramer HLA-A*0201 RMF. This peak T-cell response occurred post TCD HSCT and DLI, and coincided with disease regression. This patient has remained in complete remission for more than 3 years post transplant, with fluctuating levels of WT1-specific CD8+ T cells ranging from 0.3–1.5% still persisting. Findings from concurrent molecular chimerism studies conducted on isolated T cells post TCD HSCT suggest that the WT1-specific T cells are of donor origin. Immunohistochemical analyses of WT1 and CD138 staining in MM bone marrow specimens demonstrated consistent co-expression within malignant plasma cells. WT1 expression in the bone marrow of all 6 pts tested correlated with the extent of malignant plasma cell infiltration. In contrast, no WT1 expression was observed when disease was low or absent. Taken together, our findings suggest a correlation between the emergence of WT1-specific T cells post DLI, and disease regression in pts being treated for relapsed MM. The present data support the development of adoptive immunotherapeutic approaches utilizing WT1-specific T cells for pts with MM. Disclosures: No relevant conflicts of interest to declare.

Abstract Wilms tumor protein (WT1) is a transcription factor selectively over expressed in several types of leukemia and solid tumors, making it a promising potential target antigen for immunotherapy. Several open clinical trials use native or altered peptide sequences derived from the WT1 protein in order to overcome the weak immunogenicity of the self-antigen. Here we report a new strategy to circumvent tolerance by designing peptides that incorporate non-natural amino acids into the native sequence of WT1 peptides. Starting from the nonamer sequences WT1 187–195 and WT1 235–243, eight peptides containing natural amino acids and nine peptides in which different chemical modifications (fluorination, photo-reactive azido groups or benzophenone groups) were introduced at major histocompatibility complex (MHC) and T cell receptor binding positions, were synthesized. The new non-natural peptides could stabilize MHC class I molecules better than the native sequences and were also able to elicit strong specific T-cell responses. Photo-reactive peptides were additionally modified with biotin handles to allow streptavidin-biotin pull down and western blot analysis of kinetics of binding and catabolism. Upon UV irradiation, these peptides covalently bound to MHC molecules on the live cells; clearance of the peptide-MHC covalent complex occurred over 24 hours, consistent with the T2 thermo-stabilization data for the same peptide. Further catabolic studies may elucidate the important or novel cellular proteins involved in antigenic peptide processing and cross presentation and should aid in vaccine development. We are investigating whether covalent interaction with the MHC may lead to alterations in immune responses as well. T cells stimulated with one of the synthetic peptides (WT1J-W4WF) cross-reacted with the native WT1J sequence and were able to kill WT1 positive HLA-A0201 matched acute lymphoblastic leukemia cell lines. In conclusion, this study shows that peptides with non-natural amino acids can be successfully incorporated into T cell epitopes to provide increased immunogenicity and novel biological information.

Chromosome translocations found in neoplasms often result in the creation of hybrid genes encoding chimeric proteins. Desmoplastic small round cell tumor (DSRCT) is a recently described aggressive malignancy associated with a unique chromosomal translocation t(11;22)(p13; q12). This translocation has recently been characterized, revealing the rearrangement and fusion of the WT1 gene on chromosome 11 to the EWS gene on chromosome 22. Fusion of these two genes results in the production of a putative oncogenic protein composed of the zinc finger DNA-binding domains of WT1 linked to the potential transcriptional regulatory domains of EWS. The typical chimeric transcript consists of the first 7 exons of EWS and the last 3 exons of WT1. We report here the first case of DSRCT with a variant EWS-WT1 chimeric product that includes 9 exons of EWS and 3 exons of WT1.

Abstract Abstract 2599 The Wilms' tumor oncogene protein (WT1) is an intracellular, oncogenic transcription factor that is over-expressed in leukemias and a wide range of cancers. WT1 may be expressed in leukemia stem cells. RMFPNAPYL, “RMF”, a WT1-derived CD8 T cell epitope presented by HLA-A0201, is a validated target for T cell-based immunotherapy, used in multiple clinical trials of vaccines and cellular therapies. Therefore, we hypothesized that a “TCR-like antibody” specific for the WT1 peptide/HLA-A0201 complex might be an effective therapeutic agent. Using phage display technology, we generated 2 lead, high avidity (Kd < 0.2nM), fully human monoclonal antibodies (mAb) specific for the WT1 RMF peptide/HLA-A0201 complex. One version, ESK1, is a native human IgG1. A second version, ESKM, with enhanced antibody dependent human effector cell cytotoxicity (ADCC) function due to altered Fc glycolsylation was also prepared. ESK mAb bind to leukemia lines and other cancer cell lines, as well as primary leukemia cells that are both WT1+ and HLA-A0201+. In vitro, both ESK mAb mediated ADCC against CML cells, at concentrations below 3 ug/ml, but ESKM was about 8–10 fold more potent and could kill targets with far fewer peptide/MHC complexes expressed on the cell surface. At therapeutic doses of ESKM, there was no difference in biodistribution between the 2 mAb in C57BL6 mice or in mice that were transgenic for HLA-A0201. Low doses of ESK (25–100ug twice) effectively treated an established disseminated, ALL or human bcr/abl + lymphoid leukemias in a NSG mouse model (T, B and NK deficient) and prolonged survival. In mice, ESKM was slightly more effective than ESK1. An F(Ab')2 version of the antibody had no anti-tumor effect, indicating that an Fc-mediated mechanism plays the major role in therapy. When combined with a single infusion of human CD34-, CD3-, human NK and monocyte effectors in the NSG mice, therapeutic effects of the mAb were more pronounced and more durable. There was no therapeutic effect of either mAb on WT1 low/A0201 negative disseminated Daudi ALL in mice. There was no observed toxicity in HLA-A0201 transgenic mice at the therapeutic mAb dose and schedule. ESK mAb are potential therapeutic agents for ALL, CML, other leukemias and cancers over-expressing the WT1 oncoprotein. Its expression in early leukemia cells may allow for elimination of the progenitors. The data also provide proof of concept for developing therapeutic mAb targeting important intracellular oncogenic proteins. Disclosures: Yan: Eureka: Employment, Equity Ownership. Liu:Eureka: Employment, Equity Ownership.

Abstract The Wilms’ Tumor 1 (WT1) gene is highly expressed in bone marrow progenitor cells, and is downregulated during the differentiation towards mature blood cells. Several lines of evidence suggest that WT1 plays an important role in leukemia development. WT1 overexpression can be detected in more than 80% of acute leukemia’s and an inverse correlation has been found between the expression levels of WT1 and the overall survival of patients. In addition, in about 9% of AML cases and 3% of ALL cases, WT1 is mutated and the presence of these mutations may have an adverse effect on the survival of the patients. So far, little is known about the biological activities of the wildtype and the WT1-mutant proteins during hematopoiesis and the presence of different isoforms with different biological activities has hampered a clear interpretation of the results so far. In a comprehensive study, we have investigated the function of all four major WT1 isoforms in primary mouse bone marrow cells using in-vitro and in-vivo assays. In addition, we have studied for each isoform the effect of mutations on the biological activity. In-vitro studies: 4 wildtype WT1 isoforms, 6 mutants and an empty vector control were retrovirally transduced into primary murine bone marrow cells. Subsequently, the transduced cells were FACS-sorted and used for various assays. WT1 inhibited the in-vitro colony formation (CFU-GM) by 60–95%, depending on the expressed isoform. In contrast, expression of the corresponding WT1 mutant proteins had no effect on colony formation. To study the underlying mechanism, we cultured the WT1-transduced bone marrow cells and analyzed the cells each day for proliferation (Cell count & DNA histograms), differentiation (Mac1, Gr1) and apoptosis (Annexin V) using FACS analysis. In agreement with the colony assays, the expression of all 4 wildtype WT1 isoforms induced growth arrest and resulted in accelerated differentiation. Target genes: To investigate which genes may be involved in the observed phenotypes, we quantitatively analyzed the expression levels of 34 putative WT1-target genes in the transduced murine bone marrow cells. Briefly, primary mouse bone marrow cells were retrovirally transduced with 4 different wildtype WT1 isoforms, 4 different mutant isoforms or an empty vector control. Sixteen hours after transduction, the transduced cells were FACS-sorted and RNA was extracted for quantitative real-time RT-PCR analysis. We have identified a number of putative WT1-target genes that are differentially regulated by the 4 wildtype WT1 isoforms but not by the WT1 mutant proteins: E-cadherin, syndecan, NGF-receptor, Egr-1, TGF-b, c-Myc, Vitamin D-receptor, insulin-receptor thrombospondin and the taurine transporter. In-vivo studies: To study the effect of WT1 on more immature bone marrow stem cells, we have transplanted WT1-transduced primary mouse CD45.2 bone marrow cells together with empty-vector-transduced primary mouse CD45.1 bone marrow cells into ablatively irradiated syngenic CD45.1/CD45.2 heterozygous mice. Six weeks after transplantation, 5-colour FACS analysis of peripheral blood indicated that the expression of WT1 promotes myeloid differentiation (Mac1 & Gr1) and inhibits the formation of B- (IgM/B220) and T-cells (CD4 & CD8).

Abstract The Wilms’ tumor oncogene protein (WT1) is an intracellular, oncogenic transcription factor that is over-expressed in a wide range of leukemias and solid cancers. RMFPNAPYL (RMF), a WT1-derived CD8 T cell HLA-A0201epitope, is a validated target for T cell-based immunotherapy. We generated a high affinity, fully human IgG1 mAb specific for the RMF/HLA-A0201 complex. The mAb shows potent anti-leukemia activity both in vitro and in vivo in mouse models. Bi-specific T cell engaging antibodies (BiTE) have been used effectively to target cell surface proteins and kill cancers. We have developed a new, potent form of the ESK mAb that is a bi-specific T cell engaging antibody (BiTE) specific for tumor cells coexpressing the intracellular oncoprotein, WT-1 and HLA A0201. ESK-BiTE and an irrelevant control BiTE were constructed with ESK1 scFv or irrelevant ScFv on one arm, and anti-CD3 ScFv fragment as the other arm. The BiTE constructs were expressed in CHO cells. Both the ESK-BiTE and the control BiTE bind human CD3 T cells. The ESK-BiTE selectively bind to leukemia cells that express both WT1 and HLA-A0201. The ESK-BiTE activated human resting T cells and EBV-specific T cells retargeting potent cytotoxicity against WT-1+ HLA A0201+ human leukemia cells in vitro. In an NSG mouse xenograft model, injection of ESK-BiTE (20 ug/ml) twice a week, following I.V. administration of 2x107 human EBV-specific T cells, once a week, significantly inhibited the growth of a previously established disseminated HLA A0201+, WT-1+ human Ph+ ALL, BV173 expressing luciferase, as measured by bioluminescence imaging. In a second NSG mouse model mice injected I.V. with an aggressive human AML, SET-2, on day 0, were treated on day 4 with ESK-Bite for 6 days consecutively at 20 ug/day together with EBV-specific T cells given twice a week. In this setting, the ESK-BiTE and T cells resulted in undetectable leukemic growth for 14 days post-leukemia inoculation, with a minimal tumor burden detected by day 18, while all control groups showed massive increases in leukemia burden by day 14. Mice bearing SET-2 leukemia, that received ESK-BiTE and T cells also showed longer survival and delayed limb paralysis. As expected, the human T cells, which were EBV-specific, did not induce signs of GVHD in mice. Our data provide evidence that ESK-BiTE is a potent and specific therapeutic agent against aggressive human leukemias expressing WT1 and HLA-A0201. This is the first study showing efficacy of a TCR-like BiTE antibody targeting an intracellular tumor antigen expressed at low density. Supported by the Leukemia and Lymphoma Society, NIH R01CA55349 and P01 23766. Disclosures: No relevant conflicts of interest to declare.

Abstract The Wilms’ tumor 1 (WT1) gene encodes a Krupple-like zinc finger transcription factor, which can act as a transcriptional enhancer or repressor. Mutations in WT1 were originally identified in Wilms’ tumor, but are also present in other types of cancer. WT1 has been implicated as tumor suppressor gene when mutated in AML, but as oncogene when aberrantly high expressed. Mutations in WT1 are present in approximately 10% of AML and were recently implicated as an adverse prognostic marker for patients with AML. In AML, WT1 mutations cluster predominantly within exons 7 and 9, and appear as missense mutations or insertions-deletions mutations usually resulting in frameshifts that generate premature termination codons (PTCs). In this study, we analyzed a representative cohort of 262 AML cases by RT-PCR, WAVE dHPLC followed by sequence analyses for mutations in exons 6 to 9. A relatively low number of missense and in-frame insertion mutations were detectable in the WT1 mRNA transcripts in these AML cases (2.7%). Interestingly, we subsequently screened 351 AML cases by applying genomic DNA PCR amplification, WAVE dHPLC followed by sequence analyses of WT1 exon 7 and 9 and detected WT1 mutations in 5.7% of all cases. Mutations were absent in patients older than 60 years. WT1 mutations were present in AML patients normal karyotypes (6.3%), but also in patients carrying chromosomal aberrations, such as inv(16), t(8;21) and t(15;17). Survival analyses in AML patients younger than 60 years did not reveal any significant difference in overall (OS) and event-free survival (EFS) between patients with or without WT1 mutations. These survival analyses were performed on all AML patients, except those with favourable cytogenetics (n = 233) (OS p = 0.09 and EFS p = 0.08) or AML patients with normal karyotypes (n = 134) (OS p = 0.35 and EFS p = 0.25). Although these analyses did not reveal a significant association, a trend for adverse outcome for patients carrying WT1 mutations was apparent. Missense, in-frame insertion and nonsense insertions-deletions WT1 mutations were present in our AML cohort. The majority of the detected WT1 aberrations were insertion-deletion mutations that introduced PTCs. However, by Western blot analyses we showed that the affected AML cases did not express the expected truncated WT1 proteins, whereas full-length WT1 was expressed. Nonsense-mediated RNA decay (NMD) degrades mRNAs bearing PTCs. We hypothesized that the mutant WT1 mRNA transcripts might be sensitive to NMD. The involvement of NMD in the AML cases was confirmed by treatment of primary AML cells carrying out-of-frame WT1 mutations with the translation inhibitor emetine. Treatment with emetine stabilized the mutant WT1 mRNA transcripts to levels similar or even higher than wild-type WT1. In conclusion, AML cases with WT1 missense and in-frame insertion mutations express mutant WT1 mRNA at WT1 wild-type levels. These transcripts most probably encode proteins that affect WT1 function since mutations are located within the WT1 zinc fingers. In addition, mutant WT1 mRNA transcripts that carry PTCs are sensitive to NMD in AML. In these AML cases mutations in WT1 result in haploinsufficiency rather than expression of dominant negative truncated WT1 proteins. Of note, similar mechanisms may also be important in other types of malignancies carrying WT1 mutations.

Abstract Background With the advent of monoclonal antibodies such as rituximab, the duration of remission and overall survival in B-cell non-Hodgkin’s lymphoma has improved significantly. However, new therapeutic approaches are needed for patients with refractory disease. Active immunizations using dendritic cells (DCs) loaded with ideotype proteins or tumor lysates have shown safety with limited efficacy. Wilms’ tumor 1 (WT1) gene is frequently overexpressed in lymphoma and is considered to be one of the ideal tumor antigens for cancer immunotherapy. In the present pilot study, we treated lymphoma patients with WT1 peptide-pulsed DC and examined safety, clinical and immunological responses to the vaccination. Methods 5 HLA-A*24:02(A24)+patients (4 males, 1 female; aged 51-90 years; 2 with diffuse large B cell lymphoma, 1 with mantle cell lymphoma, 1 with follicular lymphoma and 1 with peripheral T cell lymphoma) with relapsed or refractory disease were enrolled in the present study. Autologous DCs were generated from patients' peripheral blood monocytes which were separated by an adherence technique from mononuclear cells collected by apheresis. Monocytes were cultured with GM-CSF and IL-4 followed by maturation with OK432 and PGE2. HLA-A24-restricted, modified 9-mer WT1 peptide-loaded mature DCs were administered intradermally every 2 weeks 5-7 times in combination with OK432. Induction of vaccine-induced T cell responses was monitored by a HLA-tetramer assay and a flow cytometry analysis. Cytotoxicity against WT1 peptide pulsed target cells was tested in a LDH release assay. Results The treatment was well tolerated and none of the patients experienced more than grade 2 adverse events during the treatment period. The most common adverse events were mild, transient erythema at injection sites and low grade fever. Of 5 patients, 1 had CR, 1 had PR, 2 had SD and 1 had PD following one course of the treatment. Survival of patients achieving CR, PR or SD (responder) after one course of DC vaccination was longer than those who did not respond to the treatment. Duration of survival in responders was 56, 32, 24 and 19 months after initiation of therapy. Three responders are still alive in remission. Increase in positivity of WT1-specific CD8+ T cells was observed in responders after one course of treatment; the percentage of HLA-A24 restricted WT1 tetramer positive cells was 0.12±0.12% before vaccination, it increased to 6.82±10.2% after the first course. WT1 tetramer positivity in one of the responding patients who received 3 courses of vaccination increased from 0.06% to 24.5% following one course of vaccination. High level of positivity persisted thereafter (14.6% and 14.6% after second and third course, respectively). Cytotoxic T cells (CTLs) generated by in vitro stimulation with WT1 peptide showed cytotoxic activity in an LDH release assay; percent specific lysis against WT1 peptide-pulsed HLA-A24+ target was 64.8% at effector/target ratio 30 to1. CTL did not lyse irrelevant HIV peptide-pulsed HLA-A24+ target and WT1 peptide-pulsed HLA-A24- target, demonstrating antigen specificity and HLA restriction. Absolute number of lymphocytes, CD3+ T cells, CD4/CD8 ratio and NK cells increased by 1.10±0.38, 2.03±1.78, 1.27±0.36, 1.31±0.74 fold, respectively, following the first course of DC vaccination in responders. On the other hand, absolute number of CD4+CD25+Foxp3+ regulatory T cells (Treg) decreased by 50.7%. Lin-CD33+HLA-DR- myeloid-derived suppressor cells (MDSC) decreased from 17.8%±10.1 to 9.0±2.5% (38.3% reduction). Similarly, monocytic MDSC (CD14+HLA-DRlow/-) and granurocytic MDSC (CD15+CD33+) decreased by 34.4% and 34.0%, respectively, indicating that DC vaccination may contribute to the reversal of immunosuppression by Treg and MDSC. Conclusions The present study demonstrated that a vaccination with WT1 peptide-pulsed DC was well tolerated and no serious adverse event was observed. DC vaccination elicits both innate and acquired cellular immune responses correlated with clinical effects. These results suggest WT1 peptide-pulsed DC vaccination might be a promising novel strategy for the treatment of malignant lymphoma patients with relapsed or refractory disease. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2171 Poster Board II-148 Chronic Myelogenous Leukemia is the prototype of myeloproliferative disorder characterized by a reciprocal chromosomal translocation, involving the chromosomes 9 and 22 —t(9;22)-. The molecular consequence of this translocation is the generation of the Bcr-Abl oncogene that encodes the chimeric Bcr-Abl protein with constitutive tyrosine kinase activity. Its expression in hematopoietic cells induces uncontrolled and growth factor independent cellular proliferation, alteration in cell-cell and cell-matrix adhesion, resistance to apoptosis which altogether are leukemogenesis landmarks. Although it is well established that Bcr-Abl-expressing leukemic cells are highly resistant to apoptotic cell death induced by chemotherapeutic drugs and a number of signaling molecules have been shown to be activated by the Bcr-Abl kinase, the antiapoptotic pathway/s triggered by this oncogene has not been fully understood. The numerous experimental evidences collected in the last years highlight the crucial role played by alternative splicing in the control of apoptosis. Several pre-mRNAs for cell death factors are alternatively spliced, yielding isoforms with opposing functions during programmed cell death. A clear example is Bcl-x transcript which is alternatively spliced to produce the antiapoptotic Bcl-x(L) or the proapoptotic Bcl-x(S). Identical features are shared by another member of the Bcl-2 family, the myeloid cell leukemia-1 (MCL-1) gene which encodes two alternative splicing variants MCL-1S and MCL-1L displaying pro- and anti-apoptotic effects, respectively. CML-derived cell lines overexpress the antiapoptotic protein Bcl-x(L) and their erythroid differentiation is inhibited by Bcl-x(L). The data so far collected indicate that there is an extensive cross-talk among BCL-2 family members by virtue of their protein-protein interactions and the ratio of pro-apoptotic to anti-apoptotic proteins has been shown to be a major detereminant of the cell propensity to undergo apoptosis. Furthermore, it is well established that accelerated and blastic phases of the disease are characterized by deregulated WT1 expression. WT1/KTS- gene encodes for a transcription factor but the WT1/KTS+ isoform has been reported to localize into nuclear speckles, the major sub-nuclear structures enriched pre-messenger RNA and splicing factors. Based on the above premise we started investigating the possibility of an active involvement of Bcr-Abl as candidate regulator of splicing events affecting Bcl-x pre-mRNA. By means of an interactomic approach, based on proteomic strategy using GST-Pull Down assay with an array of SH2 containing proteins, we attempted to gain insight into the role played by adapter molecules and Bcr-Abl in splicing assembling machinery. The data presented aims to demonstrate the presence of quaternary complex involving the SH2-SH3 containing adapter protein Nck-beta, the oncogenic tyrosine kinase Bcr-Abl, the RNA binding protein Sam68, the spliceosome ribonucleprotein hnRNPA1 and WT1. The experimental evidences we have collected support the hypothesis of an Imatinib-dependent interaction occurring between Nck-beta and Bcr-Abl. Furthermore, Pull Down experiments indicate an intermolecular interaction between Nck-beta, Sam68, and hnRNPA1 supporting the idea of a novel complex Bcr-Abl/Nck-beta/Sam68/hnRNPA1/WT1. Biochemical analysis carried-out by Pull-Down experiments has been further corroborated by immunofluorescence staining. RNA Pull Down assay suggest that the quaternary complex Nck-beta/Sam68/hnRNPA1/Bcr-Abl/WT1 might modulates splicing process of Bcl-x gene, whose function has been recently described as crucial in myeloproliferative disorders. Astoningshly, the data collected so far indicates that other mRNAs are pulled-down together with the quaternary complex. Taken together these results represent the first experimental evidences showing an interaction between the oncogene Bcr-Abl and Sam-68 leading to speculate a novel putative role played by Bcr-Abl in the intriguing and complex pre-mRNA splicing scenario. Disclosures: No relevant conflicts of interest to declare.

Abstract Introduction: Acute myeloid leukemia (AML) and other hematological malignancies that constitutively express MHC class II molecules represent the ideal target for leukemia-specific CD4+ T helper (Th) cells. CD4+ Th cells are central to the functioning of the immune system. They regulate adaptive immunity against infections and drive pathogenic responses in many autoimmune diseases. Our preclinical studies indicate that adoptively transferred antigen specific Th cells efficiently eradicate even advanced tumors in mice, showing functional advantage over their better characterized classical CD8+ cytotoxic counterparts. Unfortunately, the production of human class II-restricted Th cells is complex and challenging. Consequently, the clinical activity of tumor-specific Th cells has not been systematically evaluated and very little is known about their therapeutic potential in humans. Striking evidence of their enormous power comes from some anecdotal clinical reports of complete regression of metastatic cancers upon transfer of antigen-specific Th cells. Methods and Results: We investigated a reliable GMP-compatible method for in vitro expansion of antigen-specific CD4+ T cells targeting common leukemia associated antigens (LAAs) Willm's Tumor antigen 1 (WT1) and Preferentially Expressed Antigen in Melanoma (PRAME) for future adoptive immunotherapy in the setting of allogeneic stem cell transplantation (SCT). We hypothesized that the naïve CD4+ T cell compartment, rather than the bulk Th cell population could be a superior source for generating a tumor antigen specific cell product. To test this hypothesis bulk and naïve CD4+ T cells were isolated from the peripheral blood of normal donors by magnetic bead separation. Purified T cells were stimulated in vitro with autologous dendritic cells (DCs) pulsed with overlapping 15 amino-acid long peptide (pepmixes) spanning the length of both proteins. Two rounds of stimulation were performed in presence of IL-7, IL-15 and later +/- addition of IL-1, IL-6 and IL-23. IL-2 in low concentration was supplemented during the second round of stimulation. At the end of the second expansion the cells were tested for reactivity by intracellular cytokine production using flow cytometry (FACS) upon stimulation with cognate LAAs or irrelevant control pepmixes. In T cell cultures derived from naïve CD4+ T cells we observed robust induction of PRAME reactivity from majority of tested normal donors, while reactivity against WT1 was donor dependent. The frequency of LAAs in bulk CD4+ T cells was significantly lower in all cases, supporting the notion that pre-exiting memory Th cells have a competitive advantage over the LAA-specific precursor. T cell cultures supplemented with inflammatory cytokines demonstrated further enhancement of antigenic reactivity. Next we tested if LAA pepmix -stimulated T cells can recognize tumor targets. Naïve-derived PRAME and WT1 Th cells generated from normal SCT sibling donors produced IFN-γ, IL-2 and TNF-α upon exposure to fully HLA-matched AML blasts while no reactivity was seen in control CMV pp65-specific Th cells from the same donors. This observation suggests that LAA-specific CD4+ T cells induced with pepmixes have the ability to recognize physiologically-relevant tumor antigens. Conclusions: Here we report the feasibility of generating naïve-derived anti-leukemia CD4+ T cells from majority of normal donors. Removal of competing memory Th cells unmasks the LAA-specific reactivity, thus improving the reliability of the process. Importantly, these Th cells demonstrate highly-specific recognition of the tumor epitopes naturally processed by HLA-matched leukemic blasts, establishing the foundation for a future adoptive immunotherapy clinical trial in patients with hematological malignancy. Disclosures No relevant conflicts of interest to declare.

Wilms tumor protein 1 (WT1) is a suitable target to develop an immunotherapeutic approach against high risk acute myeloid leukemia (AML), particularly their relapse after allogeneic hematopoietic stem cell transplantation (HSCT). As an intracellular protein traversing between nucleus and cytoplasm, recombinant expression of WT1 is difficult. Therefore, an induction of WT1-specific T-cell responses is mostly based on peptide vaccination as well as dendritic cell (DC) electroporation with mRNA encoding full-length protein to mount WT1-derived peptide variations presented to T cells. Alternatively, the WT1 peptide presentation could be broadened by forcing receptor-mediated endocytosis of DCs. In this study, antibody fusion proteins consisting of an antibody specific to the human DEC205 endocytic receptor and various fragments of WT1 (anti-hDEC205-WT1) were generated for a potential DC-targeted recombinant WT1 vaccine. Anti-hDEC205-WT1 antibody fusion proteins containing full-length or major parts of WT1 were not efficiently expressed and secreted due to their poor solubility and secretory capacity. However, small fragment-containing variants: anti-hDEC205-WT110-35, anti-hDEC205-WT191-138, anti-hDEC205-WT1223-273, and anti-hDEC205-WT1324-371 were obtained in good yields. Since three of these fusion proteins contain the most of the known immunogenic epitopes in their sequences, the anti-hDEC205-WT191-138, anti-hDEC205-WT1223-273, and anti-hDEC205-WT1324-371 were tested for their T-cell stimulatory capacities. Mature monocyte-derived DCs loaded with anti-hDEC205-WT191-138 could induce ex vivo T-cell responses in 12 of 16 blood samples collected from either healthy or HSC transplanted individuals compared to included controls (P < 0.01). Furthermore, these T cells could kill WT1-overexpressing THP-1 leukemia cells in vitro after expansion. In conclusion, alongside proving the difficulty in expression and purification of intracellular WT1 as a vaccine protein, our results from this work introduce an alternative therapeutic vaccine approach to improve an anti-leukemia immune response in the context of allogeneic HSCT and potentially beyond
Für die Entwicklung eines immuntherapeutischen Ansatzes zur Behandlung von hoch Risikopatienten mit akuter myeloischer Leukämie (AML) und insbesondere zur Vorbeugung von Rezidiven nach allogener Stammzelltransplantation (SZT) stellt das Wilms-Tumor-Protein 1 (WT1) ein geeignetes Angriffsziel dar. Die rekombinante Expression von WT1, welches als Transkriptionsfaktor vom Zytosol in den Zellkern transloziert, gestaltet sich äußerst schwierig. WT1-spezifische T-Zellantworten werden daher hauptsächlich mittels Peptidvakzinierung oder Transfektion dendritischer Zellen (DC) mit mRNA, welche das vollständige WT1-Protein kodiert, herbeigeführt. Letzterer Ansatz bietet den Vorteil, dass passierenden T-Zellen eine größere Vielfalt an WT1-Peptidvarianten präsentiert werden kann. Eine verbesserte Peptidpräsentation kann außerdem über eine Optimierung der Rezeptor-vermittelten Endozytose der DCs erzielt werden. Ziel der folgenden Arbeit war es, ein rekombinantes DC-gerichtetes WT1-Vakzin zu entwickeln. Dazu wurden anti-hDEC205-WT1-Fusionsproteine, bestehend aus einem Antikörper gegen den humanen DEC205-Endozytoserezeptor und verschiedenen WT1-Fragmenten, konstruiert. Während sich Fusionsproteine, die das vollständige WT1-Protein oder große Teile dessen beinhalteten, aufgrund ihrer schlechten Löslichkeit und schwachen Sekretion kaum exprimieren und aufreinigen ließen, lieferte die Produktion der Fusionsproteine mit kürzeren WT1-Fragmenten, anti-hDEC205-WT110-35, anti-hDEC205-WT191-138, anti-hDEC205-WT1223-273 und anti-hDEC205-WT1324-371, sehr gute Ausbeuten. Da letztere drei Proteine die meisten bislang bekannten immunogenen WT1-Peptide in ihrer Sequenz enthalten, wurde anschließend ihre Fähigkeit zur T-Zellstimulation untersucht. Dabei konnte in 12 von 16 Blutproben, die entweder von gesunden Spendern oder SZT-Patienten stammten, gezeigt werden, dass mit anti-hDEC205-WT191-138 beladene, reife, aus Monozyten generierte DCs ex vivo signifikant stärkere T-Zellantworten auslösen als die jeweils mitgeführten Kontrollen (P < 0.01). Nach Expansion waren die so aktivierten WT1-spezifischen T-Zellen sogar in der Lage, die WT1-überexprimierende AML-Zelllinie THP-1 in vitro zu lysieren. In der vorliegenden Arbeit konnten daher nicht nur die bereits bekannten Schwierigkeiten der WT1-Expression und Aufreinigung bestätigt werden, sondern darüber hinaus konnte eine alternative therapeutische Vakzinierungsmethode zur Optimierung der anti-leukämischen Immunantwort im Rahmen einer allogenen SZT entwickelt werden